Heat Shock Protein 70 and the Relationship to Glutamine in the Critically Ill Child

Home , C7orf25, MLSTD2, SLC2A11, SLC2A14, SSBP4, Translocator protein

Heat Shock Protein 70 and the relationship to glutamine in the critically ill child

Luise Marino BSc (Hons) Cat. & App. Nutrition, Post Grad Diploma Dietetics, MMed Sci Nutrition Paediatric Research Dietitian Section of Paediatrics Department of Infectious Diseases Faculty of Medicine Imperial College London St. Mary’s Campus Norfolk Place London

Dedication

To my angel, Zarah

Declaration

In accordance with Imperial College London regulations, I declare that all work presented in this thesis has been undertaken by me. Technical and analytical help that I received from my colleagues and collaborators is listed in the acknowledgements section.

Luise Marino July 2012

Abstract

Abstract Background: Specific nutrients such as glutamine have an important role in the management of critically ill patients, modulating the host response to stress, improving outcome. The mechanisms of action of glutamine are thought to be through the release of heat shock protein 70 (HSP70), which helps orchestrate an appropriate inflammatory response to critical illness. HSP70 release stimulates immune activation, maintenance of cell homeostasis, cellular protection, preventing apoptotic cell death and is associated with increased survival following severe trauma. However, much of the work considering the use of glutamine and HSP70 in critical illness has been completed in adults. The focus of the current research was therefore to investigate HSP70 release and its relationship to glutamine and inflammatory markers in healthy adults and paediatric volunteers using a whole blood endotoxin model. In addition to this, describing relationships between glutamine, HSP70 and inflammatory mediators, in a cohort of critically ill children.

Methodology: An in vitro whole blood endotoxin stimulation model using lipopolysaccharide (LPS) was adapted for use in healthy adult (n=18) and paediatric volunteers (n=25) to investigate the effect of glutamine supplementation on the release of HSP70 and inflammatory mediators (IL-6, IL-8, IL-1β, IL-10, TNF-α). Plasma from children with acute meningococcal disease (MD) (n=143) and during convalescence (n=78) was analysed for levels of HSP70, inflammatory cytokines (IL-6, IL-8, IL-10, TNF-α) and glutamine. Following a clinical folder review, transcriptomic analysis of differential gene expression between acute disease and convalescence was carried out in a subset of children.

Results: A whole blood stimulation model was effective in measuring the inflammatory response to endotoxin in vitro over an incubation period of 4 to 24 hours. Glutamine supplementation significantly increased HSP70 expression over time in healthy adult and paediatric volunteers compared to unsupplemented controls (p<0.05). HSP70 levels in vitro rose over time and was correlated with inflammatory mediators (IL-1β, TNF-α, IL-8) in adults, and IL-6, TNF-α in paediatric samples at 4 hours, following glutamine supplementation. There was no relationship between HSP70 and IL-10 in either adults or children. Glutamine supplementation significantly attenuated the release of IL-8 at 24 hours (p<0.05) and in the paediatric in vitro endotoxin model significantly increased TNF-α release at 4 hours

(p<0.005). Although there were no other significant differences in the release of inflammatory mediators (IL 6, TNF-α, IL10) between conditions, glutamine supplementation appeared to attenuate the release of inflammatory mediators in adults in contrast to promoting the release of inflammatory mediators in children.

In patients with MD, HSP70 was significantly increased (p=<0.0001) in the acute phase [26.7ng/ml; SD± 79.95 (median 5.7ng/ml; range 0.09 – 600.5ng/ml)] vs. convalescence [mean 3.16ng/ml ±SD; 5.67 (median 1.045ng/ml range 0.001 – 37.31ng/ml)]. Mean glutamine levels during the acute phase were 0.31mmol/l; ±SD 0.13 (median 0.31mmol/l; range 0.50 – 0.64) and for convalescence 0.40mmol/l; ±SD 0.14 (median 0.40mmol/l; range 0.72 – 0.64). During the acute phase of illness, glutamine levels were 52% below the normal range, (using 0.6mmol/l as the lower reference range). In convalescent samples, taken on average 55 days later (35 – 135 days), glutamine levels were found to be 26% below normal range. Results from the transcriptomic analysis show that genes associated with HSP70 and inflammatory signalling pathways, glycolysis and gluconeogenesis are significantly upregulated and those relating to adaptive immunity are downregulated during the acute phase of meningococcal disease compared to convalescence. There were numerous correlations between plasma glutamine and HSP70 with genes associated with p38MAPK signalling pathway during the acute phase of meningococcal disease, suggesting that glutamine and HSP70 could mediate the inflammatory response to infection.

Conclusion: Glutamine supplementation increased the release of HSP70 in an in vitro endotoxin model in children and adults. Children with MD during the acute phase of their illness have significant glutamine depletion, occurring early during the course of the disease, with a concomitant increase in plasma levels of HSP70 and inflammatory mediators. A transcriptomic analysis of differential gene expression showed that genes associated with HSP70 (HSPA1A and HSPA1B) were differentially upregulated during the acute phase of illness. HSP70 genes and plasma levels of HSP70 were correlated, which was suggestive of a role in meningococcal disease. Relationships were found with plasma levels of HSP70 and glutamine and genes associated with p38MAPK signalling pathway during the acute phase of meningococcal disease, suggesting that glutamine and HSP70 could mediate the inflammatory response to infection. Future work would aim to understand the effect of

6 glutamine supplementation on gene expression during critical illness, especially relative to the differential expression of genes associated with HSP70, inflammatory signalling pathways and those relating to the antigen presentation pathway.

Acknowledgements I would like to thank the Laventis Trust for providing the funding for this research project, which I hope will lead to the use of novel nutrition therapy as part of the medical management of critically ill children in the future.

To my supervisor, Dr. Parviz Habibi for providing this amazing opportunity, along with supervisors Dr. Nazima Pathan and Dr. Rosan Meyer for their support, encouragement and guidance in the completion of this research project.

Professor Michael Levin for generously providing access to samples from the meningococcal disease biobank.

Dr. Victoria Wright for her assistance with laboratory techniques and related technical advice for the transcriptomic analysis of the meningococcal disease database.

Professor Paul Langford for providing excellent academic mentorship and pragmatic advice.

Dr Margaret Hancock and her team, Charing Cross Hospital, for their expertise and assistance in analysing the plasma glutamine samples.

Yvonne Clements, Meso Scale Discovery with regards to training for MSD multiplex assays.

Statistical Support Services at Imperial College for their assistance in the statistical analysis plan for the data arising from this project.

To the healthy adult volunteers and St Mary’s Hospital Imperial NHS Healthcare Trust patients for partaking in this study.

To my family and friends for their unwavering support and faith, but more especially to my wonderful daughter for her patience and understanding beyond her years. Finally, in memoriam to Professor Joe Ireland, Paediatric Gastroenterology, Red Cross War Memorial Children’s Hospital, Cape Town for being an inspirational mentor.

Table of Contents Abstract 5 Acknowledgements 8 Table of contents 9 List of Tables 18 List of Figures 20 Abbreviations 24 References 166 Appendices 194

Chapter 1: Introduction 26 1.1. Epidemiology and health economic burden of sepsis 27 1.2 Definition of SIRS, sepsis and septic shock 28 1.3 Causative pathogens 29 1.4 Pathophysiology of sepsis 30 1.5 Infectious process 31 1.6 Innate immune response during sepsis 32 1.7 Cytokine release 34 1.7.1 IL – 6 35 1.7.2 TNF-α 36 1.7.3 IL-8 36 1.7.4 IL-10 36 1.7.5 Cytokine release in N. meningitidis 37 1.7.6 Cytokine pathogenesis 38 1.8 HSP70 and the relationship to the immune and inflammatory response 38 1.8.1 Clinical consequences of upregulated HSP70 release 42 1.8.2 Upregulation and release of HSP70 into the extracellular milieu 43 1.8.3 The effect of the “model” on the inflammatory response 44 mediated by HSP70 1.9 Glutamine and the upregulation of the heat shock response in sepsis 47 1.9.1 Glutamine requirements in infancy 51 1.9.2 Glutamine and anabolism 51

1.9.3 Enteral vs. parenteral administration of glutamine 51 1.9.4 Use of glutamine in children 54 1.9.5 Mechanisms for glutamine supplementation on HSP70 expression 56 1.10 Novel therapeutic strategies in sepsis 57 1.11 Conclusion 59 1.12 Hypothesis 61 1.13 Research Aims 61

Chapter 2: Materials and Methods 63 2.1 Ethical consideration 64 2.1.1 In vitro experiments 64 2.1.2 Children with meningococcal disease 64 2.2 In vitro experiments 64 2.2.1 Optimisation phase 65 2.2.2 Experiment 1: To examine the effects of endotoxin and glutamine 66 using a whole blood model on HSP70 expression in healthy adult volunteers and its association with markers of inflammatory response in vitro 2.2.3 Experiment 2: To examine the effects of endotoxin, glutamine and 67 a HSP70 inhibitor KNK437, on HSP70 expression, using a whole blood model, in healthy adult volunteers and its association with markers of inflammatory response in vitro 2.2.4 Experiment 3: To examine the effects of endotoxin and glutamine 68 on HSP70 expression, in whole blood obtained from healthy paediatric volunteers and its association with markers of inflammatory response in vitro 2.2.5 Experiment 4: To examine the relationship between clinical 68 variables, plasma HSP70, glutamine and inflammatory mediators in vivo and genome-wide transcriptional profiles in children with acute meningococcal disease compared to convalescence. 2.2.5.1 Study population 69 2.3 Techniques for protein measurement, clinical variable and transcriptomic 70

Analysis 2.3.1 Method a: Analysis of levels of inflammatory mediators by 70 multiplex ELISA (Meso Scale Discovery) multiplex assay kit 2.3.2 Method b: Measurement of HSP70 Stressgen EKS-715 High 71 Sensitivity ELISA Kit 2.3.3 Method c: Analysis of plasma glutamine 72 2.3.4 Method d: Transcriptomic analysis 73 2.3.4.1 Study population 73 2.3.4.2 RNA extraction, amplification and labelling 73 2.3.4.3 Data analysis 74 2.3.4.4 GeneSpring – normalisation and filtering 74 2.3.4.5 Ingenuity Pathway Analysis 74 2.3.4.6 Internal and external validation of transcriptomic analysis 75 2.3.5 Methods e: Analysis of clinical variables from folder review 75 2.4 Data analysis 76 2.4.1 Statistical analysis plan for the in vitro model of sepsis 76 examining the effect of glutamine on the release of HSP70 and inflammatory mediators 2.4.2 Statistical analysis plan to examine relationships between 77 HSP70, glutamine and inflammation in children with acute meningococcal disease compared to convalescence 2.4.3 Statistical plan for analysis of gene expression of HSP70, 78 glutamine and pathways associated with inflammation during acute and convalescence in children with meningococcal disease

Chapter 3: In vitro models to consider the effect of glutamine 79 supplementation on endotoxin stimulated release of HSP70 and inflammatory mediators 3.1 Introduction 80 3.2 HSP70 activation of inflammatory signalling pathways 80 3.3 Inhibition of heat shock protein 70 and the effect of inflammatory 80 Mediators 3.4 The effect of glutamine on inflammatory mediators via HSP70 release 81 3.5 Aims 83 3.6 Results 83 3.6 Optimisation of in vitro HSP70 release with glutamine supplementation in 83 endotoxin stimulated whole blood 3.7 An in vitro whole blood endotoxin stimulation assay to investigate the 84 relationship between glutamine, HSP70 and inflammatory mediators 3.7.1 Glutamine supplementation promotes HSP70 release in 84 endotoxin stimulated whole blood from healthy adult volunteers at 4 hours and 24 hours 3.7.2 Glutamine supplementation in endotoxin stimulated whole 85 blood appears to attenuate the release of inflammatory cytokines 3.7.3 Glutamine supplementation in endotoxin stimulated whole 85 blood from healthy adult volunteers appears to attenuate the release of IL-1β 3.7.4 Glutamine supplementation in endotoxin stimulated whole 86 blood from healthy adult volunteers attenuates the release of IL-8 3.7.5 Glutamine supplementation in endotoxin stimulated whole 87 blood from healthy adult volunteers attenuates the release of TNF-α 3.7.6 Glutamine supplementation in endotoxin stimulated whole 88 blood from healthy adult volunteers attenuates the release of

IL-6 3.7.7 Glutamine supplementation in endotoxin stimulated whole 89 blood from healthy adult volunteers attenuates the release of IL-10 3.7.8 Relationships between HSP70 and inflammatory mediators in 89 adult in vitro endotoxin stimulated whole blood following glutamine supplementation (2mM) 3.7.9 Summary of results for glutamine supplementation (2mM) in 90 endotoxin stimulation model and the effect on the release of HSP70 and inflammatory mediators 3.8. Results of the in vitro model examining the inhibitory effects of KNK437 on 91 HSP70 release and markers of the inflammatory responses following endotoxin stimulation and glutamine supplementation (2mM) in healthy adult volunteers 3.8.1 With the addition of KNK437 to endotoxin stimulated whole 91 blood the significant effect of glutamine supplementation (2mM) on extracellular HSP70 release at 24 hours is abrogated 3.8.2 The effect on glutamine supplementation (2mM) on 92 intracellular HSP70 release at 4 hours and 24 hours in endotoxin stimulated whole blood is abrogated with the use of KNK437 3.8.3 The addition of KNK437 diminishes the release of inflammatory 93 mediator in endotoxin stimulated whole blood 3.8.4 KNK437 attenuates the release of extracellular IL-6 in endotoxin 93 stimulated whole blood 3.8.5 KNK437 attenuates the release of intracellular IL-6 in endotoxin 94 stimulated whole blood 3.8.6 KNK437 attenuates the release of extracellular TNF-α in 95 endotoxin stimulated whole blood 3.8.7 KNK437 attenuates the intracellular release of TNF-α in 96 endotoxin stimulated whole blood

3.8.8 KNK437 attenuates the release of extracellular IL-10 in 97 endotoxin stimulated whole blood 3.8.9 KNK437 attenuates the release of intracellular IL-10 in 98 endotoxin stimulated whole blood 3.8.10 Summary of KNK437 results from in vitro adult endotoxin 98 stimulation model 3.9 An in vitro model examining the effects of endotoxin and glutamine on 99 HSP70 release and markers of the inflammatory responses in healthy paediatric volunteers 3.9.1 Glutamine supplementation upregulates HSP70 release in 99 endotoxin stimulated whole blood from healthy paediatric volunteers 3.9.2 The effect of glutamine supplementation on release of 100 inflammatory cytokines using endotoxin stimulated whole blood from healthy paediatric volunteers 3.9.3 The release of IL-10 in endotoxin stimulated whole blood from 100 healthy paediatric volunteers was not influenced by glutamine supplementation (2mM) 3.9.4 The release of IL-6 in endotoxin stimulated whole blood from 101 healthy paediatric volunteers was not influenced by glutamine supplementation (2mM) 3.9.5 The release of TNF-α in endotoxin stimulated whole blood from 102 healthy paediatric volunteers was augmented with glutamine supplementation (2mM) 3.9.6 Relationships between HSP70 and inflammatory mediators in 103 paediatric in vitro endotoxin stimulated whole blood following glutamine supplementation (2mM) 3.9.7 Summary of experimental results of in vitro paediatric 103 endotoxin stimulation model 3.10 Comparison between HSP70 in a whole blood endotoxin stimulation 104 model with healthy paediatric volunteers and healthy adult volunteers

3.10.1 Comparison between inflammatory mediator (IL-6, IL-10 and 104 TNF-α) release in a whole blood endotoxin stimulation model with healthy paediatric volunteers and healthy adult volunteers 3.11 Discussion 105 3.12 Conclusion 110 Chapter 4: Heat shock protein 70, glutamine and inflammatory markers during 112 the course of meningococcal disease and the relationship to clinical indices 4.1 Introduction 113 4.2 Glutamine and critical illness 113 4.3 Aims 114 4.4 Results 115 4.4.1 Patient demographics 115 4.4.2 Nutrition Support 117 4.5 Results of the effect of freeze thaw cycles on glutamine stability 118 4.6.1 Plasma glutamine levels in children with acute meningococcal 119 disease compared to convalescence 4.6.2 HSP70 levels in critically ill children with meningococcal disease 120 during the acute phase of their illness compared to convalescence 4.6.3 Inflammatory mediators in children with acute meningococcal 120 disease compared to convalescence 4.6.4 Relationships between HSP70, glutamine and inflammatory 121 Mediators 4.6.5 Relationships with HSP70 levels and clinical indices in 121 meningococcal disease cases during the acute phase of illness 4.6.6 Relationships with glutamine and clinical measures in 122 meningococcal disease cases during the acute phase of illness 4.6.7 Relationships within clinical variables of children with acute 122 meningococcal disease 4.6.8 Factors predicting the use of steroid administration in children 123 with acute meningococcal disease 4.8 Discussion 124

4.10 Conclusions 129 Chapter 5: Transcriptomic profiling during the acute phase of meningococcal 130 disease and the associations with HSP70, glutamine and inflammatory mediators 5.1 Introduction 131 5.2 Endotoxin induced activation of inflammatory signalling pathways 131 5.3 HSP70 release and the regulation of MAPK and NF-κB signalling pathways 132 5.4 Glutamine supplementation and the regulation of MAPK and NF-κB 133 signalling pathways 5.5 Glutamine and HLA expression during critical illness 134 5.6 Aims 134 5.7 Results 135 5.8.1 Immune pathways were dysregulated during the acute phase of 136 meningococcal disease adaptive compared to convalescence 5.8.2 Comparison of signalling pathways of SDE upregulated and 138 downregulated genes 5.8.3 Upregulated genes, during the acute phase of meningococcal 138 disease, were associated with pathways associated with cell to cell signalling and activation of the inflammatory response 5.8.4 Downregulated genes, during the acute phase of meningococcal 139 disease, were associated with apoptosis 5.8.5 Summary of the results from signalling pathway analysis 140 5.9.1 Gene network analysis of dataset with 1,780 SDE transcripts 140 focused on HSP70 in acute meningococcal disease compared to convalescence and the role of HSP70 5.9.2 Gene network analysis focused on 933 upregulated SDE 142 transcripts in acute meningococcal disease compared to convalescence and the role of HSP70 5.9.3 Gene network analysis focused on 492 downregulated SDE 143 transcripts in acute meningococcal disease compared to convalescence and the role of HSP70

5.9.4 Summary of network analysis considering the differential 144 expression of HSP70 in acute meningococcal disease compared to convalescence 5.10.1 Relationships between protein levels of HSP70, glutamine, 145 inflammatory mediators (IL-8, IL-6, IL-1β and TNF-α) and SDE transcripts associated with inflammatory and immune functions 5.10.2 Plasma protein levels of HSP70 are positively correlated with 147 genes associated with HSP70 during the acute phase of meningococcal disease 5.11.1 Internal validation of SDE genes, using ToppGene show signalling 149 pathways represent dysregulated adaptive immune function, the antigen presentation pathway and inflammatory signalling pathways in acute meningococcal disease compared to convalescence 5.11.2 External validation of SDE genes represented in signalling 149 pathways represent dysregulated adaptive immune function, the antigen presentation pathway and inflammatory signalling pathways in acute meningococcal disease compared to convalescence 5.12 Discussion 150 5.13 Conclusion 157

Chapter 6: General Discussion and Conclusions 159 6.1 Discussion 160 6.2 Conclusion 165

7: References 166 7.1 References 167

List of Tables Chapter 1: Table 1.1: Definitions of paediatric SIRS, infection, sepsis and septic shock 28 Table 1.2: Principle pathogens in paediatric sepsis 30 Table 1.3: Members of the HSP70 family 40 Table 1.4: Heat shock protein 70 expression in health and acute, chronic and 42 autoimmune disease Table 1.5: Organ specific function of glutamine therapy in critical illness 54 Table 1.6: Clinical trials of therapeutic agents used to mediate sepsis 58 Chapter 2: Table 2.1: Mechanistic steps for cation exchange chromatography system using 72 Ninhydrin Chapter 3: Table 3.1 Relationships between adult in vitro endotoxin stimulated HSP70 90 release and inflammatory mediators following glutamine supplementation (2mM) Table 3.2 Relationships between paediatric in vitro endotoxin stimulated HSP70 103 release and inflammatory mediators following glutamine supplementation (2mM) Table 3.3: A comparison of HSP70 release in adult and paediatric samples 104 Chapter 4: Table 4.1: Patient demographics and PICU support of children with acute 115 meningococcal disease Table 4.2: Patient demographics and clinical values of children with acute 116 meningococcal disease Table 4.3: Feeding characteristics with glutamine intake and plasma levels 117 Table 4.4: Nutrition Support, Route & Complications 117

Table 4.5: Factors predicting the use of steroid administration in children with 123 acute meningococcal disease Chapter 5: Table 5.1: Protein levels of HSP70, glutamine and inflammatory mediators in a 135 18

sub-cohort of children with acute meningococcal disease and during convalescence Table 5.2: SDE gene transcripts associated with p38MAPK pathway in children 146 with acute meningococcal disease compared to convalescence Table 5.3: Correlations between protein levels and gene transcripts related to 148 inflammation and immune function during the acute phase of meningococcal disease and convalescence

List of Figures Chapter 1: Figure 1.1: A graph showing cytokine release into the circulation over time, 35 following endotoxin inoculation in a human endotoxin model Figure 1.2: Effect of HSP70 on inflammatory and immune response 46 Figure 1.3: An overview of the intracellular metabolism of glutamine 48 Figure 1.4: Proposed mechanism for glutamine mediated upregulation of heat 57 shock protein 70 Chapter 2 Figure 2a: A schematic outline of the experimental plan for in vitro whole 65 blood stimulation assays using endotoxin from healthy adult and paediatric volunteers Figure 2b: A schematic outline of the in vivo experimental plan for sample 70 analysis of plasma obtained from blood samples of children with meningococcal disease during acute illness and in convalescence Chapter 3: Figure 3.1: The effect of various doses of glutamine supplementation on HSP70 83 release using 10µg/ml of endotoxin in whole blood from healthy adult volunteers Figure 3.2: The effect of glutamine supplementation (2mM) on endotoxin 84 stimulated HSP70 release in whole blood from healthy adult volunteers Figure 3.3: The effect of glutamine supplementation on endotoxin stimulated 85 release of IL-1β at 4 hours and 24 hours in whole blood from healthy adult volunteers Figure 3.4: The effect of glutamine supplementation (2mM) on LPS stimulated 86 release of IL-8 at 4 hours and 24 hours in whole blood from healthy adult volunteers Figure 3.5: The effect of glutamine supplementation (2mM) on endotoxin 87 stimulated release of TNF-α at 4 hours and 24 hours in whole blood from healthy adult volunteers

Figure 3.6: The effect of glutamine supplementation (2mM) on LPS stimulated 88 release of IL-6 at 4 hours and 24 hours in whole blood from healthy adult volunteers Figure 3.7: The effect of glutamine supplementation (2mM) on LPS stimulated 89 release of IL-10 at 4 hours and 24 hours in whole blood from healthy adult volunteers Figure 3.8: The effect of KNK437 on extracellular HSP70 release at 4 hours and 91 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) Figure 3.9: The effect of KNK437 on intracellular HSP70 release at 4 hours and 92 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) Figure 3.10: The effect of KNK437 on extracellular IL-6 release at 4 hours and 93 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) Figure 3.11: The effect of KNK437 on intracellular IL-6 release at 4 hours and 24 94 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) Figure 3.12: The effect of KNK437 on extracellular TNF-α release at 4 hours and 95 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) Figure 3.13: The effect of KNK437 on intracellular TNF-α release at 4 hours and 96 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) Figure 3.14: The effect of KNK437 on extracellular IL-10 release at 4 hours and 97 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation Figure 3.15: The effect of KNK437 on intracellular IL-10 release at 4 hours and 98 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation Figure 3.16: The effect of glutamine supplementation (2mM) on LPS stimulated 99 HSP70 release at 4 hours and 24 hours in whole blood from

healthy paediatric volunteers

Figure 3.17: The effect of glutamine supplementation (2mM) on LPS stimulated 100 IL-10 release at 4 hours and 24 hours in whole blood from healthy paediatric volunteers Figure 3.18: The effect of glutamine supplementation (2mM) on LPS stimulated 101 IL-6 release at 4 hours and 24 hours in whole blood from healthy paediatric volunteers Figure 3.19: The effect of glutamine supplementation (2mM) on LPS stimulated 102 TNF-α release at 4 hours and 24 hours in whole blood from healthy paediatric volunteers Chapter 4: Figure 4.1: Effect of freeze-thawing on plasma glutamine levels in healthy adult 118 volunteers Figure 4.2: Glutamine levels in children with acute meningococcal disease and 119 during convalescence Figure 4.3: HSP70 levels on admission in children with acute meningococcal 120 disease compared to convalescence and healthy paediatric controls

Figure 4.4: Log10 of inflammatory mediators (IL-6, TNF-α, IL-10, IL-8 and IL-1β) 121 in children with acute meningococcal disease compared to convalescence Chapter 5: Figure 5.1a and b: Top signalling pathways represented by the significantly 137 differentially expressed genes between patients with meningococcal disease compared to convalescence Figure 5.2: Top signalling pathways represented by the 933 SDE upregulated 139 genes between patients with meningococcal disease compared to convalescence Figure 5.3: Top signalling pathways represented by the 492 SDE differentially 140 downregulated genes between patients with meningococcal disease compared to convalescence Figure 5.4: Representative gene network within the 1,780 SDE transcripts 142

(upregulated and downregulated) in acute meningococcal disease compared to convalescence and the role of HSP70 Figure 5.5: Representative gene network within the 933 upregulated SDE 143 transcripts in acute meningococcal disease compared to convalescence and the role of HSP70 Figure 5.6: Representative gene network within the 492 downregulated SDE 144 transcripts in acute meningococcal disease compared to convalescence and the role of HSP70 Figure 5.7: Validation of SDE gene transcripts using ToppGene confirms 149 dysregulated adaptive immune function and inflammatory signalling pathways in meningococcal disease Figure 5.8: Significant terms for signalling pathways comparing an independent 150 validation dataset with SDE gene transcripts for acute meningococcal disease 8: Appendices 194 Appendix 8.1: Differential fold changes of gene transcripts in children with 195 acute meningococcal disease compared to convalescence Appendix 8.2: Signalling pathways from 1780 SDE gene transcripts 263

Appendix 8.3: Signalling pathways from 933 SDE upregulated gene transcripts 276 Appendix 8.4: Signalling pathways from 492 SDE downregulated gene 289 Transcripts

Abbreviations ARDS Acute respiratory distress syndrome ATP Adenosine triphosphate ASPEN American Society of Parenteral and Enteral Nutrition Ang-2 Angiopoietin 2 APC Antigen presenting cells ANS Autonomic nervous system RANTES Regulated upon Activation, Normal T cell expressed, and Secreted alias for CCL5 Chemokine (C-C motif) ligand 5 CARS Compensatory anti-inflammatory response syndrome CRP C-reactive protein DAMPs Danger associated molecular patterns DIC Disseminated intravascular coagulopathy ELAM-1 Endothelial leucocyte adhesion molecule 1 ICAM-1 Intracellular adhesion molecule 1 ESPEN European Society of Parenteral and Enteral Nutrition GFAT Glutamine fructose-6-phosphate amidotransferase GIT Gastrointestinal tract Gln Glutamine GA Glutaminase GS Glutamine synthetase G-CSF Granulocyte colony stimulating factor GlcNac UPD-N-acetylglucosamine HSF Heat shock factor HSE Heat shock element HSP Heat shock protein HBP Hexosamine biosynthetic pathway HMBG-1 High mobility box group 1 HLA Human leucocyte antigen HR Hydrophobic repeat Ig Immunoglobulin iNOS Inducible nitric oxide IKB Inhibitory kappa beta ICU Intensive care unit IL Interleukin IL-1RA Interleukin 1 receptor antagonist IRAK Interleukin-1 receptor associated kinase IKK I Kappa B Kinase IV Intravenous IKKγ IκB kinase-gamma LPS Lipopolysaccharide LBP Lipopolysaccharide binding protein LPA Lipoteichoic acid MD Meningococcal disease

MIF Macrophage inhibitory factor MIP-1 Macrophage inhibitory factor-1 MHC Major histocompatibility complex mRNA Messenger ribonucleic acid MAPK Mitogen activated protein kinase MCP-1 Monocyte chemotactic protein-1 MYD88 Myeloid differentiation primary response protein 88 NEMO NF-κB essential modulator NF-ΚB Nuclear factor kappa B NOD-LRR Nucleotide oligomerisation domain leucine rich repeat proteins O-GlcNAcase O-N-acetylglucosaminidase PICU Paediatric intensive care unit PAMPs Pathogen associated molecular patterns PRR Pattern recognition receptors PBMC Peripheral blood mononuclear cells PAF Platelet activating factor OGT Polypeptide O-β-acetylglucosamineamyltransferase PEPCK Phosphoenolpyruvate carboxykinase PKA Protein kinase A PKC Protein kinase C PP Protein phosphatase ROS Reactive oxygen species RIG-1 Retinoic acid inducing gene 1 RLH's Retinoic acid inducing like helicase Rt-PCR Real time polymerase chain reaction SCCM Society of Critical Care Medicine sTNFR Soluble tumour necrosis factor receptor SD Standard deviation SDE Significantly differentially expressed SE Standard error SEM Standard error mean SIRS Systemic inflammatory response syndrome SVR Systemic vascular resistance TIR Toll interleukin-1 receptor TLR Toll like receptors TGF-β Transforming growth factor beta TNF Tumour necrosis factor TNF – α Tumour necrosis factor alpha TRAF-6 Tumour necrosis factor receptor associated factor 6 VWF Von Willebrand factor UK United Kingdom

Chapter 1: Introduction

Chapter 1: Introduction 1.1 Epidemiology and health economic burden of sepsis Globally, sepsis and septic shock remain a leading cause of mortality in adults and children (1, 2). Mortality from severe sepsis in children is reported to be between 10.3 – 14% (3-5), which is lower than the mortality rates reported in adults of between 27 - 54% (6).

Prevalence studies estimate that paediatric sepsis occurs on average in 0.6 cases per 1000 population (4) with severe sepsis being more common in neonates (5.2 cases per 1000 population) (3) compared to children 5 – 14 years of age, where the incidence is 0.2 cases per 1000 population (3). Recently published data shows that amongst children admitted to a paediatric intensive care unit (PICU) 82% had evidence of systemic inflammatory response syndrome (SIRS), with 23% of these meeting the criteria for sepsis, of which 4% had severe sepsis and 2% had septic shock (7). An investigation into readmission rates and late mortality after severe sepsis in children (1 month to 18 years of age) revealed that 6.4% of children died either during their PICU stay or within 28 days of discharge. A further 6.5% died within 2 years of the initial admission and discharge from PICU, although the majority of these children had underlying disease pathology (8).

Patients with severe sepsis often spend prolonged periods of time in an Intensive Care Unit (ICU) and are significantly more expensive to treat than those without sepsis (1, 9). The median length of stay for adults admitted to ICU’s in the United Kingdom (UK) with sepsis was 9.6 days compared to 2 days for those without sepsis (9). Owing to the severity of disease, the total costs of patient care in children with meningococcal disease is significantly greater, estimated at $US 49, 626 per patient, compared to $US 5, 041 for non septic critically ill children (10). In the US, paediatric sepsis costs nearly $2billion per annum and in the UK the estimated total cost of adult care was between £7000 and £28,000 per patient per admission (9, 11). The cost of healthcare during acute disease does not account for hidden costs relating to post discharge rehabilitation following severe sepsis, especially in children and the long term impact on cognitive abilities, executive functioning and psycho/ social difficulties experienced into adulthood (12-15).

1.2 Definition of SIRS, sepsis and septic shock Consensus criteria have been developed to define SIRS, sepsis, severe sepsis and septic shock for adults, which have been modified for the use in critically ill children. Severe sepsis and septic shock in children are defined as ‘sepsis plus evidence of organ dysfunction defined around paediatric parameters’ (Table 1.1) (16).

Table 1.1: Definitions of paediatric SIRS, infection, sepsis and septic shock (16) Definition Criteria SIRS The presence of at least two of the following four criteria, one of which must be abnormal temperature or leukocyte count: Temperature

 Core temperature > 38.5°C or <36°C Tachycardia  Mean heart rate >2 standard deviations (SD) for age (excluding pain, fever, or as a result of drug therapy)  Or protracted increase in heart rate over a ½ – 4 hour time period Bradycardia  Infant < 1 year of age - mean heart rate < 10th percentile for age (excluding side effects of drug therapy or where there is congenital heart disease)  Or protracted decrease in heart rate over a ½ hour time period Respiratory rate  Mean respiratory rate >2 SD above usual for age or mechanical ventilation (excluding those with underlying predisposition e.g. neuromuscular disease or following general anaesthesia) Immune response  Leukocyte count increased or decreased for age (excluding those with chemotherapy-induced leukopenia)  Or >10% immature neutrophils associated with a high probability of infection (e.g. evidence of white blood cells in a normally sterile body fluid, chest radiograph consistent with pneumonia, petechial or purpuric rash) Sepsis  SIRS with evidence of an infection as defined below Severe  Sepsis with concomitant cardiovascular dysfunction or acute respiratory sepsis distress syndrome (ARDS)  Or two or more other organ dysfunctions

Septic shock  Sepsis and cardiovascular organ dysfunction Infection  A suspected or proven (e.g. positive culture, tissue stain, polymerase chain reaction, infection caused by any pathogen  Or a clinical syndrome associated with a high probability of infection (evidence of infection includes positive findings on clinical examination, imaging, or laboratory tests (e.g. white blood cells in a normally sterile body fluid, perforated viscous, chest radiograph results consistent with pneumonia, petechial or purpuric rash, or purpura fulminans) Adapted, with permission (16)

1.3 Causative pathogens Escherichia coli, Staphylococcus aureus and Neisseria meningitidis are the most common pathogens implicated in paediatric sepsis as outlined in Table 1.2 (5, 17-19). Severe sepsis is associated with multi-organ dysfunction syndrome (MODS) and the degree to which this occurs is dependent on factors such as age (3), gender (20, 21), ethnicity (22), immune- competence (3), genetic predisposition (23, 24) in addition to any existent co-morbidities (3, 25) as well as the specific pathogen involved (2, 26).

N. meningitidis is a gram negative pathogen which causes meningococcal disease via bloodstream invasion. There are numerous groups, of which Group B and Group C are the most common in the UK. N. meningitidis usually presents in previously healthy children as meningococcal meningitis or bacteraemia which has a low mortality rate (<5%) (27). However, in some children it presents as overwhelming meningococcal septic shock and an associated risk of high morbidity and mortality (27, 28), which ranges from 9 – 35% in those admitted to a PICU, with significant neurological sequelae in up to 29% of patients (27).

Table 1.2: Principle pathogens in paediatric sepsis (5, 17-19) Estimated prevalence Gram negative bacteria 23 – 47% E. coli 4.5 - 13.0% Pseudomonas aeuroginosa 2 - 3.2% N. meningitidis 3 - 6% Klebsiella pneumoniae 2 - 5.8% Other Enterobacter spp 3 - 5.0% Acinetobacter spp 1 – 2% Salmonella spp 1 – 3% Gram positive bacteria 27 - 39% Coagulase – negative staphylococcus 27 -30% Staphylococcus aureus 8 – 12% Staphylococcus pneumoniae 9 – 12% Streptococcus spp 1 - 4% Enterococcus spp 4 – 13% Mirococcus spp 1 – 3% Other gram positive bacteria 1 – 5% Fungus Candida albicans 9.3% Parasites 1 – 3% Viruses Others 10 - 13%

1.4 Pathophysiology of sepsis The pathogenesis of sepsis in adults differs from that in children in a number of ways. Adults usually present with vasodilatation (warm shock), low systemic vascular resistance (SVR), hypotension, average or elevated cardiac output, tachycardia and increased oxygen consumption (29). Neonates and children often present with fluid refractory shock (from decreased circulating intravascular volume, secondary to losses from diarrhoea, vomiting or capillary leak) also known as “cold shock” requiring vasodilators and aggressive fluid resuscitation (2, 29). Common to both adults and children is myocardial depression, depressed ejection fraction and ventricular dilation. Both adults and children usually require inotropes to act as either vasoconstrictors (in adults) and vasodilators (in children) (29), improving cardiac output and/or blood pressure maintenance (30).

In order to understand the physiological consequence of sepsis and septic shock, it is necessary to consider the interaction between colonisation and translocation of microbial organisms and the subsequent host response (31).

1.5 Infectious process Young infants and those with underlying medical conditions (e.g. neurodevelopmental delay, chronic lung disease and primary immunodeficiency) are at particular risk of bacterial blood stream infections due to immature mucosal barriers and immune system (including macrophages, neutrophils, immunoglobulin (Ig) and complement) (18).

Many blood stream infections in children are caused by colonising, pathogenic bacteria, which are present on the skin or mucosal surfaces such as the nasopharynx. Blood stream infections can arise when there is disruption to the mucosal-epithelia barrier allowing bacterial adherence to occur. Colonisation is a highly complex and competitive process involving numerous bacterial ligands. Commensal organisms such as N. meningitidis also need to compete with endogenous flora and associated lymphoid tissue within the mucosal immune system in order to successfully persist. N. meningitidis manages to do this by producing IgA proteases capable of breaking down IgA within the lymphoid tissue. N. meningitidis have evolved mechanisms to evade detection by complement mediated killing or opsonophagocytosis (18).

The nasopharynx and gastrointestinal tracts are common sites of bacterial carriage into the blood stream and once the mucosal barrier is breached, bacterial translocation can occur (18). As such there are a number of primary defence mechanisms, which aim to prevent bacterial colonisation and subsequent translocation including structural barriers e.g. epithelial tight junctions and mucociliary ladders, within the respiratory epithelium, which move unwanted particles away from usually sterile surroundings of the lower respiratory tract. In addition compounds including surfactants and anti-bacterial peptides (cathelicidins and defensins) are capable of neutralizing both gram positive and gram negative bacteria through pore formation (32).

The endothelial mucosal layer acts as an early detection system for pathogen invasion, stimulating host defense mechanisms by recruiting leukocytes to the blood stream entry site (18). From blood stream infection to the development of sepsis there is an interplay between the invading organism and the host response; the systemic inflammatory response is the body’s organised response to infection promoting the activation of the complement system and innate immune response (33). These mechanism are initiated through the recognition of pathogen associated molecular patterns (PAMPs) by receptors sites on the cell surface such as toll like receptors (TLR) known as pattern recognition receptors (PRRs) (34). PRRs (such as TLR’s) form the first line of cellular defence, recognising and transducing signals via ligand receptors, on the host cell surface, when they come into contact with PAMPs (17, 32, 35). There are three main PRR families which comprise (1) TLR’s, (2) nucleotide-oligomerisation domain leucine rich repeat proteins (NOD-LRR) and (3) cytoplasmic caspase activation and recruiting domain helicases including retinoic acid inducible gene 1 (RIG-1) – like helicases (RLH’s) (35).

Pathogens and the subsequent host response results in tissue and cell damage, stimulating the release of intracellular proteins, such as heat shock protein 70 (HSP70), known as alarmins, which aim to protect tissue or cells from further damage. As alarmins and PAMPs elicit similar innate and adaptive immune responses they are broadly known as damage- associated molecular patterns (DAMPs) acting to promote an inflammatory response to infection (34).

1.6 Innate immune response during sepsis Inflammation, as co-ordinated by the innate immune system, is a necessary response to infection promoting increased blood flow to the injured site (36). In the absence of an inflammatory response the host would succumb to overwhelming infection, however, an exaggerated host response results in septic shock and increased risk of mortality (32).

During homeostasis, macrophages remove cell debris originating from normal tissue function. The function of PRRs and alarmins is to promote a burst of inflammatory mediators facilitating leucocyte penetration from within the blood vessels to the site of infection. Following infection or injury, macrophages are rapidly activated by a variety of alarmins

32 which signal via PRRs upregulating inflammatory signalling pathways and the secretion of cytokines and chemokines (37). The production of pro-inflammatory cytokines serves to stimulate the recruitment of macrophages/monocytes and neutrophils to the site of inflammation (38). The function of these cells is to phagocytose pathogens, repair tissue damage, mediate an adaptive immune response and restore homeostasis (38, 39).

Endotoxin such as lipopolysaccharide (LPS) on the bacterial outer membrane of gram negative bacteria (such as N. meningitidis) is recognized by the host’s LPS-binding protein, which on monocytes forms a complex with CD14 signalling via TLR2/4 (40) and on neutrophils via β2-integrins and ligand receptors CD11b and CD18 (41). In contrast, lipoteichoic acid (LPA) from gram positive bacteria is recognized by lectin pathway of complement (42). This process of PAMP-PRR recognition via TLR2/4, initiates a signalling cascade via adaptor protein MD-2, down through the intracellular Toll/IL-1 receptor (TIR) domains, recruiting myeloid differentiation primary response protein 88 (MyD88), which in turn activates IL-1 receptor associated kinase (IRAK). IRAK phosphorylation causes a disassociation from MyD88-TLR complex and subsequent binding to tumour necrosis associated factor 6 activating members of the kinase family including mitogen activated protein kinase family (MAPK) and the nuclear factor kappa beta (NF-κB) signalling pathway promoting the release of inflammatory mediators (43).

NF-κB family has five related transcription factors capable of regulating genes associated with the inflammatory response (44-46) immunity and apoptosis (47). During homeostasis NF-κB is found in an inert state within the cytoplasm, bound to inhibitory κB (IKKs) proteins IKKα and IKKβ (33, 48). In response to stress IKKs are rapidly phosphorylated and degraded, leading to the disassociation of NF-κB from NEMO (IKKγ) complex leading to the release of NF-κB protein subunits, p65 and p50. These protein subunits translocate into the nucleus activating NF-κB transcription factors and subsequent downstream inflammatory signalling pathways promoting the release of inflammatory mediators such as TNF-α (49).

During the early phase of sepsis, DAMPs are released in large amounts, both from the invading organism and the host’s damaged tissue promoting the release of inflammatory cytokines (TNF-α, IL-1, IL-6, IL-12, IL-8) in addition to free radicals and enzymes in large

33 amounts. HSP70 acts as a DAMPs and signal through CD14-TLR4 complex (50-52), eliciting rapid signal transduction via MYD88 –IKK/NF-κB pathway activating NF-κB and MAPK pathways (33, 53, 54) promoting the release pro-inflammatory mediators (TNF-a, IL-1β and 1L-6) (55-59).

1.7 Cytokine release Cytokines are small peptides, which have a pivotal role in orchestrating the inflammatory response to infection. There are more than 200 cytokines including interleukins, interferons, colony stimulating factors and chemokines. Cytokine receptors are ubiquitous and are found on leucocytes, organs, vascular and epithelial cells. The response elicited is dependent on the type of cytokine that binds to cell surface receptors, the location of the cytokine and the timing of release. Cytokine production and release is tightly regulated, with members of the interleukin family and TNF-α being some of the most well studied (60).

There are more than 27 interleukin cytokines with their principle role to promote cell signalling between various leucocytes promoting leucocyte proliferation and infiltration, in addition to facilitating systemic responses such as fever, malaise and anorexia. Members of the interleukin family have traditionally been considered to be either anti- or pro- inflammatory although it is increasingly recognised that individual cytokines such as IL-6 can have dual functions (60).

There is a characteristic pattern of cytokine release, following the activation MAPKs and NF- κβ signalling pathways (45, 46). In a human endotoxaemia model using healthy volunteers, cytokines in plasma are detected early post endotoxin inoculation. TNF-α was detected in the plasma first, 45 – 60 minutes post inoculation, which was quickly followed by inflammatory cytokines including IL-6, IL-8 and IL-10. Plasma levels declined at 3 hours as the inflammatory response resolved (Figure 1.1) (26).

Figure 1.1: A graph showing cytokine release into the circulation over time, following endotoxin inoculation in a human endotoxin model (26).

In critical illness the appearance of cytokines in the plasma follows a similar pattern although the time course is usually over several days (61, 62), rather than hours, with 50% of patients continuing to have elevated IL-6 levels at day 7 despite a resolution in clinical signs of SIRS (63).

Early sepsis results in a “cytokine storm” with increased plasma protein levels. Cytokines promote the release of endothelial leucocyte adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM) encouraging T cell and monocyte adhesion. In response, neutrophils release reactive oxygen species (ROS) and a second wave of inflammatory mediators such as leukotrienes, prostaglandins, proteases and other cytokines leading to endothelial barrier dysfunction increasing the permeability and facilitating further bacterial translocation (64).

1.7.1 IL - 6 IL-6 is a pleiotropic cytokine exerting a multitude of effects. IL-6 has a longer half life than TNF-α and IL-1β and as such has been used as a predictor of outcome in a variety of disease states such as septic shock (65), trauma (65, 66), severe acute pancreatitis (67) and cardiogenic shock (68).

During the initial stages of inflammation IL-6 exerts a pro-inflammatory response upregulating the production and maturation of neutrophils, natural killer cells, B cells, acute phase protein production in addition to stimulating the release of IL-1 and TNF-α. Over time, IL-6 also has anti-inflammatory properties attenuating the production of TNF-α and IL -1 through the upregulation of other anti-inflammatory mediators such as IL-1RA (interleukin-1 receptor antagonist) and soluble tumour necrosis factor receptor (sTNFR) (60, 69).

1.7.2 TNF-α TNF-α is seen early in the course of infection stimulating endothelial adhesion of leucocytes and neutrophil production. It also promotes hepatic production of acute phase proteins and the upregulation of other pro-inflammatory cytokines e.g. IL-6 and chemokines (60). Skeletal muscle mass is also affected by TNF-α with proteolysis and myopathy (69) and over production of TNF-α is associated with poor prognosis and diminished survival in septic patients (70). TNF-α promotes further cytokine release by signalling through TNFR I and II stimulating NF-κB to transcribe additional pro-inflammatory cytokine genes, which down regulate the usual pro-apoptotic mechanisms of TNFR and TNF-α (32).

1.7.3 IL-8 IL-8 is a member of the CXC chemokine family and is the primary activator of neutrophil chemotaxis and has been used as a outcome biomarker for sepsis as it is seen early on in the disease process and low levels in children are negatively correlated with 28 day survival (71).

1.7.4 IL-10 IL-10 is an anti-inflammatory and immunosuppressive cytokine produced by macrophages. Its principle function is to inhibit pro-inflammatory cytokine production by leucocytes. It forms a negative feedback mechanism helping to prevent excessive over production of pro- inflammatory mediators, influencing in particular TNF surface receptors (60).

The resolution of inflammation is as important as the ability to mount an appropriate stress response (72, 73). In order to counter the exaggerated pro-inflammatory response, the immune system simultaneously produces a compensatory anti-inflammatory response mediated by IL-10, IL-1RA, transforming growth factor β (TFGβ) and cytokine receptors

36 including soluble tumour necrosis factor receptor sTNFR (74). An equally excessive release of these anti-inflammatory mediators, especially IL-10 is also associated with poor outcome in sepsis, as they counter-regulate leucocyte functions (75).

1.7.5 Cytokine release in N. meningitidis High levels of endotoxin released by meningococci are associated with shock, renal failure and respiratory distress and a potent inflammatory response (76). TNF-α is one of the first cytokines released in meningococcal disease stimulating the release of other cytokines, such as IL-1, IL-6 and IL-10 (77, 78). High concentrations of IL-6 described in meningococcal disease are associated with disease severity (79, 80) and myocardial dysfunction (81). IL-6 levels remain elevated during the acute phase of the disease declining during recovery (82). IL-10 is also shown to be released in high amounts, serving to inhibit further production of TNF-α and IL-1 (79). However, high levels of IL-10 in this population group are associated with increased risk of mortality (83). A pro-inflammatory response at the outset of meningococcal disease is replaced by an anti-inflammatory response later in the course of the disease (84), with an associated increased risk of immune paresis and risk of secondary infectious insult (85), tissue damage, organ dysfunction and death (86).

Non-survivors of septic shock have a different time course of cytokine expression. There is the usual pattern of early release and initial resolution, but with a subsequent late (day 6) dynamic increase of both IL-6 and IL-10 (61). This pattern is confirmed in other studies, showing an association between significantly higher levels of IL-6 and IL-10 both of which lead to immune paresis and risk of early mortality (87-89). An increase in 1L-6:1L-10 ratio is also associated with a poor outcome and is thought to be related to an uncontrolled inflammatory response (90).

There is a significant body of work, which has helped to progress our understanding relative to the dynamics of the inflammatory response (61, 88, 89). The effective modulation and management of the inflammatory response to infection however, remains elusive, as complete abrogation of inflammation and or over stimulation result in death (91).

Although cytokines are classically categorised into being either pro or anti-inflammatory it is increasingly recognised this definition may be too simplistic. It appears the promotion of an ongoing appropriate inflammatory response by a mix of pro- and anti-inflammatory mediators is the ideal, rather than previous models of an initial pro-inflammatory response which is then countered by an anti-inflammatory response days later (92). HSP70 influences the cytokine response and resolution to inflammation and as such may be able to influence the type of cytokine released during times of stress (93).

1.7.6 Cytokine pathogenesis The release of cytokines has significant metabolic consequences causing anorexia, fever and abrogation of growth in children and weight loss in adults. Interrupted growth in children is as a result of the loss of metabolically active tissue such as muscle, fat reserves and growth factors (micronutrients). There is a complex interaction between cytokines (IL-1 and TNF-α), the hypothalamus and autonomic nervous system with increased production of glucocorticoids, catecholamines and corticotrophin releasing factor. The release of IL-1, TNF- α and IL-6 cause a net efflux of glutamine (amongst other amino acids) from skeletal muscles, increasing glutamine synthesis and protein catabolism used for immune cells, acute phase protein mediators and glucose production (94). Through their release, cytokines promote the release of substrate for use by the innate and adaptive immune system, particularly amino acids from skeletal muscle breakdown (95).

1.8 HSP70 and the relationship to the immune and inflammatory response The heat shock response was discovered in 1962 following the inadvertent overnight heating of Drosophila melanogaster (96), which resulted in the formation of a previously undescribed “puffing pattern” of the salivary chromosome (96-98). Through this discovery a new set of genes were described, which became upregulated with heat shock, environmental or cellular stress (96, 97). HSP’s are highly conserved proteins functioning in the unstressed state, as intracellular molecular chaperones, maintaining homeostatic function, including protein refolding, movement of proteins across membranes and gene regulation (99). As a result of cells being exposed to stress (acute and chronic), mechanisms have developed to allow for the detection and response to a range of stressors. Stress affects protein homeostasis resulting in misfolding and clumping of proteins (both self and

38 non-self). The HSP response has evolved with the principle task of restoring protein homeostasis; repairing damage to proteins and re-establishing normal growth conditions (33, 100). HSP’s are involved in numerous biological processes including the cell cycle, cell proliferation, differentiation and apoptosis (101, 102).

Numerous HSP’s have been described and subsequently have been classified into 6 main families; HSP27, HSP40, HSP60, HSP70, HSP90 and HSP110. The HSP70 family, (one of the most well studied), ranges from 66-78 kilodalton (kDa) (101-103). Research into HSP70 has evolved into two main pathways: firstly to determine the molecular mechanism for HSP response and secondly to characterize the structure and function of HSP70 and how this may be of benefit in various disease conditions (104).

HSP70 is constitutively expressed and required at all times for cells to function normally. HSP70 is present in various cells including; embryo cells, glial cells, dendritic cells (DC), endothelial cells, monocytes, granulocytes, macrophages, B and T lymphocytes, peripheral blood mononuclear cells (PMBC) and vascular smooth muscle cells (105). Within the cell, HSP70 comprises 1 -5 % of the cell protein content and is found in the cytoplasm, nucleus, mitochondria and endoplasmic reticulum (33).

HSP70 also occurs in an inducible form (106). The heat shock response is rapid, occurring within minutes of stress recognition (33), resulting in the ubiquitous release of HSP70 (107). HSP70 is released in response to environmental stress (e.g. heat shock, heavy metals, oxidative stress) and pathological stress (e.g. ischaemic and reperfusion injury and inflammatory response) (100). Cytoplasmic HSP70 accumulation is relative to the degree of cellular stress (33), conferring cytoprotection, in addition to acting as an extracellular chemokine to neighbouring cells. Under these circumstances inducible HSP70 can comprise up to 20% of cell protein content (108). HSP70 also acts to increase phagocytosis and uptake of foreign antigens and upregulation of co-stimulatory molecules (109).

Table 1.3 lists genes, which encode for human HSP70 protein, and inducible HSP70 expression is under the control of HSPA1A and HSPA1B, (located in the MHC class III region on chromosome 6) (107). The heat shock response is mediated by cis-acting sequences

39 known as heat shock promoter elements (HSE) (33, 110, 111). HSE are positioned in multiple copies at various sites upstream of the HSP gene (98, 100).

Table 1.3: Members of the HSP70 family (112) Name Gene Alternative name Entrez Gene ID heat shock 70kDa HSPA1A HSP70-1, HSP72, HSPA1 3303 protein 1A heat shock 70kDa HSPA1B HSP70-2 3304 protein 1B heat shock 70kDa HSPAIL Hum70t 3305 protein 1-like heat shock 70kDa HSPA2 HSP2 70 KD 3306 protein 2 heat shock 70kDa HSPA5 BIP, GRP78, MIF2 3309 protein 5 (glucose- regulated protein, 78kDa) heat shock 70kDa HSPA6 HSP6 70 KD (HSP70B’) 3310 protein 6 (HSP70B) heat shock 70kDa HSPA7* 3311 protein 7 (HSP70B) heat shock 70kDa HSPA8 HSC70, HSC71, HSP71, 3312 protein 8 HSP73 heat shock 70kDa HSPA9 GRP75, HSPA9B, MOT, 3313 protein 9 (mortalin) MOT2, PBP74, mot-2 heat shock 70kDa HSPA12A FLJI3874, KIAA0417 259217 protein 12A heat shock protein HSPA13 Stch 6782 70kDa family, member 13 heat shock 70kDa HSPA14 HSP70-4, HSP70L1, 51182 protein 14 MGC131990 *possibly a pseudogene

Although HSE’s are essential for HSP70 transcription, heat shock transcription factors (HSF’s) regulate transcriptional activation of gene expression. HSF’s bind to HSE’s inducing HSP gene expression and protein accumulation within the cell (113). Under normal conditions

HSP70 protein content of the cell is tightly controlled via a negative feedback mechanism involving HSF-1 and HSF-2 (114). Cellular stress results in the ubiquitous release of HSF-1 (100), whereas HSF-2 is more selectively released (111). During times of stress, HSF-1 and HSF-2 can form heterodimers increasing the effectiveness of HSP70 gene transcription promoting an accumulation of HSP70 within the cell, promoting homeostasis (113).

Elevated levels of HSP70 remain within the cell for several hours conferring protection from subsequent insults (115). However, ongoing HSP70 expression in an unstressed state has been found to be detrimental to cells and as a result mechanisms have evolved to ensure the timely resolution of the heat shock response (116, 117). These mechanisms include a negative regulatory feedback of heat shock factor binding protein 1 (HSBP1) at a transcriptional level (33). Once HSP70 levels within the cell exceed that of non-self proteins, a negative regulatory feedback mechanism, down regulates HSF-1 phosphorylation inhibiting the translocation of unphosphorylated HSF-1 from the cytosol into the nucleus. This action prevents HSF-1 binding with HSE in the nucleus, decreasing gene transcription of HSP70 attenuating the production of HSP70 within the cytosol (118). HSF trimers dissociate and are refolded back into their monomeric inert state (33, 98, 114).

Other self limiting mechanisms include the production of HSP70 messenger ribonucleic acid (mRNA), which has a short half life of 1 hour in cells post insult (119, 120), and is decreased further in cells where there are high levels of HSP70 (121). This is only observed in cells hours after the initial stress, following restoration of homeostasis (121). A mechanism to limit the post-transcriptional production of HSP70 is necessary in order to regulate the production of protein within the cell from already synthesized HSP70 mRNA. HSP70 protein- HSP70 mRNA interaction limits HSP70 gene expression as the HSP70 mRNA stability is reduced (allowing for selective degradation of the mRNA) and so limiting HSP70 production (115). It appears that HSP70 mRNA is only isolated for degradation when HSP70 protein levels exceed refolded proteins. HSP70 then acts on itself by either attenuating any further transcription or by increasing the degradation process relative to its own mRNA (115).

1.8.1 Clinical consequences of upregulated HSP70 release The biological significance of HSP70 release into the extracellular circulation is as part of a network of molecules arising from stressed or damaged cells which stimulate immune activation (122-125), maintenance of cell homeostasis, cellular protection (112), preventing apoptosis and death (119, 126-129). HSP70 expression has been shown to occur not only in critical illness but also in a wide variety of conditions (Table 1.4).

Table 1.4: Heat shock protein 70 expression in health and acute, chronic and autoimmune disease Physiological condition Health  exercise (130)  healthy adults (131)  aging process (132, 133) Acute disease  ischaemic reperfusion injury (134, 135)  burns (136, 137)  trauma (138)  lung injury (139)  infection (140)  septic shock (141, 142)  critical illness (143)  head injury (144)  fever (145-147) Chronic disease  cardiac disease (109, 148-150)  renal disease (151, 152)  atherosclerosis/ inflammation (153, 154)  vascular disease (155)  hypertension (156)  sickle cell disease (157) Auto immune disease  Crohn’s disease (106, 158)  rheumatoid arthritis (59, 159, 160)  multiple sclerosis (161)

Experimental burn model in rats and severe burns in adults have shown that upregulation of HSP70 expression (136, 137) protects against organ damage, leading to increased survival (127, 162). In humans, HSP70 expression occurs within minutes of the stress/insult with detectable levels in the plasma within the first few hours post insult (138, 141, 142, 163),

42 with peaks between day 2 - 6 (164, 165) (although elevated levels are still detected at day 28) (136, 137). In adults HSP70 levels ≥15 ng/ml is associated with increased survival following severe trauma (138).

Conversely, high levels of HSP70 is associated with an increased risk of mortality in traumatic brain injury in adults (144), severe sepsis in both adults (166) and children (141). However, these high levels may be more reflective of overwhelming sepsis and pervasive HSP70 release from dying or necrotic cells rather than a cytotoxic effect of HSP70 (141, 166), especially since overall enhanced expression of HSP70 in adults (143, 167) and in experimental animal models of sepsis appear to have a protective effect (41, 162, 168). Further work is required to determine the specific nature of HSP70 release and the clinical relevance, especially in critically ill children. There is paucity of data regarding HSP70 levels in critically ill children. Wheeler et al describe high levels of HSP70 on admission in children with septic shock (141), however, in this cohort convalescent or controls samples were not assessed over time.

1.8.2 Upregulation and release of HSP70 into extracellular milieu Dendritic cells that are virally infected, (stressed or necrotic) produce danger signals, however healthy or apoptotic cells do not act in the same way (169, 170). During apoptosis, the cell contents are encapsulated into pro-apoptotic bodies, which are then scavenged by phagocytes. As a result, the cellular content is not released into the external milieu. Conversely, the intracellular contents of necrotic cells are expelled into the extracellular milieu, significantly increasing circulating levels of proteins such as HSP70 (171, 172). This release of HSP70 contributes to the circulating HSP70 levels during critical illness (141), trauma (138) and following coronary artery bypass graft (173-175), where it can subsequently activate the immune system (55, 176).

It was thought that HSP70 release only occurred via this passive mechanism from necrotic cells (108, 170) especially as HSP70 does not have a consensual secretory signal allowing it to reach the extracellular environment (125). However, more recent work has shown that HSP70 is present in plasma of normal healthy individuals (177), psychologically stressed

43 animals (178) as well as in the absence of cell death (125) suggesting there is an active mechanism for release.

Subsequent work showed that inducible HSP70 is secreted from the cytosol of intact cells via exosomes or by an endo-lysosomal-dependent pathway (179). Glial cells (180), B cells (181), natural killer cells (182) and peripheral blood mononuclear cells (PBMC’s) (105) all have these exosomes (179, 182) or an endo-lysosomal-dependent pathway (183). HSP70 is inserted into the plasma membrane before being released into the extracellular milieu via membrane associated structures from intact stressed cells either by inverse evagination, exocytosis or membrane shedding (125). As prolonged intracellular exposure to HSP70 is cytotoxic, some of the mechanisms relating to extracellular release may be a protective mechanism to limit internal exposure (117, 184). HSP70 can become cell membrane bound during active release and in this form HSP70 is capable of activating macrophages through antigen presenting cells (APC), acting as both a chaperone and a DAMP (185-187).

1.8.3 The effect of HSP70 on the inflammatory and immune response The inflammatory response has been described as “a process that begins following a sublethal injury and ends with complete healing” (188) and is involved in both defence and maintenance (189). HSP70 performs a range of immunoregulatory and inflammatory functions and is capable of promoting either a pro- or anti-inflammatory response dependent on its location (within and external to the cell)the receptor sites it binds to e.g. TLR 2/4 (185) and the type of T cells stimulated, which ultimately influences the mix of cytokines released (108).

Under normal circumstances HSP70 is detectable in plasma of healthy individuals (who have no evidence of inflammation), suggesting that during times of homeostasis HSP70 does not promote an inflammatory response and its immunoregulatory/inflammatory functions are tightly controlled (108). During times of stress HSP70 is able to interact with APC’s performing two distinct functions, the first of which is to present HSP:protein complexes, which are taken up by APC for processing via MHC Class II receptors activating adaptive immunity. The second function relates to HSP70 being able to stimulate the innate immune response and the secretion of cytokines and chemokines from APC cells, such as TNF-α, IL-

10, IL-6, IL-1β (amongst others) by macrophages and dendritic cells (189, 190) (Figure 1.2). As the stress response resolves, HSP70 acts to dampen down the inflammatory and immunoregulatory response, restoring homeostasis (190, 191).

Endotoxin has been used extensively to understand the pathophysiological response to sepsis/stress (192). To understand the effect of HSP70 on cytokine release, endotoxin has been used as a stimulant in both in vivo and in vitro models (41, 193-197) and is known to be able to activate and deactivate NF-κB (48, 194) and MAPK signalling pathways (198, 199), mediating a pro- or anti-inflammatory response to stress (108).

When healthy adult volunteers are administered a dose of endotoxin intravenously, HSP70 is released along with pro-inflammatory cytokines TNF-α and IL-6 (197). However, other work in vitro (using cDNA transfected HSP70 macrophages) has shown that overexpression of HSP70 can inhibit the release of inflammatory mediators (IL-10, IL-1β, TNF-α, IL-12) following endotoxin stimulation. It is hypothesised that HSP70 may play an important role in mediating the release of pro-inflammatory cytokine following exposure to endotoxin (93) and perhaps plays a crucial role in directly and indirectly preventing an over exuberant inflammatory response (93, 189).

Figure 1.2: Effect of HSP70 on inflammatory and immune response. HSP70 signals via ligands such as TLR 2 and 4, which upregulates NF-κB signalling pathway via MYD88 and stimulates IKK disassociation. This leads to the release of inflammatory cytokines, chemokines, nitric oxide production, which concomitantly upregulates the expression of major histocompatibility complexes (MHC) receptor and other co-stimulatory molecules. With respect to the adaptive immune response HSP70 chaperoned peptide complexes are presented and processed via MHC class II via endocytosis. TNF-α, tumour necrosis factor alpha; IL, interleukin; NF-κB, nuclear factor kappa B (190). (Reproduced with permission)

HSP70 therefore appears to play an important role in the modulation of both immune and inflammatory response in sepsis. However, laboratory mechanisms for stimulating HSP70 release such as heat shock (96), sodium arsenite (200) and adenoviral transfection (201, 202) are not appropriate for use within a clinical setting, due to the negative consequences these agents have on the host. Glutamine, however, can be used as a vehicle to increase the expression of HSP70 (203-205). In adults, glutamine administration significantly increased HSP70 levels, which was associated with a decreased length of ICU stay and fewer ventilator days (143). As glutamine is safe, non-toxic and easy to administer, it could represent a potentially attractive modulator of the heat shock response (206-210). 46

1.9 Glutamine and the upregulation of heat shock response in sepsis Nutrition support, especially in children who have limited metabolic reserves, has traditionally been seen as a means to prevent malnutrition by providing substrate e.g. protein, fat and carbohydrate (211-213). Recent advances in the field of nutrition research have resulted in the realisation that specific nutrients such as glutamine, may be of benefit to critically ill patients (214) with immune modulating effects, altering the host response to stress (215). However, much of the work considering the use of glutamine in critical illness has been completed in adults (206-210), with little data available from paediatric critical illness (214).

Glutamine is the most abundant extracellular amino acid in the plasma with a concentration of between 0.6 - 0.7mmol/l (216, 217). Conversely, glutamate is usually the most abundant intracellular amino acid with a concentration of 2 – 20mmol/l depending on the cell type (218). Glutamate facilitates de novo amino acid transamination, donating an amino group or ammonia. In some organs e.g. liver and cells e.g. astrocytes glutamate and ammonia are combined by glutamine synthetase to form glutamine, which is then exported from the cell. Organs and skeletal muscle are the main source of endogenous glutamine with high intracellular concentrations of 20 – 25mmol/L glutamine (216, 219, 220). Blood glutamine levels reflect the balance between synthesis, release and consumption by all types of cells (217). Glutamine serves as a metabolic intermediate and precursor, providing carbon and nitrogen for the de novo synthesis of other amino acids, nucleic acids, fatty acids, nucleotides and proteins (221). During times of stress, skeletal muscle is an active net exporter of free glutamine (222). Glutamine’s functions within the cell can be broadly categorized into four main categories: 1) nitrogen transport, 2) maintenance of the cellular redox state through glutathione, 3) a metabolic intermediate, 4) a source of energy (221). Figure 1.3 provides an overview of the metabolism of intracellular glutamine.

Figure 1.3: An overview of the intracellular metabolism of glutamine. Glutamine is a precursor for a number of molecules within the cell. Glutamine is deaminated by glutaminase to form glutamate, which can then be converted into γ-amino butyric acid (GABA), ornithine, 2-oxoglutarate, glucose or glutathione. The organ specific metabolism of glutamate and by-products are also indicated (218). (Reproduced with permission)

Glutamine exerts an effect on immune cells and has been shown to stimulate lymphocyte proliferation and optimises macrophage and lymphocyte production of cytokines in animal models (223) and lymphocyte numbers in acute pancreatitis in adults (224). In vitro, glutamine has also been shown to upregulate the bactericidal killing activity of neutrophils from post operative and thermally injured patients (225).

Glutamine homeostasis is tightly controlled. Synthesis occurs in the cytosol and is controlled by glutamine synthetase (GS) and is metabolised by mitochondrial glutaminase (GA) (226, 227). GS transcription is upregulated by glucocorticoid release (228) and in response to plasma glutamine levels (229). Glutamine functions as an inter-organ nitrogen transporter, taking excess nitrogen from peripheral tissues and the brain, to the liver and kidney for assimilation into ammonia. The residual carbohydrate backbone is then used in 48 gluconeogenesis. As such glutamine is a readily available source of fuel, which is utilized by the intestine and immune cells (in particular macrophages) (230, 231).

Glutamine has an important role in ammoniagenesis and gluconeogenesis in the kidney. Glutamine is used as a nitrogen transporter from organs such as the brain and plays a vital role in ammonia detoxification in the kidney (218). Glutamine is deaminated in the kidney by glutaminase to glutamate, which then undergoes oxidative deamination (232, 233). Gluconeogenesis in the liver only becomes established in infants at around 6 months of age and before then is mainly restricted to renal gluconeogenesis. In infants, skeletal muscle represents 60 – 80% of the total body protein synthesis and in order to achieve protein deposition in light of ongoing protein degradation and turnover, amino acid and nitrogen losses need to be conserved. This is achieved through decreased nitrogen excretion and increased re-use of amino acids derived from ammonia than compared to adults (234).

Within the gluconeogenic pathway, glutamine exerts some control on the expression and activity of phosphoenolpyruvate carboxykinase (PEPCK) a key regulatory enzyme in the gluconeogenic pathway, PEPCK is also influenced by hormone levels in addition to other dietary factors (235). In vitro work with hepatocytes has shown that glutamine (5mM) facilitates cell swelling, which decreases PEPCK mRNA accumulation thereby downregulating gluconeogenesis. Cell shrinkage favours gluconeogenesis and proteolysis and is associated with increased activity of PEPCK mRNA and glucose-6-phosphate promoting the production of glucose from glutamine (236). Glucose production from the gluconeogenic pathway provides up to 25% of circulating plasma glucose (218).

Glutamine is used as a fuel source, by rapidly dividing cells, and involves the conversion to glutamate, which is then hydrolysed by glutaminase to form 2-oxoglutarate and glutamate dehydrogenase. These products enter the citric acid cycle where 2-oxoglutarate is converted to malate via succinate and fumarate. An NADP+ dependent malic enzyme creates pyruvate and, following an amination process involving alanine-aminotransferase, produces L-alanine. Pyruvate is converted to lactate via lactate dehydrogenase, which is either released in the circulation or taken into the citric acid cycle via pyruvate dehydrogenase so completing the oxidation of glutamine carbon (235).

The fuel derived from glutamine arises from the reduced forms of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) produced, which are subsequently used in the electron transport chain, in the mitochondria, donating electrons for adenosine tri-phosphate (ATP) synthesis (235). Following its production, L-alanine is exported from the epithelial cell via the portal vein to the liver (218).

In a healthy state, there is a large circulating pool of free glutamine, mainly arising from skeletal muscle (226) and under these conditions it is considered to be a non-essential amino acid (237). However, glutamine is released into the system to provide fuel for the accelerated metabolic functions such as RNA synthesis and perhaps as a cell primer against further injury, making it a conditionally essential amino acid during periods of severe stress (237-239).

Muscle glutamine depletion occurs within hours of severe illness by up to 72% (240). Griffiths et al have shown that in adults there is a precipitous drop in plasma glutamine levels by up to 58% with the onset of catabolism, which can last for up to 21 days, increasing mortality (209). Up to 14g of free glutamine is reported as being depleted from skeletal muscle, in uncomplicated critically ill adults (241). Furthermore, during the first few days of acute illness, nutrition support is often sub-optimal and up to 40% of children are malnourished on admission to PICU (242). Plasma glutamine levels of ≤ 0.43mmol are associated with increased mortality (243) and non-surviving adult septic patients lost up to 90% of their skeletal muscle glutamine mass (244). The extent of glutamine depletion has been correlated with mortality (241), although this is not widely accepted (245). Glutamine homeostasis has been shown to not return to normal for up to 30 days post injury (209). Intravenous glutamine supplementation has been shown to restore plasma glutamine levels. Cells throughout the body utilize exogenous glutamine, although glutamine supplementation is not able to replete skeletal muscle stores due to an unfavourable concentration gradient preventing uptake (246) e.g. muscle cell glutamine content is up 25mmol/l, which is considerably higher than the 0.7mmol/l of plasma (219, 220).

During times of stress there is a net export of glutamine from muscle stores, which become rapidly depleted (234). Protein flux and turnover in rapidly growing children is considerably

50 higher than in adults and as such the need for amino acids such as glutamine is considerably more, suggesting a preferential use by rapidly dividing cells such as enterocytes and immune cells (247). Protein kinetic studies using leucine isotopes have shown glutamine supplementation attenuates protein breakdown. However, glutamine supplementation is not able to increase the rate of protein synthesis, but does exert a protein sparing effect, in very low birth weight infants (248) and in surgical neonates (249). Increased amino acid delivery in stressed, very-low-birth-weight infants, resulted in an increase in de novo glutamine synthesis (250-252).

1.9.1 Glutamine requirements in infancy Glutamic acid and taurine are the most abundant amino acids in breast milk. Glutamine increases 20 fold as lactation becomes established and in an infant, at 3 months of age represents more than 50% of the free amino acids in breast milk providing protection to the gut mucosa and precursors for neurotransmitters. The abundance of glutamine and glutamic acid provide a protein sparing effect for essential amino acids and promote net protein synthesis (253). Infant formulas contain very little glutamine compared to breast milk (254), which is likely to become significant during critical illness with volume restriction and decreased enteral intake. Neonatal animal models show glutamine deficiency is linked to intestinal atrophy (255, 256), decrease electrolyte absorption (257) and immune function (258).

1.9.2 Glutamine and anabolism Glutamine is associated with anabolism with some research suggesting that glutamine supplementation partially attenuates muscle loss and promotes muscle synthesis in steroid induced muscle wasting (259). These findings have not been replicated in critically ill adults given enteral glutamine although the dose of glutamine may have been suboptimal at ~0.3g/kg (260).

A link exists between cell volume, glutamine levels and protein metabolism. Glutamine promotes anabolism via a sodium/potassium transport mechanism promoting an intra/extracellular glutamine concentration gradient generating a sodium flux, drawing water into the cell stimulating the production of DNA/RNA and proteins within the cell. At a

51 concentration of 0.7 – 2mmo/l, glutamine optimises the cell’s hydration status (261). The hydration status of the cell can undergo rapid change mediating effects on hormones, cell metabolism and gene expression. Cell shrinking appears to be associated with upregulation of p38MAPK and extracellular signal regulated kinase (ERK) signalling pathways whereas cell swelling has the opposite anabolic effect promoting catabolism and apoptosis (262, 263). Hydration status of the cell can be affected by HSP70 in addition to glutamine, which has been shown to have a protective effect against hypertonic challenges in vitro. The effect of glutamine and HSP70 appear to be through their influence on Na+/K+ flux across the cell membrane by maintaining intracellular ions (264) and preventing cell signalling triggering net protein breakdown (265-267), supporting protein synthesis (268, 269). Liver and muscle cells have been shown to swell by 10 – 12% within 2 minutes in the presence of glutamine and this increase in cell hydration status is maintained as long as the amino acid is present (266). It has been postulated that changes to cellular hydration through intracellular glutamine depletion is a primary mediator of muscle protein catabolism in critical illness (270) and perhaps the augmentation of this through exogenous supplementation could attenuate some of the losses seen.

Although glutamine is shown to be involved in many cellular and metabolic process, it should be noted that many of the in vitro cell line work attributing the beneficial effects have been completed using supraphysiological doses of glutamine varying between 2 and 10mM, which is in excess of plasma concentrations (0.7mmol/l), as such some of the results should be interpreted with caution (235).

1.9.3 Enteral vs. parenteral administration of glutamine The American Society of Parenteral and Enteral Nutrition (ESPEN)/ European Society of Enteral and Parenteral Nutrition (ASPEN)/ Society of Critical Care Medicine (SCCM) recommend that 0.3 – 0.5g/kg of glutamine is added to parenteral nutrition for critically ill adults, which is based on a number of meta-analysis showing benefit at this level (271-273). This dose has been shown to restore glutamine levels in critical illness (274, 275).

Healthy adults usually produce 50 – 80g per day (276) and during critical illness 20 – 30g of exogenous glutamine is required to maintain plasma levels (274, 275). Glutamine synthesis

52 primarily occurs within the skeletal muscle. It is a primary fuel for gut enterocytes and immune cells and is exported from the muscle for use within the splanchnic circulation. During critical illness, de novo glutamine synthesis is not altered but skeletal muscle is quickly depleted (especially in children), diminishing supply resulting in hypoglutaminaemia (277, 278). The benefit of intravenous compared to enteral administration is that glutamine is available for systemic circulation benefitting immune cells in addition to those within the gut. Glutamine given intravenously (IV) mimics endogenously produced glutamine as shown by its even uptake across the splanchnic area (274). On the other hand, enteral glutamine is rapidly metabolised within the upper part of the small bowel (jejunum) leaving little available for the remainder of the bowel (276, 279). Portal circulation studies also show that little glutamine makes it across the “first pass elimination” to the liver and for subsequent systemic circulation (280). Enteral administration of glutamine is not as effective at restoring plasma glutamine levels to within normal range when compared to parenteral glutamine (281, 282).

In adults, IV glutamine given as ≥ 0.3g/kg (283, 284) as part of parenteral nutrition (PN) following surgery is associated with decreased infectious morbidity (206, 283, 284), mortality (209, 285, 286), improved organ function and decreased length of hospital stay (238, 287, 288). The potential mechanisms for glutamine during critical illness include; tissue protection, immune / inflammatory modulation, maintenance of glutathione levels and metabolic functions as outlined in Table 1.5 (238, 287).

Table 1.5: Organ specific function of glutamine therapy in critical illness (238, 287)

Lung  Major source of energy for endothelial cell  May protect epithelial cells against endotoxin/ oxidant related injury  Enhanced HSP expression post stress  Preserves cell metabolism (ATP) following endotoxin injury  Reduces lactate and glucose accumulation in the lung Heart  Major source of energy via conversion to glutamate for cardiomyocytes  Protects cardiomyocyte against ischemia related injury  Enhances HSP expression post stress Liver  Supports hepatocyte glutathione biosynthesis  Regulator of ammonia metabolism Gastrointestinal  Major source of energy for enterocytes tract  Support nucleotide biosynthesis  May protect epithelial cells against endotoxin/ oxidant related injury  Enhanced heat shock protein expression post stress Immune cell  Major source of energy for proliferating cells function  Supports neutrophil killing and macrophage function  Enhances HSP expression post stress  Vital for appropriate cytokine secretion  Attenuates pathological pro inflammatory cytokine release following endotoxaemia Kidney  Acid base regulation  Ammonia metabolism

1.9.4 Use of glutamine in children In children, the use of glutamine remains largely undefined with paucity of data regarding the benefits of glutamine as a pharmacological agent in paediatric critical illness (214). Conflicting results with respect to the benefits of glutamine supplementation was found in pre-term infants (289-294), infants with gastrointestinal disease and surgery (295, 296), burns (206, 277) and malnutrition (297-299). Reasons for the lack of consistent results, following glutamine supplementation in children may be as a result of the different individual study designs, heterogeneity of the populations studied, route of enteral administration chosen e.g. enteral or IV, dosage g/kg given and duration of glutamine supplementation (300, 301).

However, beneficial effects of glutamine have been reported in children which include, decreased duration of acute diarrhoea (302), reduced severity of mucositis post bone marrow transplant (241), decreased muscle catabolism in Duchene muscular dystrophy (303) and improved growth in sickle cell anaemia (304).

The use of glutamine in pre-term infants has received considerable attention, with some results showing benefit with respect to infectious complications and time to full enteral feeds (291, 305), with other studies showing no difference (294, 306). However there are inherent difficulties with trying to interpret the data from these studies as outlined below.  Heterogeneous groups e.g. sick versus relatively well versus surgical neonates (300, 301)  L-glutamine – most of the studies used L-glutamine, which rapidly degrades to the unusable pyroglutamate (301). Adult studies have used the stable alanyl-dipeptide of glutamine with much better effects relative to morbidity and mortality at 6 months (307).  Isonitrogenous – many of the pre-term studies compared the use of total parental nutrition enriched with glutamine to isonitrogenous control, the issue with this is that it potentially leads to suboptimal delivery of other essential amino acids, especially tyrosine and phenyl alanine (301, 308).  Time taken to get to goal rate – due to the delivery method some of the studies took up to ten days to reach the goal rate of delivery e.g. 0.3g/kg (301).

A Cochrane review concluded that glutamine supplementation confers no benefits to pre- term infants and as such glutamine should not be an active research stream (289). However, other groups argue the mode, timing, duration and type of analogue used in the randomised controlled trials included in the Cochrane review influenced the outcome measures. Furthermore, the beneficial effects of glutamine supplementation especially in pre-term population may only be seen beyond the neonatal period (300), as is evident from studies at 1 year and 6 years of age respectively that showed lower incidence of atopic dermatitis and gastrointestinal infections post neonatal supplementation of glutamine (305, 309).

1.9.5 Mechanisms for glutamine supplementation on HSP expression The beneficial effects of glutamine in critical illness are postulated to be due to increased production of HSP70. In vitro glutamine supplementation upregulates HSP70 release in lung macrophages and epithelial cells (310) protecting against sepsis related injury (310, 311). The beneficial effect of glutamine appears to be HSP70 dependent, since when a knockout septic mouse model was used HSP70 (-/-) no benefit was derived from glutamine supplementation (215). In experimentally induced ARDS, glutamine supplementation improved ATP levels reversing lactate accumulation, by restoring HSP70 levels (143, 239, 312). HSP70 deficiency led to a decline in lung tissue metabolism, lung injury and organ failure in a rat model of sepsis (215).

Eliasen et al. has shown that glutamine depletion affects HSP70 production downstream of transcriptional and translational sites, affecting mRNA HSP70 stability, which in glutamine depleted cells had a significantly reduced half life, resulting in decreased HSP70 expression (313).

Glutamine has also been shown to exert an effect on HSP70 production relative to the upstream influence of glutamine on HSF-1 and HSE (314). Hamiel et al (315) hypothesise that glutamine effects on HSP70 upregulation is dependent on the hexosamine biosynthetic molecular pathway (HBP) (Figure 1.4). Glutamine is metabolised via the HBP, which appears to be part of an early cellular protective response to stress and is a key substrate required for optimal activity of the HBP. It appears that glutamine enhances HBP activity leading to O-linked glycosolation, nuclear transcription and transcriptional activation of HSF-1 and SP1 in the nucleus. HSF-1 and SP1 induces gene promoter activity leading to an increase of HSP70 protein levels within the cell. It is thought that glutamine deficiency leads to a loss of HBP activity and HBP mediated HSP expression (315, 316).

SP1 SP1 HSF- HSF-1 1

SP1 HSF-1

Figure 1.4: Proposed mechanism by Hamiel et al. (315) for glutamine mediated upregulation of heat shock protein 70 (HSP70). Glutamine (GLN) has been shown to be metabolised via the hexosamine biosynthetic pathway (HBP) to UDP-N-acetylglucosamine (GlcNac). This pathway appears to be part of an early cellular protective response to stress. Principle enzymes within the HBP are; glutamine, fructose-6-amidotransferase (GFAT), polypeptide-O-β-acetylglucosamineamyltransferase (OGT) and O-N-acetylglucosaminidase (O-GlcNAcase). It is thought that glutamine deficiency could lead to a loss of HBP activity and HBP mediated HSP expression (Reproduced with permission).

1.10 Novel therapeutic strategies in sepsis Septic patients in the ICU are resource demanding, requiring intensive multi-organ system support (32). Artificial nutrition, especially in children, is required to provide sufficient substrate with the aim of preventing endogenous catabolism of protein and preventing malnutrition (214) . Following discharge from the ICU survivors of sepsis often require long- term follow-up and rehabilitation (317).

The medical management of sepsis and septic shock uses targeted anti-microbials (318), prevention of enteric bacterial translocation and secondary bacterial infections (319). Other therapies such as the use of monoclonal antibodies (320), activated protein C (321), anti-

TNF-α therapies (322), arginine (323, 324) have all had limited, or sometimes harmful effects (32) (Table 1.6).

Table 1.6: Clinical trials of therapeutic agents used to mediate sepsis (325) Harm  Soluble tumour necrosis factor (TNF) receptor p75  Inhibition of nitric oxide with L-N-monomethyl arginine (L-NMMA) Benefit  Neutralisation of endotoxin with polyvalent anti-plasma or monoclonal antibody HA- 1A  Neutralisation of endotoxin with recombinant bactericidal permeability increasing protein (reduced morbidity)  Neutralisation of TNF by monoclonal antibody  Acceleration of removal of platelet-activating factor (PAF) with recombinant PAF acetylhydrolase  Administration of recombinant human activated protein C4 (clinical use)  Administration of pharmacological doses of corticosteroids (clinical use) Indeterminate or no effect – due to heterogeneity or underpowered study design  Neutralisation of endotoxin by monoclonal antibody, antibody to enterobacterial common antigen, or polymyxin B  Inhibition of TNF by monoclonal antibody or soluble receptor p55  Inhibition of interleukin-1 (IL-1) with recombinant IL-1 receptor antagonist  Inhibition of PAF with a receptor antagonist  Inhibition of nitric oxide with L-NMMA or methylene blue  Inhibition of bradykinin  Inhibition of prostaglandins with ibuprofen  Nonspecific suppression of inflammation with high dose corticosteroids or pentoxifylline  Administration of granulocyte colony-stimulating factor (G-CSF) or interferon-γ  Inhibition of coagulation with anti-thrombin III or tissue factor pathway inhibitor

The focus of recent research has been to understand the relationship between pro- and anti- inflammatory mediators (326) and whether multi-organ failure and mortality is as a result of a dysregulation between this balance (327). It has been postulated that mortality arising from sepsis is secondary to a prolonged immunosuppressive state (29, 40, 328) which is more common in children than adults (329) and may be as a result of reduced bacterial clearance and hypo-responsive immune system (330). However, trials using therapeutics

58 such as non steroidal anti-inflammatory agents and corticosteroids (331-333) aimed at controlling the systemic inflammatory response have not yielded promising results thus far (40, 334, 335).

Part of the issue with therapeutic and nutrition trials in sepsis, is the “one size fits all” approach. Critically ill children represent a heterogeneous cohort, however many of the medical and nutrition management strategies are homogenous. As a result many interventional studies have failed to deliver significant positive outcomes despite exciting pre-clinical findings as seen with the anti-TNF-α and activated protein C (drotrecogin) and CRISIS trials (glutamine, zinc, whey protein, metoclopramide, selenium) (325, 336). As a result, many groups are endeavouring to develop clinically relevant biomarkers to stratify treatment (337). The mediation of the heat shock response may fit into this stratification plan given recent advances in the understanding of biological mechanisms and influences of alarmins within the inflammatory, coagulation, immune and neuroendocrine response to sepsis. New management strategies are being developed with the aim of upregulating alarmins, such as HSP70, in the hope that benefit to the host will be conferred (32, 239) and with increasing understanding being derived from transcriptomic analysis, a more targeted approach to treatment may be able to be developed (338, 339).

During the last 50 years there has been a significant body of research into the role of heat shock response and its role in mediating a response to danger. The heat shock response (by heat shock proteins), confers cytoprotection by refolding damaged proteins, upregulating cellular defence mechanisms (119, 127, 340) in addition to alerting surrounding cells to danger through extracellular signalling (341). HSP have also been shown to reduce gut endothelial permeability and prevent post infectious metabolic dysfunction (342) and it is thought that through the mediation of this response using nutraceuticals such as glutamine better long term outcomes will be achieved.

1.11 Conclusion HSP70 is part of a family of highly conserved proteins that are involved in the most fundamental aspects of cellular protection and homeostasis (239). The heat shock response has been shown to be beneficial following various types of injury such as ischaemia and

59 reperfusion injury (343), acute lung injury (344) and septic shock/ sepsis (128). The release of HSP70 has also been shown to activate and attenuate the inflammatory response, which has been associated with survival post sepsis (126). In addition to this, elevated levels of HSP70 post trauma are associated within improved outcome (138). Previously there had been no practical way of upregulating the expression of HSP70 as methods used in vitro and in animal models were of detriment to humans e.g. sodium arsenite, glutamate, heat shock etc (239).

Glutamine has been used in critically adults to upregulate HSP70 release with some evidence to suggest survival benefits (143). Glutamine depletion is associated with increased cellular stress (313), programmed cell death (apoptosis) (345), pro-inflammatory response (86) and immune paresis (346, 347) and some of these effects are thought to be mediated by downregulated HSP70 production (314, 348).

Although the development of stratified treatment plans requires investment (methodological, validation and application processes) (337), it is possible in the future that treatment modalities may include the targeting of endogenous danger signals such as heat shock proteins with therapeutic agents such as glutamine to improve outcomes within clearly defined groups of patients (1, 207).

Whilst elevated HSP70 levels have been described in critically ill children (141), to our knowledge, the effect of glutamine on HSP70 release and the influence on the inflammatory response has not been described. Understanding the effects of glutamine supplementation on HSP70 release will enable the development of a greater understanding as to whether or not severely ill children could benefit from glutamine supplementation early in the course of their disease, through regulation of the immune and inflammatory response, and so improving outcome.

The focus of the current research was therefore to investigate HSP70 release and its relationship to glutamine and inflammatory markers in healthy adults and paediatric volunteers using a whole blood endotoxin model. In addition, this study also aimed to describe relationships between glutamine, HSP70 and inflammatory mediators, in a cohort of critically ill children.

1.12 Hypothesis During times of stress, glutamine promotes the release of HSP70.

1.13 Research Aims 1. To set up an in vitro model of sepsis examining the effect of glutamine on the release of HSP70 and inflammatory mediators

 To investigate the effect of glutamine on HSP70 release using a whole blood in vitro endotoxin stimulation model in healthy adult volunteers, and its association with markers of an inflammatory response.

 To investigate the effect of KNK437 (HSP70 inhibitor), on the release of HSP70 and inflammatory mediators in response to endotoxin stimulation of whole blood, from healthy adult volunteers.

 To investigate the effects of endotoxin and glutamine on HSP70 in healthy children using whole blood, and its associations with markers of the inflammatory response.

2. To measure levels of HSP70, glutamine and markers of inflammation in critically ill children with acute meningococcal disease compared to convalescence.

 To investigate relationships between HSP70, glutamine and inflammation in critically ill children with acute meningococcal disease compared to convalescence.

 To investigate relationships between clinical variables, HSP70, markers of inflammation and glutamine in critically ill children with acute meningococcal disease compared to convalescence.

3. Use of gene expression analysis of whole blood to examine relationships between genes associated with HSP70 and inflammatory signalling in children with meningococcal disease

 Investigating relationships between HSP70, glutamine and inflammatory mediators and significantly differentially expressed gene transcripts in critically ill children with acute meningococcal disease compared to convalescence.

Chapter 2: Materials and Methods

Chapter 2: Materials and Methods 2.1 Ethical consideration 2.1.1 In vitro experiments In vitro aspects of this study, including all methods were approved by the Local Research Ethics Committee (reference number 09/H0712/98). Inclusions and exclusion criteria for healthy adult and paediatric volunteers were based on signed informed consent. Participants were free to withdraw their consent at any point without any prejudice.

2.1.2 Children with meningococcal disease Following informed parental consent, and with approval of the Local Research Ethics Committee, (reference number EC3263), venous blood was collected from children admitted to St. Mary’s Imperial College NHS Trust, PICU with suspected meningococcal disease at the time of admission to the intensive care. The diagnosis was confirmed by isolation of the organism from blood, CSF, or by PCR amplification of meningococcal DNA from peripheral blood (349).

2.2 In vitro experiments Experiments 1 - 3 were designed to consider the effect of endotoxin stimulation on the release of HSP70 and inflammatory markers using a whole blood model as outlined in Figure 2.1a.

Experiment 1 was designed to optimise the use of a whole blood in vitro model examining the effects of endotoxin and glutamine on the release of HSP70 and markers of inflammation in healthy adult volunteers, using a variety of doses of endotoxin and glutamine.

Experiment 2 was designed to investigate the in vitro effects of KNK437, endotoxin and glutamine on HSP70 release and markers of inflammatory response, using a whole blood model, in healthy adult volunteers.

Experiment 3 was designed to investigate the in vitro effects of endotoxin and glutamine on HSP70 release, using a whole blood model, in healthy paediatric volunteers and its association with markers of inflammatory response.

Figure 2.1a: A schematic outline of the experimental plan for whole blood stimulation assays using endotoxin from healthy adult and paediatric volunteers

2.2.1 Optimisation phase A whole blood in vitro endotoxin model was used to replicate conditions of sepsis seen in vivo. The Department of Paediatrics at Imperial College London has previously demonstrated that this is a valid model to study the process of inflammation in sepsis (350). HSP70 is ubiquitously present and produced by a large variety of cells and as such an in vitro whole blood endotoxin model was used to replicate the septic in vivo cell mix. Endotoxin derived from E.coli (0111:B4) was chosen as a well established stimulant in previous models investigating the release of HSP70 in vitro (193, 194, 351, 352). Glutamine free culture

65 medium (RPMI -1640) was used throughout the experimental phase, enabling the effect of subsequent glutamine supplementation on HSP70 release following endotoxin stimulation to be examined.

Optimisation of the whole blood endotoxin stimulation assay in adults, where blood volume was less limited, evaluated a range of doses of endotoxin (0.01μg/ml - 1μg/ml), glutamine (2mM – 3.4mM – 6.8mM), KNK437 (50 -200μM) and different time points (0, 1, 2, 4, 6 and 24 hours). Following this, a more limited model was developed for use in blood samples obtained from children where blood volume was more restricted.

Optimisation of sample dilutions were completed using Meso Scale Discovery (MSD) 4-plex II ultra sensitive plate (1:10 dilution) and HSP70 ELISA plates (1:2 dilution). To control for variance between ELISA plates (HSP70 and inflammatory mediators) a positive plasma control from endotoxin stimulated blood (1µg/ml endotoxin at 24 hour timepoint) from one volunteer was used in duplicate on all of the ELISA plates. The results derived from the positive control on all ELISA plates were evaluated. An acceptable coefficient of variance from the mean was 20% either side of the positive control mean value. Samples on plates where the positive control was outside this range were adjusted.

2.2.2 Experiment 1: To examine the in vitro effects of endotoxin and glutamine using a whole blood model on HSP70 expression in healthy adult volunteers and its association with markers of inflammatory response The experimental design analysed the effect of time, glutamine and endotoxin dose on the release of HSP70 in vitro.

Whole blood (8ml) with 60 USP Units of Sodium Heparin was diluted 1:1 with an equal volume of warmed RPMI 1640 without glutamine (SIGMA Aldrich). A time zero aliquot of whole blood (500µl) was centrifuged for 10 minutes at 1200g. The supernatant was aliquoted and immediately stored at -80OC.

Whole blood (180µl) and 20µl endotoxin (0µg/ml, 0.01µg/ml, 0.1µg/ml or 1µg/ml) was aliquoted in duplicate into a 96 well plate, representing four conditions. The plate was

o incubated at 37 C, 5% CO2 for 2 hours. After 2 hours 2mM of glutamine was added to half the conditions and incubated up to 4 and 24 hours. At each time point glutamine/-glutamine blood was aspirated and centrifuged for 10 minutes at 1200g.

Plasma (60µl) from each condition was aliquoted in duplicate into a 96 well plate and mixed equal measure (60µl) with HSP70 Assay Buffer (Catalogue No. 80-1599) in preparation for completing a high sensitivity HSP70 ELISA plate (Enzo Life Sciences Catalogue No. EKS-715 CA; USA).

Plasma (25μl) was aliquoted in duplicate into a 96 well plate and mixed with 225μl of 10% phosphate buffer solution and 0.05% Tween 20 (PBS-T) in preparation for completing MSD Human pro-inflammatory – 4 and 3 II ultra sensitive kit, measuring inflammatory mediators (IL-6, IL-8, IL-1β, TNF-α,) and (IL-6, TNF-α, IL-10) respectively. Following processing, all samples were immediately stored at -80oC until the ELISA kits were processed and analysed.

2.2.3 Experiment 2: To examine the in vitro effects of endotoxin, glutamine and a HSP70 inhibitor KNK437, on HSP70 expression, using a whole blood model, in healthy adult volunteers and its association with markers of inflammatory response Experiment 2 was based on methods in experiment 1 using one dose of endotoxin (1μg/ml) in addition to KNK437. Cells were lysed to investigate differences between intracellular and extracellular release of HSP70 and inflammatory mediators following the addition of KNK437.

A range of KNK437 doses were evaluated (50μM – 200μM), with a dose of 200μM KNK437 being used in the inhibition model. To diluted whole blood aliquots of 180µl, 20μl of 200μM of KNK437 was added to half of the conditions and incubated at 37⁰C, 5% CO2 for an hour, after which to half of the conditions, 1µg/ml of endotoxin was added, followed by 2mM of glutamine 2 hours thereafter. Samples were incubated at 37⁰C, 5% CO2 for up to 4 and 24 hours. Samples were centrifuged for 10 minutes at 1200g. Plasma was harvested, diluted and stored as outlined in experiment 1.

Any remaining supernatant was discarded and the cell pellet was resuspended and washed with Dulbecco’s Phosphate Buffered Saline (DPBS) and centrifuged for 5 minutes at 450g and repeated twice.

Cells were lysed according to the manufacturer’s instruction using CellLytic M (Catalogue number C2978; Sigma Aldrich) and a protease inhibitor cocktail (Catalogue number P8340; Sigma Aldrich). To lyse 400μl of cells, 50μl of CellLytic M and 40μl of the protease inhibitor cocktail were used. The cell pellet was resuspended in CellLytic M reagent and protease inhibitor cocktail, and incubated at room temperature for 15 minutes on a shaker at a vigorous speed of 250g. Cells were subsequently centrifuged for 15 minutes at 3,000g pelleting cell debris. The protein containing lysate was harvested, diluted and stored as outlined in experiment 1.

2.2.4 Experiment 3: To examine the in vitro effects of endotoxin and glutamine on HSP70 expression, in whole blood obtained from healthy paediatric volunteers and its association with markers of inflammatory response. After obtaining informed parental consent, 25 healthy children were prospectively enrolled from a general paediatric outpatient clinic. As the available blood sample volume was considerably smaller compared to amounts available from adults, the experimental conditions were rationalised to those with the most significant effect. As such there were two conditions, (a) 0µg/ml LPS, with and without glutamine and (b) 1μg/ml LPS, with and without glutamine cultured for 4 and 24 hours. All of the volunteers were well on the day of phlebotomy and a total of 3ml of blood was collected from each volunteer via venipuncture into lithium heparin tubes. Plasma was harvested, diluted and stored as outlined in experiment 1.

2.2.5 Experiment 4: To examine the in vivo relationship between clinical variables, plasma HSP70, glutamine and inflammatory mediators and genome-wide transcriptional profiles in children with acute meningococcal disease compared to convalescence. To examine the in vivo relationships between clinical variables, plasma HSP70, glutamine, inflammatory mediators and genome-wide transcriptional profiles in children with acute meningococcal disease (n=143) compared to convalescence (n=78), a variety of techniques

68 were used to analyse the samples. These included a) inflammatory mediators using MSD, 4- plex kit on a smaller cohort (acute n=34, convalescence n=8) and 3-plex (acute n=109, convalescence n=70) b) HSP70 using ELISA kit (Enzo Life Sciences), c) glutamine analysis using AminoTac 500, in addition to d) a transcriptomic analysis of differentially expressed genes considering relationships between HSP70, glutamine inflammatory mediators and e) analysis of clinical variables from a clinical notes folder review as outlined in Figure 2.1b.

2.2.5.1 Study population Venous blood was collected from paediatric Caucasian patients admitted to PICU at St Mary’s Hospital with suspected meningococcal sepsis, meningococcal meningitis or both. Samples were collected in EDTA tubes and processed. Whole blood was centrifuged for 10 minutes at 1200g, plasma was harvested and aliquoted (400µl) and stored at -800C freezers in anonymised Ependorf tubes, (Department of Paediatric, Imperial College, St. Mary’s Campus). The remaining cell pellet was similarly stored for later use in real time polymerase chain reaction (rt-PCR) microarray analysis. Convalescent samples were collected in children during outpatient follow-up on average 55 days [range 35 – 153] post discharge from the PICU and processed as above. Samples included in the analysis were selected using the following inclusion criteria:

Inclusion criteria:  length of PICU stay for ≥ 3 days,  mechanical ventilation  PRISM score (353) of ≥ 10  Caucasian Exclusion criteria:  length of PICU stay for ≤ 3 days,  breathing in room air  PRISM score (353) of ≤ 10  Non-Caucasian

Samples were identified using inclusion criteria and in batches of forty were brought to room temperature. During processing, samples were kept on ice. Aliquots for ELISA’s were diluted as outlined in experiment 1 and re-frozen within 30 minutes of being processed.

Figure 2.1b: A schematic outline of the experimental plan for sample analysis of plasma obtained from blood samples of children with meningococcal disease during acute illness and in convalescence.

2.3 Techniques for protein measurement, clinical variable and transcriptomic analysis

2.3.1 Method a: Analysis of levels of inflammatory mediators by multiplex ELISA (Meso Scale Discovery) multiplex assay kit Pro- and anti-inflammatory mediators selected were based on previous work in meningococcal disease (77, 78) and available validated multiplex plates from Meso Scale Discovery (MSD). MSD Human inflammatory 4-plex II ultra sensitive kits were used to measure a range of inflammatory mediators (IL-1β, IL-6, IL-8, TNF-α) in samples, from Experiments 1 and 4. MSD Human inflammatory - 3 plex II ultra sensitive kits were used to measure a range of inflammatory mediators (IL-6, IL-10, TNF-α) in samples, from Experiments 2 - 4. MSD platform uses electrochemiluminescence technology which emits light following electrochemical stimulation of the electrode surfaces in the multi-spot plates and is a well described technique for measuring inflammatory mediators (354-356). Due to 70 the limited plasma volumes available from paediatric patients there was insufficient samples to run independent plates for all of the cytokines.

Standards, samples and diluents (No.2, No.3) were thawed to room temperature. Diluents, antibodies, read buffer and plate analysis were completed as per the manufacturer’s instructions. Twenty five µl of diluent 2 was pipetted onto the 96 well MSD plate, ensuring equal coverage. The plate was shaken vigorously (700rpm) for 30 minutes, after which 25µl of reference kit standards, samples, positive control and blank were aliquoted in duplicate. The sealed plate was shaken vigorously for 2 hours followed by PBS-Tween (PBS-T) wash x 3. A detection antibody (25µl) was added to each well, followed by vigorous shaking for a further 2 hours and subsequent PBS-T wash x 3. One hundred and fifty microlitres of “read buffer” was added in duplicate allowing the plate to be analysed using the MSD Multiplex platform Sector Image 4000. The mean value was used for all duplicates if the coefficient of variance < 20%.

2.3.2 Method b: Measurement of HSP70 Stressgen EKS-715 High Sensitivity ELISA Kit HSP70 high sensitivity EKS-715 kit, measuring inducible HSP70 release in plasma samples is described by others (141, 143, 151, 152) and was used in all experiments as per the manufacturer’s instructions.

Samples and reagents were thawed to room temperature. One hundred microlitres of reference kit standards, samples, positive control and blank were aliquoted in duplicate onto the HSP70 ELISA plate, which was sealed and shaken at 50g for 2 hours, followed by a 3 x plate wash using HSP70 wash buffer. Following that the addition of 100µl high sensitivity polyclonal antibody was aliquoted into each well (except for the blank). The plate was shaken for 1 hour and washed x 3. A further 1 hour incubation with 100µl IgG followed and washed x 3. One hundred microlitres of substrate solution was added for 30 minutes followed by 100µl of a stop solution. The optical density for each duplicate sample was measured at 450nm using a spectrophotometer, calibrating against the substrate blank. The mean value was used for all duplicates if the coefficient of variance < 20%.

2.3.3 Method c: Analysis of plasma glutamine Plasma glutamine levels were measured using ninhydrin cation exchange chromatography, in collaboration with Dr Margaret Hancock, Department of Chemical Pathology, Charing Cross Hospital.

Samples were thawed to room temperature, 160µl of sample were aliquoted and then refrozen to -80oC. Glutamine was measured in supernatant using AminoTac JLC500/V (Amino Acid Analyser). AminoTac JLC500/V uses a cation exchange chromatography system, using a post column sorting with ninhydrin (2,2-Dihydroxyindane-1,3-dione). Ninhydrin is used to detect primary and secondary amines in addition to ammonia. Ninhydrin reacts with these free amines to form a deep blue or purple colour which is known as Ruhemann’s purple leading to the identification of indivdual aimo acids.Amino acids are separated according to their net charge determined by the pKa of their ionised groups. The mobile phase incorporates 5 lithium citrate buffers used in an incremental process of increasing pH and molarity maximised through the use of a temperature gradient, involving five mechanistic steps (Table 2.1) (357).

Table 2.1: Mechanistic steps for cation exchange chromatography system using ninhydrin Step 1 alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + water Step 2 alpha-amino acid + water ---> alpha-keto acid + ammonia Step 3 alpha-keto acid + ammonia ---> aldehyde + carbon dioxide Step 4 & 5 alpha-amino acid + 2 ninhydrin ---> carbon dioxide + aldehyde + final complex (blue) + water

Analysed glutamine samples met quality assurance and control standards as defined by the European Network of Inherited Disorders of Metabolism plasma amino acid quantification. Glutamine results were expressed as the sum total of glutamine and glutamic acid, to account for the breakdown of glutamine to glutamic acid on storage (358).

To investigate the effect of freeze-thaw cycles on glutamine levels in stored plasma samples from children with meningococcal disease, a series of freeze-thaw cycles on whole blood samples from adults were completed. Following venipuncture 8ml of blood from adult

72 volunteers (n=3), was aliquoted into equal 1ml samples with time zero sample stored at - 80oC. The remaining samples were spiked with 2mM of glutamine and underwent the following experimental conditions: a) baseline sample with 2mM glutamine added b) stored at room temperature for 1 hour, c) frozen at -80oC, then brought to room temperature and refrozen at -80oC (a freeze thaw cycle), d) 2 freeze thaw cycles e) 3 freeze thaw cycles, f) 4 freeze thaw cycles g) 5 freeze thaw cycles h) 6 freeze thaw cycles.

2.3.4 Methods d: Transcriptomic analysis 2.3.4.1 Study population Venous blood was collected from paediatric Caucasian patients admitted to PICU at St Mary’s Imperial NHS Trust Hospital between December 2002 and May 2005 with suspected meningococcal sepsis, meningococcal meningitis or both. All patients had a clinical diagnosis of meningococcal disease, with a confirmed Group B positive sample (blood, CSF or bacterial DNA PCR amplification) identified in 33 out of 39 patients. Six of thirty nine cases had unconfirmed meningococcal disease (4 were ‘negative’ and 2 were ‘not known’).

Thirty-nine patients were successfully arrayed. Twenty eight patients had meningococcal sepsis (MS), 3 with meningococcal meningitis (MM) and 8 with both. The 28 sepsis only and the 8 meningococcal meningitis and sepsis patients were combined to form a ‘sepsis’ cohort (n=36), of which there were 32 acute samples (15 acute only), 21 convalescent (4 convalescent only) and 17 paired acute and convalescent samples.

2.3.4.2 RNA extraction, amplification and labelling Total ribonucleic acid (RNA) extraction, amplification, hybridisation and microarray data analyses were previously completed by Dr Victoria Wright, Department of Paediatrics, Imperial College London and the data arising from the analyses was used to complete the transcriptomic analysis. Human RNA was extracted from whole blood collected into PAXgene tubes according to manufacturer’s instructions (PreAnalytix/Qiagen), reverse transcribed to cDNA and biotinylated amplified cRNA generated by in vitro transcription using Illumina TotalPrep RNA Amplification kit (Ambion) according to the manufacturer’s instructions. After purification, 500ng of cRNA was hybridised to an Illumina Human Ref 8 V3 Beadchip (containing probes to 24,526 RefSeq (Build 36.2, Release 22) gene sequences) at 55°C for 18

1/2 hours, washed, blocked and stained with streptavidin-Cy3 according to the manufacturer’s instructions. The arrays were scanned with an Illumina Bead Array reader confocal scanner.

2.3.4.3 Data analysis Standard normalisation procedures for 1-colour array data were used with GeneSpring GX software (version 11.02, Agilent). Fold induction values were normalised by log transformation and differences in gene expression in the patients compared to convalescence determined using unpaired Mann-Whitney and corrected for multiple testing using Bonferroni Family Wise Error Rate.

2.3.4.4 GeneSpring – normalisation and filtering Data was normalised by a shift to the 75th percentile, baseline transformed to the median of all samples, and flags that were “present” used (defined with the lower cut off for a “present” flag of 0.9899, and the default upper cut off for an absent flag of 0.6). The probe must be flagged as ‘present’ in 90% of the samples in any 1 out of 2 conditions (i.e. acute vs. convalescence). All filtering was unsupervised and the Mann Whitney unpaired tests was used for significance testing with the most stringent multiple comparison test, Bonferroni Family Wise Error Rate (BFWER), as well as the least stringent, Benjamini Hochberg (BH). A fold change of ≥2 were the cut-offs.

2.3.4.5 Ingenuity Pathway Analysis Ingenuity Pathway Analysis application ((IPA), Ingenuity Systems, Redwood City, CA, USA) is a tool enabling the discovery of signalling pathways and gene networks within an uploaded gene list (359, 360).

IPA generates gene networks by mapping each gene identifier to its corresponding gene object within the IPA Knowledge base. Focus genes are then overlayed into a global molecular network that is generated from information within the Knowledge Base. The focus genes are then assigned into networks using algorithms based on their connectivity. Each IPA network can be set to have a maximum of 35 focus genes, to which a significance score is assigned (based on a P value). This is representative of the likelihood that the focus gene

74 within the network is there by random chance. A high number of focus genes within a dataset results in a higher network score, which is equal to the negative exponent of the P value e.g. score of 47 is equal to 10E-47 (359, 360).

2.3.4.6 Independent cohort validation of transcriptomic analysis Since Illumina array render highly reproducible results, rt-PCR was not completed in this cohort. Validation of the significantly differentially expressed (SDE) transcripts within the dataset was completed functionally using plasma protein levels of glutamine, HSP70 and inflammatory mediators (IL-1β, TNF-α, IL-6 and IL-8). An independent gene ontology database (ToppGene) (361), was used to determine the most significant functional signaling pathways from the dataset and to corroborate pathway findings identified by IPA e.g. antigen presentation, adaptive immunity and inflammatory signaling pathways.

An independent cohort of children with meningococcal septicaemia (n=6) compared to age matched controls from Gene Expression Omnibus (362) was used as a validation cohort. A log2 fold change of ≥2 was used as the cut off with P values adjusted using Benjamini- Hochberg multiple comparison testing. The top 250 SDE gene probes (from analysis using IPA) were used for validation of the biological functions and signaling pathways in the 1,780 SDE transcripts from the acute meningococcal cohort.

2.3.5 Methods e: Analysis of clinical variables from clinical notes folder review For those children with identified plasma samples, clinical and nutrition variables were collected using a password encrypted anonymised data-base. Paediatric specific criteria was used to determine organ failure, severity of injury score and mortality was followed for 28 days after enrolment (16). Although this study was not powered to examine the significance of clinical associations between the changes in biomarkers, genome-wide expression patterns and disease severity, relationships were investigated for associations between transcriptomic data, clinical biomarkers and nutrition indices. The following clinical and nutritional data was collected for all patients:

Clinical Data  Demographic data: age, gender  Diagnosis: Clinical and microbiological diagnosis obtained from blood culture, serology, latex agglutination or PCR  Inflammation: SIRS criteria (363), CRP and white cell count  PRISM and PIM2 score on admission as a predictor of mortality (364, 365)  Worst daily lactate and glucose (366, 367)  Duration of mechanical ventilation and PICU stay

Nutritional data:  Anthropometry: Weight  Route of feeding e.g. nasogastric, nasojejunal, total parenteral nutrition  Type and volume of feed e.g. name  Days to goal rate of feeds  Glutamine g/kg at goal rate of feeds  Gastric aspirates e.g. ml/kg  Complications e.g. abdominal distension, constipation, vomiting, diarrhoea

2.4 Data analysis The Imperial College Statistical Advisory Service was engaged for this study. Statistical analysis of clinical variables, HSP70, glutamine and cytokine data was completed using statistical analysis package Graphpad Prism 4.0 for Windows (Graphpad Software, San Diego, CA) and Statistical Package for Social Sciences 19.0 (SPSS: An IBM Company, Chicago, IL). Anthropometric data was analysed using Epi InfoTM Version 3.5.3 (CDC, USA).

2.4.1 Statistical analysis plan for the in vitro model of sepsis examining the effect of glutamine on the release of HSP70 and inflammatory mediators Continuous variables (HSP70 and cytokines) were tested for normality and linearity and were found to be significantly skewed with a non-Gaussian distribution as a result the variables were analysed using non-parametric tests.

To determine statistical significance with respect to the release of HSP70 and inflammatory mediators at comparative time points, (e.g. varying doses of endotoxin, KNK437, with and without glutamine), Mann-Whitney t-test (non-parametric) were used (368).

One way ANOVA’s were used to compare HSP70 and inflammatory mediators between groups e.g. adults and paediatric using Krauskal-Wallis test (non-parametric) to test for significant with a post hoc analysis of Dunn’s to compare all groups for significant differences (368).

Results are presented in this study as means and standard deviations (± SD) and median with range. Statistical significance was achieved with a p-value < 0.05.

2.4.2 Statistical analysis plan to examine relationships between HSP70, glutamine and inflammation in children with acute meningococcal disease compared to convalescence Applying statistical methods described in similar work (369), a range of statistical methods were used as follows:

Continuous variables (HSP70, glutamine and inflammatory mediators) were tested for normality and linearity. Variables were significantly skewed with a non-Gaussian distribution and as such were analysed using non-parametric tests. Difference between acute and convalescent samples were analysed using Mann-Whitney t-test (non-parametric) to determine statistical significance.

Spearman rank order correlation (non-parametric correlation) coefficient (r) was used to determine the correlation between plasma protein measurements of HSP70, glutamine and inflammatory mediators. Pearson correlation coefficient (parametric) was used to analyse normalised log10 transformed variables of HSP70, glutamine and inflammatory mediators. Correlation was deemed to be low when r = +/- 0.10-0.29, medium +/- 0.30-0.49 and high +/- 0.50 – 1.0 (368).

To explore further the relationships between clinical variables of interest and glutamine, HSP70 or cytokines, logistic regression was used, allowing multiple comparisons between

77 variables. Within the clinical database, categorical dependent variables e.g. steroid (yes/no) were used to predict the likelihood of a relationship between continuous clinical variables such as glutamine, HSP70 and cytokines. Using this technique does not imply causality but it does inference about the possibility of certain relationships (368).

Results in this study, are presented as means and standard deviations (±SD), median with the range, correlation coefficients (r) and R2 and adjusted R2 values for regression models. Statistical significant was achieved with a p-value < 0.05 (368).

2.4.3 Statistical plan for analysis of gene expression of HSP70, glutamine and pathways associated with inflammation during acute and convalescence in children with meningococcal disease. Pearson correlation coefficient was used to analyse transcriptomic data and plasma levels of HSP70, glutamine and inflammatory mediators. Correlation was deemed to be low when r = +/- 0.10-0.29, medium +/- 0.30-0.49 and high +/- 0.50 – 1.0 (368).

Results of this study are presented as means and standard deviations (±SD), median with the range, correlation coefficients (r). Statistical significant was achieved with p-value < 0.05 (368).

Chapter 3: In vitro models to consider the effect of glutamine supplementation on endotoxin stimulated release of HSP70 and inflammatory mediators

3.1 Introduction In vitro whole blood endotoxin stimulation models mimic the effect of sepsis, allowing for the study of cellular responses to infection, providing insight into the host response (26, 193, 370). Endotoxin stimulation models have been used for the study of cytokine (371, 372) and HSP70 release (197) in addition to increasing the understanding of the overlap between pro- and anti-inflammatory mediator release following immune activation (26). The use of endotoxin stimulation models have also provided the opportunity to study any potentially unwarranted side effects of a potential therapeutic strategy (26). However, there are limitations to the use of such models in that they cannot completely replicate the individual response to infections as confounding factors also influence individual disease pathogenesis and the specific pathogen involved, as discussed in Chapter 1 (2, 26).

3.2 HSP70 activation of inflammatory signalling pathways As discussed in Chapter 1, HSP70 release primes immune cells to a potential attack from pathogenic organisms promoting the rapid release of bactericides such as nitric oxide and inflammatory mediators. If no attack ensues, the cell can quickly “stand down” with the rapid decline in extracellular HSP70 levels (373). In vitro work by Campisi et al. has shown that when HSP70 forms a complex with endotoxin, there is a greater immunostimulatory effect on pro-inflammatory cytokine release than with either HSP70 or endotoxin alone (176). It has been suggested that the priming of a cell may determine the type of immune response elicited following stress (176). It has been questioned which occurs first i.e. the release of pro-inflammatory cytokines stimulating the release of HSP70 or the converse where the release of HSP70 promotes signalling via TLR2/4 – CD14 and activation of inflammatory pathways such as NF-κB (93, 108, 352).

3.3 Inhibition of heat shock protein 70 and the effect of inflammatory mediators In order to understand the effect of HSP70 on endotoxin release of inflammatory mediators, a range of HSP70 inhibitors have been used (93, 374-376), such as KNK437 (N-formyl-3,4-

80 methylenedioxy-benzylidene-γ-butyrolactam), a benzylidene lactam (198, 375, 377), quercitin (376, 378) and HSP70 anti-sense oligomers (93).

Quercitin is part of the flavanoid family. Flavanoids are phenolic compounds found in some plants which have a wide number of biological actions including antioxidant, antimicrobial, anti-inflammatory and anti-allergenic effects (379). In vitro work using cell lines has shown that quercitin is an effective HSP70 inhibitor (380, 381) although the mechanisms of action are not understood. However, it is known that quercitin also inhibits several kinases including CK2 and MAPK2/ERK (376). As the kinase family are known to be inflammatory mediators (382), effective blocking of their action has an indirect effect on HSP70. CK2 has been shown to upregulate HSP70 production through HSF-1 phosphorylation, therefore by blocking CK2, quercitin inhibits HSP70 production (383). As such the use of quercitin within a whole blood model may not effectively elucidate the effect HSP70 may have on the upregulation of inflammatory mediators.

Unlike quercitin, KNK437 does not have any effect on the kinase family. It inhibits HSP70 production (374, 384-386) by blocking HSF-1 phosphorylation (198) and transcription (387) and is more effective than quercitin (385). The use of KNK437 in an in vitro endotoxin stimulation model, using monocytes, inhibited the release of HSP70 attenuating the release of TNF-α, suggesting that HSP70 facilitates the upregulation of TNF-α via the NK-κB signalling pathway (377).

Anti-sense oligomers have been successfully used in cell line work but not in whole blood. Transfection work shows that endotoxin induced HSP70 mediates inflammatory cytokine release. Conversely when HSP70 production is inhibited, mRNA expression of TNF-α, IL-10, IL-1β and IL-12 is diminished (93). Inhibition of HSP70 only partially prevented cytokine release as the accumulation of TNF-α, IL-10, IL-1β, IL-6 and IL-12 was not completely abrogated in the presence of endotoxin stimulation, suggesting that there are other factors that mediate the release of inflammatory cytokines (93). These results suggest that HSP70 influences the cytokine milieu released mediating an optimal inflammatory response on dependent on the level of stress (93), an effect supported by others (191, 388).

3.4 The effect of glutamine on inflammatory mediators via HSP70 release Glutamine has been shown to attenuate the release of pro-inflammatory cytokines in animal models of sepsis through the inhibition of IKK (IκKα) and the subsequent downregulatory effect on the NF- κB pathway and MAPK pathways (86). In an endotoxin stimulation model TNF-α upregulation is suppressed by glutamine supplementation (2 – 10mM) (389, 390) and in rats pre-treatment with glutamine (through increased HSP70 release) resulted in decreased expression of IL-6 and TNF-α following endotoxin stimulation compared to untreated controls (391). Other endotoxin and injury animal models show similar results, with glutamine attenuating the release of IL-6, TNF-α (86, 390, 392-394), IL -8 (395), IL 1β (393) and IL-10 (392), all of which are potentially mediated by upregulated HSP70 production and provide examples of the dampening effect that HSP70 upregulation can have on the inflammatory response (390). The beneficial effect of glutamine supplementation is lost in the absence of HSP70 gene upregulation in HSP70.1/3 knock-out mice with a subsequent uncontrolled release of TNF-α and IL-6, increasing the rate of mortality compared to normal wild type mice (396). Glutamine depletion in lymphocytes is associated with impaired expression of HSP70 (397) . However, in many of the in vitro sepsis models supraphysiological doses of glutamine e.g. up to 10mM were administered (390, 398, 399) and in other studies where doses of glutamine administered to septic rat models ranged from 0.5g/kg - 0.75g/kg the effect on inflammatory mediators were less pronounced (86, 392, 394, 395, 400).

Thus HSP70 is able to act as a DAMP, upregulating the inflammatory response (401, 402) in addition to acting as a “DAMPERs” resolving inflammation (191, 401, 403), and restoring cell homeostasis (191). As such, the maintenance of an appropriate HSP70 response may diminish the risk of immune paralysis and overwhelming inflammation seen in the severely ill child. Effective therapeutic strategies that harness endogenous defence mechanisms, reducing sepsis and septic shock morbidity are still to be elucidated. However, the use of glutamine to stimulate HSP70 release is an attractive proposition (207, 237).

3.5 Aims In order to understand the effect of glutamine supplementation on HSP70 release and the association with inflammatory markers, we sought to mimic the effects of sepsis through the use of an in vitro endotoxin whole blood model.

3.6 Results A series of experiments were completed to optimise a whole blood endotoxin stimulation model. With respect to glutamine, there were no significant differences in endotoxin (10µg/m) induced HSP70 release between glutamine doses of 2mM, 3.4mM and 6.8mM. However, there was a trend towards significance (p=0.063) in HSP70 release with glutamine supplementation of 2mM at 4 hours (mean 0.51ng/ml, ±SD 0.36) compared to 24 hours (mean 0.78ng/ml, ±SD 0.39), suggesting this dose of glutamine effectively promoted HSP70 release in response to endotoxin stimulation of whole blood in vitro. Therefore, a dose of 2mM of glutamine supplementation was chosen for the remaining whole blood endotoxin stimulation assays (Figure 3.1).

3.6 Optimisation of in vitro HSP70 release with glutamine supplementation in endotoxin stimulated whole blood

0 hours 1 hour 1.4 4 hours 1.3 24 hours 1.2 1.1 1.0 * 0.9 0.8 0.7 0.6

HSP70 (ng/ml)HSP70 0.5 0.4 0.3 0.2 0.1 0.0 No LPS 2mM 2mM 2mM 3.4mM 3.4mM 3.4mM 6.8mM 6.8mM 6.8mM

Glutamine

Figure 3.1: The effect of various doses of glutamine supplementation on HSP70 release using 10µg/ml of endotoxin in whole blood from healthy adult volunteers (n=5). Results are expressed as mean; ± standard error of mean (SEM). Gln = glutamine * NS p=0.063 83

3.7 An in vitro whole blood endotoxin stimulation assay to investigate the relationship between glutamine, HSP70 and inflammatory mediators

3.7.1 Glutamine supplementation promotes HSP70 release in endotoxin stimulated whole blood from healthy adult volunteers at 4 hours and 24 hours

2.5 4 hours No Gln ** 4 hours 2mM Gln 24 hours No Gln 2.0 ** ** 24hr 2mM Gln ** ** **

1.5

** 1.0 HSP70 (ng/ml)HSP70

0.5

0.0

No LPS 0.01g/ml LPS 0.1g/ml LPS 1g/ml LPS Figure 3.2: The effect of glutamine supplementation (2mM) on endotoxin stimulated HSP70 release in whole blood from healthy adult volunteers (n=16) Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin *p<0.05, **p<0.005

There was a significant difference in HSP70 release between groups (p<0.0001). HSP70 release was significantly increased with glutamine supplementation (2mM) in endotoxin stimulated blood at 24 hours in all conditions compared to controls without glutamine, including those conditions with no endotoxin. In addition there was a significant difference in HSP70 release with glutamine supplementation at 4 hours with 1µg/ml endotoxin compared to control condition without glutamine. The release of HSP70 also significantly increased over time from 4 hours to 24 hours, including those conditions with no endotoxin. The relative similarity in HSP70 release between conditions are in contrast to those pertaining to the release of inflammatory mediators, indicating this phenomenon is not as a result of endotoxin contamination and is more likely to be reflective of a physiological response to the experimental conditions (Figure 3.2).

3.7.2 Glutamine supplementation in endotoxin stimulated whole blood appears to attenuate the release of inflammatory cytokines

The effect of glutamine supplementation on endotoxin stimulated release of inflammatory cytokines (IL-6, IL-8, IL-1β and TNF-α) was investigated.

3.7.3 Glutamine supplementation in endotoxin stimulated whole blood from healthy adult volunteers attenuates the release of IL-1β at 4 hours and 24 hours

* 13000 12000 11000 *** 10000 ** 4hours No Gln 9000 4 hours 2mM Gln 8000 24hrs No Gln

7000 24hrs 2mM Gln (pg/ml)

 6000

IL-1 5000

4000 3000

2000 1000 0

No LPS 0.01g/ml LPS 0.1g/ml LPS 1g/ml LPS

Figure 3.3: The effect of glutamine supplementation on endotoxin stimulated release of IL- 1β at 4 hours and 24 hours in whole blood from healthy adult volunteers (n=12) Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin *p<0.05, **p<0.005, *** p<0.0005

There was a significant difference in IL-1β release between groups (p<0.0001). As expected endotoxin stimulation significantly increased IL-1β release over time with (p<0.005) and without (p<0.0005) glutamine supplementation at 24 hours with 1µg/ml endotoxin and at 0.01µg/ml 24 hours with glutamine supplementation compared to 4 hours (p<0.05). Although there were no other significant differences glutamine supplementation (2mM) appears to attenuate 1L-1β release at 24 hours compared to those without glutamine supplementation, although this effect was not significant (Figure 3.3).

3.7.4 Glutamine supplementation in endotoxin stimulated whole blood from healthy adult volunteers attenuates the release of IL-8 at 4 hours and 24 hours

45000 *

40000

35000 ** * 4hours No Gln 30000 4 hours 2mM Gln 24hrs No Gln 25000 24hrs 2mM Gln

20000 IL-8 (pg/ml) 15000

10000

5000

0 No LPS 0.01g/ml LPS 0.1g/ml LPS 1g/ml LPS Figure 3.4: The effect of glutamine supplementation (2mM) on LPS stimulated release of IL-8 at 4 hours and 24 hours in whole blood from healthy adult volunteers (n=12). Results are expressed as mean; ± SEM. Gln = glutamine. LPS = endotoxin *p<0.05, **p<0.005

There was a significant difference in IL-8 release between groups (p<0.0001). Glutamine supplementation (2mm) significantly attenuated the release of IL-8 at 24 hours 1µg/ml endotoxin compared to control conditions with no glutamine (p<0.05). Although there were no other significant differences in glutamine supplemented conditions at 24 hours with 0.01 – 0.1 µg/ml endotoxin compared to control conditions, glutamine appeared to attenuate the release of IL-8. Over time, IL-8 release was significantly increased in 0.1µg/ml (p<0.05) and at 1µg/ml endotoxin groups (p<0.005) at 24 hours compared to 4 hours without glutamine supplementation (Figure 3.4).

3.7.5 Glutamine supplementation in endotoxin stimulated whole blood from healthy adult volunteers attenuates the release of TNF-α at 4 hours and 24 hours

8000

7000

6000 4hours No Gln 4 hours 2mM Gln 5000 24hrs No Gln 24hrs 2mM Gln

(pg/ml) 4000 

TNF- 3000

2000

1000

No LPS 0.01g/ml LPS 0.1g/ml LPS 1g/ml LPS

Figure 3.5: The effect of glutamine supplementation (2mM) on endotoxin stimulated release of TNF-α at 4 hours and 24 hours in whole blood from healthy adult volunteers (n=12). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin.

There was a significant difference in TNF-α release between groups (p<0.0001). Although, there was no significant effect on TNF-α release with glutamine supplementation (2mM), TNF-α release at 4 hours and 24 hours appeared to be attenuated in glutamine conditions compared to those without glutamine supplementation (Figure 3.5).

3.7.6 Glutamine supplementation in endotoxin stimulated whole blood from healthy adult volunteers attenuates the release of IL-6 at 4 hours and 24 hours

65000 60000 55000 50000 * 4hours No Gln 45000 4 hours 2mM Gln 40000 24hrs No Gln 35000 24hrs 2mM Gln 30000

IL-6 (pg/ml) 25000 20000 15000 10000 5000 0

No LPS 0.01g/ml LPS 0.1g/ml LPS 1g/ml LPS Figure 3.6: The effect of glutamine supplementation (2mM) on LPS stimulated release of IL-6 at 4 hours and 24 hours in whole blood from healthy adult volunteers (n=12). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin *p=0.05

There was a significant difference in IL-6 release between groups (p<0.0001). Endotoxin stimulation significantly increases IL-6 release at 24 hours compared to 4 hours with and without glutamine supplementation (p<0.05). There were no significant differences between conditions with and without glutamine supplementation (2mM). Although not statistically significant, glutamine supplementation appears to attenuate 1L-6 release at 4 hours and 24 hours compared to those without glutamine supplementation (Figure 3.6).

3.7.7 Glutamine supplementation in endotoxin stimulated whole blood from healthy adult volunteers attenuates the release of IL-10 at 4 hours and 24 hours

2000 4 hrs No Gln * 4 hr Gln ** 24hr No Gln 24hr Gln 1500

1000 IL-10 (pg/ml) IL-10

500 *

No LPS 1g/ml LPS Figure 3.7: The effect of glutamine supplementation (2mM) on LPS stimulated release of IL-10 at 4 hours and 24 hours in whole blood from healthy adult volunteers (n=18). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin *p<0.05, **p<0.005

There was a significant difference in IL-6 release between groups (p<0.0001). As expected, endotoxin stimulation significantly increases IL-10 release at 24 hours compared to 4 hours with and without glutamine supplementation (p<0.05). There was a significant difference between IL-10 release at 4 hours with glutamine compared to control conditions (p<0.05). Although not significant at 24 hours glutamine supplementation appears to attenuate 1L-10 release compared to control conditions (Figure 3.7).

3.7.8 Relationships between HSP70 and inflammatory mediators in adult in vitro endotoxin stimulated whole blood following glutamine supplementation (2mM) Spearmen’s correlation was used to investigate whether there were relationship between HSP70 and inflammatory mediators (IL-1β, TNF-α, IL-6, IL-8, IL-10) in adult in vitro endotoxin stimulated whole blood following glutamine supplementation (2mM). Interestingly there

89 were no relationships between IL-6 or IL-10 or HSP70 release at any of the endotoxin doses or time points, following glutamine supplementation. Positive and inverse correlations were found at 4 hours for IL-1β, TNF-α, IL-8 and IL- (Table 3.1) but none at 24 hours.

Table 3.1 Relationships between adult in vitro endotoxin stimulated HSP70 release and inflammatory mediators following glutamine supplementation (2mM) IL-1β (pg/ml) IL-1β (pg/ml) TNF-α (pg/ml) IL-8 (pg/ml) 0.01μg/ml LPS 1μg/ml LPS 0.1μg/ml LPS 0.1μg/ml LPS 4 hours Gln 4 hours Gln 4 hours Gln 4 hours Gln HSP70 (ng/ml) r=0.51 4 hours Gln p=0.03 0.01μg/ml LPS n=16 HSP70 (ng/ml) r=-0.56 r=-0.52 4 hours Gln p=0.02 p=0.03 0.1μg/ml LPS n=16 n=16 HSP70 (ng/ml) r=0.47 4 hours Gln p=0.04 1μg/ml LPS n=16

3.7.9 Summary of results for glutamine supplementation (2mM) in endotoxin stimulation model and the effect on the release of HSP70 and inflammatory mediators Glutamine supplementation (2mM) significantly upregulates HSP70 release in endotoxin stimulated whole blood at 24 hours compared to conditions without glutamine. There were some positive and inverse correlations between HSP70 release and inflammatory mediators at 4 hours (IL-1β, TNF-α, IL-8) although there were no correlations between HSP70 and IL6 or IL-10. Glutamine supplementation significantly attenuated the release of IL-10 at 4 hours and IL-8 at 24 hours compared to conditions without glutamine. In endotoxin stimulated blood there were no significant differences in the release of IL-6, TNF-α and IL-1β with glutamine supplementation at 4 and 24 hours. However, glutamine supplementation (2mM) appeared to attenuate the release of inflammatory mediators (IL-1β, IL-6, TNF-α), although this effect was not statistically significant.

3.8 Results of the in vitro model examining the inhibitory effects of KNK437 on HSP70 release and markers of the inflammatory responses following endotoxin stimulation and glutamine supplementation (2mM) in healthy adult volunteers

To investigate whether HSP70, in part, mediates the release of inflammatory markers, an experiment was designed using KNK437 with the aim of inhibiting HSP70 release.

3.8.1 With the addition of KNK437 to endotoxin stimulated whole blood the effect of glutamine supplementation (2mM) on extracellular HSP70 release at 24 hours is abrogated

4 4hr No Gln 4hr 2mM Gln ** 24hr No Gln * 24hr 2mM Gln 3 4hr KNK437 200M No Gln 4hr KNK437 200M Gln 2mM

4hr KNK437 2 200M No Gln

4hr KNK437

HSP70 (ng/ml)HSP70 200M Gln 2mM

No LPS 1g/ml LPS

Figure 3.8: The effect of KNK437 on extracellular HSP70 release at 4 hours and 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) (n=18). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin, *p<0.05, **p<0.005

There was a significant difference in extracellular HSP70 release between groups (p<0.0001). The addition of KNK437 significantly inhibited extracellular HSP70 release in conditions at 24 hours with glutamine supplementation (2mM) (p<0.05) and without glutamine (p<0.005), suggesting the beneficial effect of glutamine on HSP70 release is lost with the use of KNK437 at 24 hours but not at 4 hours. In addition, in the endotoxin stimulated group with the

91 addition of KNK437 there were no significant differences between extracellular HSP70 release in conditions with and without glutamine (p>0.05) (Figure 3.8).

3.8.2 The effect on glutamine supplementation (2mM) on intracellular HSP70 release at 4 hours and 24 hours in endotoxin stimulated whole blood is abrogated with the use of KNK437

** * 22.5 * * 4hr No Gln 4hr 2mM Gln 20.0 24hr No Gln 17.5 24hr 2mM Gln 4hr KNK437 200M No Gln 15.0 4hr KNK437 200M Gln 2mM 12.5 4hr KNK437 200M No Gln 10.0 4hr KNK437

HSP70 (ng/ml)HSP70 200M Gln 2mM 7.5

5.0

2.5

0.0

No LPS 1g/ml LPS

Figure 3.9: The effect of KNK437 on intracellular HSP70 release at 4 hours and 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) (n=18). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin. *p=<0.05, **p=<0.005

There was a significant difference in intracellular HSP70 release between groups (p<0.0001). In the endotoxin stimulated group the addition of KNK437 significantly inhibited the intracellular HSP70 release at 4 hours and 24 hours with glutamine supplementation (2mM) (p<0.005). In addition there were significant differences between the intracellular release of HSP70 at 4 hours (p<0.005) and 24 hours (p<0.05) without glutamine supplementation. These results suggest the release of intracellular HSP70 in endotoxin stimulated whole blood is inhibited following the addition of KNK437, when compared to endotoxin stimulation 92 alone. In addition, in the endotoxin stimulated group with the addition of KNK437 there were no significant differences between intracellular HSP70 release in conditions with and without glutamine (p>0.05). The effect of glutamine on HSP70 release also appears to be lost following the addition of KNK437 (Figure 3.9).

3.8.3 The addition of KNK437 diminishes the release of inflammatory mediator in endotoxin stimulated whole blood To consider the effect of HSP70 on in endotoxin stimulated whole blood following the addition of KNK437, the release of inflammatory markers were investigated.

3.8.4 KNK437 attenuates the release of extracellular IL-6 in endotoxin stimulated whole blood

** * ** 225000 * 4hr No Gln 4hr 2mM Gln 200000 24hr No Gln 175000 24hr 2mM Gln 4hr KNK437 200M No Gln 150000 4hr KNK437 200M Gln 2mM 125000 4hr KNK437 200M No Gln 100000 4hr KNK437 IL-6 (pg/ml) 200M Gln 2mM 75000

50000

25000

No LPS 1g/ml LPS

Figure 3.10: The effect of KNK437 on extracellular IL-6 release at 4 hours and 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) (n=18). Results are expressed as mean; ± SEM. Gln = glutamine. *p<0.05, **p<0.005

There was a significant differences in extracellular IL-6 release between groups (p<0.0001). Following the addition of KNK437 in endotoxin stimulated whole blood, the release of IL-6 was significantly attenuated compared to conditions without the addition of KNK437. Extracellular release of IL-6 was significantly inhibited with the addition of KNK437 at 4 hours with glutamine supplementation (2mM) (p<0.05) and without (p<0.005), in addition to 24 hours with and without glutamine (p<0.05). In the endotoxin stimulated condition there was no significant difference between IL-6 release with glutamine supplementation (2mM) at 4 or 24 hours compared to conditions without glutamine supplementation (Figure 3.10).

3.8.5 KNK437 attenuates the release of intracellular IL-6 in endotoxin stimulated whole blood

* * * 14000 * 4hr No Gln 13000 4hr 2mM Gln 12000 24hr No Gln 11000 24hr 2mM Gln 10000 4hr KNK437 200M No Gln 9000 4hr KNK437 200M Gln 2mM 8000 4hr KNK437 7000 200M No Gln

6000 4hr KNK437 IL-6 (pg/ml) 5000 200M Gln 2mM 4000 3000 2000 1000 0 No LPS 1g/ml LPS

Figure 3.11: The effect of KNK437 on intracellular IL-6 release at 4 hours and 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) (n=18). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin. *p<0.05,

**p<0.005

There was a significant differences in intracellular IL-6 release between groups (p<0.0001). In the endotoxin stimulated group KNK437 had a significant inhibitory effect on intracellular IL- 6 release in conditions with and without glutamine at 4 hours (p<0.05) and 24 hours with 94 glutamine (p<0.05) and without glutamine (p<0.005), suggesting that HSP70 inhibition affects the intracellular release of IL-6. There was no significant difference between endotoxin stimulated groups with glutamine supplementation (2mM) compared to the control condition without glutamine at either time point (Figure 3.11).

3.8.6 KNK437 attenuates the release of extracellular TNF-α in endotoxin stimulated whole blood

* 1000 * 4hr No Gln * 4hr 2mM Gln * 24hr No Gln 24hr 2mM Gln 750 4hr KNK437 200M No Gln 4hr KNK437 200M Gln 2mM

4hr KNK437

(pg/ml) 500 200M No Gln  4hr KNK437

TNF- 200M Gln 2mM

250

No LPS 1g/ml Figure 3.12: The effect of KNK437 on extracellular TNF-α release at 4 hours and 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) (n=18). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin * p<0.05

There was a significant differences in extracellular TNF-α release between groups (p<0.0001). KNK437 had a significant inhibitory effect on extracellular TNF-α release with and without glutamine (p<0.05) at 4 hours and 24 hours, suggesting that HSP70 inhibition affects the release of TNF-α. In the endotoxin stimulated conditions with KNK437 there were no significant differences between conditions with and without glutamine at 4 and 24 hours (Figure 3.12).

3.8.7 KNK437 attenuates the release of intracellular TNF-α in endotoxin stimulated whole blood

*** ** 2250 *** 4hr No Gln 4hr 2mM Gln 2000 24hr No Gln 1750 *** 24hr 2mM Gln 4hr KNK437 200M No Gln 1500 4hr KNK437 200M Gln 2mM 1250 4hr KNK437

(pg/ml) 200M No Gln  1000 4hr KNK437

TNF- 200M Gln 2mM 750

500

250

No LPS 1g/ml LPS

Figure 3.13: The effect of KNK437 on intracellular TNF-α release at 4 hours and 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (2mM) (n=18). Results are expressed as mean; ± SEM. Gln = glutamine, LPS=endotoxin. **p<0.005, ***p<0.0001

There was a significant difference in intracellular TNF-α release between groups (p<0.0001). In the endotoxin stimulated group KNK437 had a significant inhibitory effect on intracellular TNF-α release with glutamine (2mM) (p<0.005) and without glutamine (p<0.0001) at both 4 hours and 24 hours with and without glutamine (p<0.0001), suggesting that HSP70 inhibition affects the release of TNF-α. In the endotoxin stimulated conditions with KNK437 there was no significant difference between glutamine supplemented groups compared to control groups (Figure 3.13)

3.8.8 KNK437 attenuates the release of extracellular IL-10 in endotoxin stimulated whole blood

650 * ** 4hr No Gln 600 4hr 2mM Gln 550 24hr No Gln 24hr 2mM Gln 500 4hr KNK437 450 200M No Gln 4hr KNK437 400 200M Gln 2mM

350 4hr KNK437 200M No Gln 300 4hr KNK437 IL-10(pg/ml) 250 200M Gln 2mM 200 150 100 50 0

No LPS 1g/ml LPS

Figure 3.14: The effect of KNK437 on extracellular IL-10 release at 4 hours and 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (n=18). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin *p<0.05, **p<0.005

There were significant differences in extracellular IL-10 release between groups (p<0.0001). In endotoxin stimulated conditions the addition of KNK437 had a significant inhibitory effect on extracellular IL-10 release at 4 hours without glutamine (p<0.05), in addition to 24 hours with (p<0.005) and without (p<0.05) glutamine, suggesting the inhibition of HSP70 with KNK437 affects the release of IL-10. There was no significant difference between glutamine supplementation conditions in endotoxin stimulated blood with the addition of KNK437 (Figure 3.14).

3.8.9 KNK437 attenuates the release of intracellular IL-10 in endotoxin stimulated whole blood

* *

1250 ** * 4hr No Gln 4hr 2mM Gln 24hr No Gln 1000 24hr 2mM Gln 4hr KNK437 200M No Gln 4hr KNK437 750 200M Gln 2mM

4hr KNK437 200M No Gln

500 4hr KNK437 IL-10 (pg/ml) 200M Gln 2mM

250

0 No LPS 1g/ml LPS

Figure 3.15: The effect of KNK437 on intracellular IL-10 release at 4 hours and 24 hours in endotoxin stimulated whole blood with and without glutamine supplementation (n=18). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin *p<0.05, **p<0.005

There were significant differences in intracellular IL-10 release between groups (p<0.0001). In the endotoxin stimulated group KNK437 had a significant inhibitory effect on intracellular IL-10 release without glutamine at 4 hours (p<0.05) in addition to with and without glutamine at 24 hours (p<0.05), suggesting that with KNK437 the release of IL-10 is inhibited. In the endotoxin stimulated KNK437 group there were no significant differences between conditions with and without glutamine (Figure 3.15).

3.8.10 Summary of KNK437 results from in vitro adult endotoxin stimulation model The addition of KNK437 to the endotoxin stimulation model appeared to significantly inhibit the intracellular and extracellular release of HSP70 and inflammatory mediators (IL-6, IL-10, TNF-α) abrogating the effect of glutamine supplementation.

3.9 An in vitro model examining the effects of endotoxin and glutamine on HSP70 release and markers of the inflammatory responses in healthy paediatric volunteers

The effect of glutamine supplementation on HSP70 and inflammatory mediator release in endotoxin stimulated blood from children was investigated. Samples were processed using methods described in Chapter 2.

3.9.1 Glutamine supplementation upregulates HSP70 release in endotoxin stimulated whole blood from healthy paediatric volunteers

20 *** 4 hr No Gln ** 4 hrs 2mM Gln 24 hr No Gln *** 24 hrs 2mM gln 15 *** ***

10 HSP70 (ng/ml) HSP70

No LPS 1g/ml LPS

Figure 3.16: The effect of glutamine supplementation (2mM) on LPS stimulated HSP70 release at 4 hours and 24 hours in whole blood from healthy paediatric volunteers (n=25). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin **p<0.005, ***p<0.0005

There were significant differences in HSP70 release between 4 hours and 24 hours (p<0.0001). Glutamine supplementation significantly upregulates HSP70 release at 24 hours in 1μg/ml endotoxin stimulated whole blood (p<0.05) compared to conditions without glutamine supplementation. As expected the release of HSP70 significantly increased over

99 time at 4 hours and 24 hours in all conditions (p<0.0005) As with the adult endotoxin model the release of HSP70, similarly increased over time from 4 hours to 24 hours in conditions with no endotoxin. As the release of inflammatory mediators, within the paediatric model indicated low levels of release with no endotoxin it is likely the methods are robust and the release of HSP70 in unstimulated blood is in response to the experimental conditions (Figure 3.16).

3.9.2 The effect of glutamine supplementation on release of inflammatory cytokines using endotoxin stimulated whole blood from healthy paediatric volunteers

The effect of glutamine supplementation on the release of inflammatory cytokines IL-6, TNF- α and IL-10, in endotoxin stimulated whole blood using one dose of LPS, 1µg/ ml LPS without and without glutamine supplementation was investigated.

3.9.3 The release of IL-10 in endotoxin stimulated whole blood from healthy paediatric volunteers was not influenced by glutamine supplementation (2mM)

2500 4 hr No Gln *** *** 4 hrs 2mM Gln 24 hr No Gln 2000 24 hrs 2mM Gln

1500

1000 IL-10 IL-10 (pg/ml)

* 500 **

No LPS 1g/ml LPS Figure 3.17: The effect of glutamine supplementation on endotoxin stimulated release of IL-10 in whole blood from healthy paediatric volunteers at 4 hours and 24 hours (n=25). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin. *p=0.05, **p<0.005, ***p<0.0005

100

There were significant differences in IL-10 release between groups (p<0.0001). There was a significant difference between IL-10 release with glutamine supplementation (2mM) with no endotoxin at 24 hours (p<0.05) but not in the endotoxin stimulated conditions. As would be expected there were significant differences in the release of IL-10 in the endotoxin stimulation group at 4 hours and 24 hours with and without glutamine supplementation (p<0.0005). Glutamine supplementation had no significant effect on the release of IL-10 (Figure 3.17).

3.9.4 The release of IL-6 in endotoxin stimulated whole blood from healthy paediatric volunteers was not influenced by glutamine supplementation (2mM)

125000 *** 4 hr No Gln *** 4 hrs 2mM Gln 24 hr No Gln 100000 24 hrs 2mM Gln

75000

50000 IL- 6 (pg/ml)

25000

No LPS 1g/ml LPS Figure 3.18: The effect of glutamine supplementation on endotoxin stimulated release of IL-6 in whole blood from healthy paediatric volunteers at 4 hours and 24 hours (n=25). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin ***p<0.0005

There were significant differences between IL-6 groups (p<0.0001). As expected in endotoxin stimulated whole blood there was a significant increase in the release of IL-6 at 24 hours compared to 4 hours with and without glutamine supplementation (2mM) (p<0.0005). There was no significant difference in IL-6 release following glutamine supplementation in endotoxin stimulated whole blood, at 4 or 24 hours, though at the latter timepoint there

101 appeared to be a slightly higher level of measured IL-6 in glutamine supplemented wells compared to non-supplemented controls Figure 3.18).

3.9.5 The release of TNF-α in endotoxin stimulated whole blood from healthy paediatric volunteers was augmented with glutamine supplementation (2mM)

* 2750 4 hr No Gln 2500 4 hrs 2mM Gln 24 hr No Gln 2250 24 hrs 2mM Gln 2000

1750 **

1500 (pg/ml)

 1250

TNF- 1000

750 * 500

250

No LPS 1g/ml LPS Figure 3.19: The effect of glutamine supplementation on endotoxin stimulated release of TNF-α in whole blood from healthy paediatric volunteers at 4 hours and 24 hours (n=25). Results are expressed as mean; ± SEM. Gln = glutamine, LPS = endotoxin *p<0.05, **p<0.005

There were significant differences between TNF-α groups (p<0.0001). As to be expected there were statistically significant differences in TNF-α release in endotoxin stimulated blood at 4 hours and 24 hours with (p<0.005) and without glutamine (p<0.05). Glutamine supplementation in endotoxin stimulated conditions stimulated TNF-α release at 4 hours (p<0.05), suggesting under these conditions glutamine promoted a pro-inflammatory response (Figure 3.19).

102

3.9.6 Relationships between HSP70 and inflammatory mediators in paediatric in vitro endotoxin stimulated whole blood following glutamine supplementation (2mM) Spearmen’s correlation was used to investigate whether there was a relationship between HSP70 and inflammatory mediators in paediatric in vitro endotoxin stimulated whole blood following glutamine supplementation (2mM). There were relationships between HSP70 and IL-6, TNF-α at 4 hours following glutamine supplementation but no relationship between HSP70 and IL-10 (Table 3.2).

Table 3.2 Relationships between paediatric in vitro endotoxin stimulated HSP70 release and inflammatory mediators following glutamine supplementation (2mM) IL-6 (pg/ml) TNF-α (pg/ml) 1μg/ml LPS 0.1μg/ml LPS 4 hours Gln 4 hours Gln HSP70 (ng/ml) r=0.61 r=0.60 4 hours Gln p=0.004 p=0.005 1μg/ml LPS n=25 n=25

3.9.7 Summary of experimental results of in vitro paediatric endotoxin stimulation model Glutamine supplementation significantly increases HSP70 release in paediatric endotoxin stimulation model at 24 hours. The effect of glutamine supplementation on the release of inflammatory mediators was less clear. There were positive correlations with IL-6 and TNF-α in glutamine supplemented conditions at 4 hours, but no relationships between HSP70 and IL-10 following glutamine supplementation at either time point. In endotoxin stimulated whole blood there was a significant difference in the release of TNF-α at 4 hours with glutamine supplementation compared to conditions without glutamine. In these circumstances glutamine supplementation appeared to promote the release of TNF-α suggesting a pro-inflammatory effect. A similar effect was seen with IL-6, although this was not significant. Glutamine supplementation appeared to have no effect on the release of IL- 10.

103

3.10 Comparison between HSP70 in a whole blood endotoxin stimulation model with healthy paediatric volunteers and healthy adult volunteers There were significant differences in HSP70 release between healthy adult volunteers (n=18) and healthy paediatric volunteers (n=25) (p<0.0001). HSP70 release in paediatric samples was statistically significantly higher than in adults in all comparable conditions with and without glutamine supplementation (Table 3.3)

Table 3.3: A comparison of HSP70 release in adult and paediatric samples Conditions HSP70 Adult ng/ml HSP70 Paediatric Statistical mean (±SD) ng/ml mean (±SD) Significance 4 hours no LPS without 0.53 (0.31) 4.50 (2.9) p<0.01 glutamine 4 hours no LPS with 0.50 (0.47) 4.65 (2.5) p<0.01 glutamine 24 hours no LPS 1.05 (0.76) 10.20 (5.57) p<0.001 without glutamine 24 hours no LPS with 1.18 (0.65) 12.97 (6.92) p<0.001 glutamine 4 hours 1µg/ml LPS 0.45 (0.32) 3.79 ( 3.6) p<0.01 without glutamine 4 hours 1µg/ml LPS 0.83 (0.69) 4.62 (2.3) p<0.01 with glutamine 24 hours 1µg/ml LPS 1.26 (1.37) 13.1 (4.4) p<0.001 without glutamine 24 hours 1µg/ml LPS 1.91 (1.41) 16.01 (6.7) p<0.001 with glutamine

3.10.1 Comparison between inflammatory mediator (IL-6, IL-10 and TNF-α) release in a whole blood endotoxin stimulation model with healthy paediatric volunteers and healthy adult volunteers Using Kruskal-Wallis one way ANOVA of variance there was a significant difference in the release of inflammatory mediators (IL-6, IL-10, TNF-α) between comparable conditions in the healthy adult and paediatric groups (p<0.0001). However, a post-hoc test using Dunn’s multiple comparisons showed there were no significant differences between comparative adult and paediatric samples (p>0.05). In endotoxin stimulated blood, HSP70 release in children was significantly higher than in adults, although there was no significant difference in the release of inflammatory mediators.

104

3.11 Discussion The use of in vitro endotoxin stimulation models to study the effects of sepsis are well described (26, 193, 370), and have been used to investigate the release of HSP70 (93, 197) and inflammatory mediators (371, 372), furthering the understanding of the inflammatory response following immune activation (26). In experimental models of sepsis, HSP70 release has been shown to improve outcome (129), decreasing mortality (310), which is associated with diminished release of inflammatory mediators such as TNF-α (404).

The use of endotoxin from E. coli 0111:B4 has been used in other in vitro models considering HSP70 release (41, 128, 193-196) and doses of 0.1μg – 10μg/ml have been shown to be effective to upregulate the release of HSP70 (93, 390, 405-407). The results from the in vitro stimulation model showed a range of endotoxin doses (0.1μg – 1μg/ml) were effective in promoting HSP70 release in both children and adults, although these levels of endotoxin are in excess of any described during critical illness (0.03 – 0.3μg/ml) (372, 408).

Glutamine supplementation (2mM) in the adult and paediatric in vitro endotoxin stimulation model significantly increased HSP70 release at 24 hours, compared to conditions without glutamine, including those conditions with no endotoxin. There was a significant release of HSP70 in unstimulated whole blood control wells although, this was unlikely to be as a result of endotoxin contamination given that inflammatory mediator levels were low at each timepoint in control wells. Instead it is likely to be a physiological response to the experimental conditions, and has previously been described in vitro and in vivo studies (215, 310, 311, 409). Glutamine has been shown to effectively promote HSP70 release in experimental sepsis (in vivo and in vitro) (310, 311, 345, 390) and during critical illness (143), as described in Chapter 1. The concomitant administration of endotoxin and glutamine (0.75g/kg) together in a septic rat model was shown to significantly increase the release of HSP70, improving ADP/ATP ratio reducing metabolic dysfunction (311). The dose of glutamine supplementation (2mM) used in this experimental model was modest compared to those reported in other in vitro models, considering HSP70 release, where between 0.5mM – 10mM glutamine have been used (203, 314, 390, 407). Doses of less than 2mM glutamine did not significantly increase HSP70 release (390). The endotoxin stimulated release of HSP70 was significantly higher with glutamine supplementation in children (mean

105

16.01ng/ml; ±SD 6.7) compared to adult (mean 1.91ng/ml; ±SD 1.41). This pattern of release has previously been described in critically ill adults (143) and children with septic shock (141). HSP70 transcription decreases with age and the release in adults and mature animal models is considerably less than observed in youth (135, 410) as confirmed by the results seen in the paediatric and adult in vitro endotoxin stimulation model.

The clinical concern, relative to this phenomenon, would be the effect of significantly higher HSP70 levels on the inflammatory response and the potential to over stimulate the release of inflammatory mediators in response to sepsis (141), especially since an excessive release of inflammatory mediators is associated with increased risk mortality (63, 87-89). Even though, the in vitro release of HSP70 between adults and children was significantly different, this did not appear to result in a heightened release of inflammatory mediators in the paediatric model, and there were no significant differences relative to the release of TNF-α, IL-6 and IL-10 between the two comparable models. As such, although HSP70 levels were considerably higher in endotoxin stimulated whole blood from healthy children this did not appear to result in a significantly elevated release of inflammatory mediators.

Glutamine supplementation significantly attenuated the in vitro release of IL-10 at 4 hours and IL-8 at 24 hours in endotoxin stimulated adult whole blood, which has been described in experimental rat endotoxin models (392, 411) and from human duodenal biopsies (412). There was however, no significant difference in endotoxin stimulated release of IL-1β, IL-6 and TNF-α following glutamine supplementation, although glutamine did appear to attenuate the release of inflammatory mediators. Results from other in vitro work has shown that glutamine supplementation in doses greater than 2mM significantly attenuates the release of TNF-α and IL-10 in human blood peripheral mononuclear cells (86) and in a rat model of sepsis (392), in addition to IL-6 and IL-8 in human intestinal epithelial and cell lines (203, 398, 412, 413). As a result it is likely that glutamine supplementation in excess of 2mm is required to significantly attenuate the release of inflammatory mediators.

Glutamine supplementation in endotoxin stimulated blood from paediatric volunteers significantly increased the release of TNF-α at 4 hours. Although, at 24 hours the release of IL-6 and TNF-α in endotoxin stimulated blood was not significantly different, glutamine

106 supplementation appeared to promote the release of these inflammatory mediators. Glutamine supplementation significantly increased IL-10 release at 24 hours in endotoxin free conditions, but had no discernible effect on IL-10 at 4 or 24 hours following endotoxin stimulation. Glutamine supplementation has been reported to promote a pro-inflammatory response in other in vitro work using human macrophages (399, 407). As glutamine is rapidly consumed by immune cells, it could be argued that glutamine supplementation provided substrate for optimal HSP70 production supporting an appropriate inflammatory response (409), which in the paediatric model may be pro-inflammatory. It could also be that in the paediatric model where HSP70 release is significantly higher compared to adults, a larger dose of glutamine may be required to mediate a dampening or anti-inflammatory effect on the release of inflammatory mediators (203, 398, 412, 413). Further to this, doses of between 4 – 10mM of glutamine are required to attenuate TNF-α, IL-8 and IL-6 release following HSP70 upregulation (86, 314, 348) and although a dose of 2mM of glutamine was able to significantly upregulate the release of HSP70 it may not have been sufficient to attenuate the release of inflammatory mediators as described elsewhere (390).

The amount of HSP70 released may influence the temporal mix of inflammatory mediators. However, the relationship relative to the amount of HSP70 protein released and subsequent inflammatory response is not well explored. There is some work from an in vitro (PBMC’s) model of sepsis showing over production of HSP70 limits the release of IL-1β, TNF-α, IL-10 and IL-12, however, the relative amount of HSP70 released is not provided (93) and it may still be lower than recorded in the in vitro paediatric model. It could argued that in the adult model, glutamine supplementation attenuated the release of the inflammatory mediator relative to the low HSP70 release (1.91ng/ml; ±SD 1.41) when compared to HSP70 levels in children (16.01ng/ml; ±SD 6.7) and it may be that the difference in the patterns of HSP70 release influence the mix of inflammatory mediators.

In this work, HSP70 levels in vitro rose over time and were correlated with inflammatory mediators (IL-1β, TNF-α, IL-8) in adults, and IL-6, TNF-α in paediatric samples at 4 hours, following glutamine supplementation (2mM). The effects seen suggest the release of HSP70 influences the inflammatory response, as described in other work (93), although further work would be required to investigate these relationships to establish causality. Interestingly

107 there were no relationships between HSP70 and IL-10 in either adults or children following glutamine supplementation, which may be relative to the small sample size.

It has been hypothesised that, in the presence of an infection, glutamine is rapidly consumed reducing the amount available for stimulating HSP70 production and the subsequent influence on the inflammatory response (409). Glutamine (2mM) is the equivalent to 0.3g/kg glutamine, and it may be that a larger dose of 4mM glutamine is required (0.6g/kg) to significantly attenuate the release of inflammatory mediators (390), which is also reflected in the result from glutamine clinical trials in critically ill adults (237). The dichotomy of these results, with respect to the release of inflammatory mediators are surprising, however results from other endotoxin in vitro work show the release of HSP70 can mediate both a pro- (399, 407) or anti-inflammatory (414-416) response. Furthermore, glutamine supplementation has been also been shown to stimulate a pro-inflammatory response in vitro (399) but in vivo is shown to attenuate pro-inflammatory cytokine release during sepsis (86, 399, 417, 418), which is likely to be as a result of increased HSP70 release (390).

In order to understand the influence of HSP70 on the release of inflammatory mediators, a HSP70 inhibitor (KNK437) was used. KNK437 has been shown to be effective at preventing HSP70 release in vitro (387) and in vivo models (419) by inhibiting HSF-1 transcription (198) significantly reducing the amount of HSP70 release (385). Other work using quercitin as the HSP70 inhibitor, show the effects of glutamine on HSP70 release are almost completely inhibited (311). Results from the adult endotoxin stimulation model show KNK437 significantly inhibited the extracellular release of HSP70 at 24 hours in conditions with and without glutamine supplementation (2mM). In addition, the intracellular release of HSP70 in endotoxin stimulated blood was significantly inhibited at 4 hours and 24 hours in conditions with and without glutamine supplementation compared to comparable endotoxin stimulated whole blood, a finding reported in other similar models (420, 421). The effect of glutamine supplementation, following the addition of KNK437, on HSP70 release in the adult endotoxin stimulation model was abrogated, similar to findings described by Singleton et al. (311).

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In endotoxin stimulated blood KNK437 had a significant inhibitory effect on the intracellular and extracellular release of inflammatory mediators (IL-6, IL-10, TNF-α) at 4 hours and 24 hours in conditions with and without glutamine compared to similar endotoxin stimulated blood. There was no significant difference in the release of inflammatory mediators between glutamine and unsupplemented conditions in endotoxin stimulated blood with KNK437. Lee et al. showed the use of KNK437 in endotoxin pre-treated rat macrophages, inhibited the expression of HSP70, partially preventing the release of TNF-α (422). In a hyperthermic mouse model, the addition of KNK437 inhibited the release of HSP70 and IL-6 (423). The inhibitory effect of KNK437 on HSP70 release, suggests that in part, HSP70 orchestrates the release of inflammatory mediators in response to stress, which is reported in other models (93).

Within the context of a whole systems perspective, it is hypothesised that during an infection HSP70 forms complexes with pathogens, promoting a pro-inflammatory response, activating innate immune cells, upregulating TLR signalling and inflammatory pathway signalling. As pathogenic organisms are eliminated and supra-physiological levels of alarmins are no longer required, HSP70 acts to downregulate the immune and inflammatory response promoting homeostasis (191). Glutamine is likely to play an important role in the response to infection and has been shown to reduce the risk of sepsis in pre-term infants (424) and decrease the incidence of gram-negative bacteraemia in severely burned patient (206) and multiple trauma (281). By contrast, glutamine depletion in sepsis is associated with increased risk of mortality (243). It may be that providing glutamine supplementation potentiates an appropriate immune response to infection (409), which may differ in adults compared to children but involves a timely resolution to the inflammatory response within both age groups.

There are a number of limitations to this work which include the investigation of only one dose of glutamine (2mM), which significantly upregulated HSP70 release in both adult and paediatric endotoxin stimulation models. The effect of glutamine supplementation on the release of inflammatory mediators is less clear. In adults, glutamine supplementation significantly attenuated the release of IL-8 but in children significantly promoted the release of TNF-α. It may be that a large dose of glutamine (≥2mM) is required in both adult and

109 paediatric models to significantly mediate the release of inflammatory mediators. Although a range of pro- and anti-inflammatory mediators were selected for study based on previous work in meningococcal disease, it was not possible to examine all those of interest (TNF-α, IL-8, IL-6, IL-10) within a single validated multiplex plate. Due to the limitation on blood volume available from paediatric samples there was insufficient to be able to run separate plates for all of the inflammatory mediators of interest. The inflammatory mediators (TNF-α, IL-6, IL-10) chosen for study in the paediatric in vitro stimulation model have been extremely well studied in meningococcal disease (77, 78) and were chosen for this reason. Further to this, the directional release of inflammatory mediators in the adult in vitro stimulation model is the converse to the paediatric model (e.g. in the adult model inflammatory mediator release including IL-8 appears to be attenuated following glutamine supplementation). It could therefore be postulated that IL-8 release in the paediatric model would increase following glutamine supplementation as seen with IL-10, TNF-α and IL-6. However, in order to confirm, this future assasys in children would be required to examine the relationship between HSP70 and IL-8 release following glutamine supplementation. Other limitations in the in vitro models of this study, was the lack of discrimination between live and dead cells, which may be a criticism with respect to the source of HSP70 release (e.g. passive release or as a result of necrosis).

3.12 Conclusion These results would suggest that glutamine supplementation (2mM) promotes HSP70 release in an in vitro endotoxin stimulation model in adults and children. When HSP70 production was partially inhibited through the use of KNK437 the release of inflammatory mediators (IL-6, IL-10, TNF-α) was attenuated. This would suggest that glutamine given at therapeutic doses ≥0.3g/kg (≥2mM) could augment HSP70 production, although a large dose of 0.6g/kg (4mM) may be required to influence the mix of cytokines released. As glutamine depletion is common in sepsis (425), the mediation of HSP70 release during critical illness, may make it a useful therapeutic strategy. A randomised clinical trial administering glutamine, as a nutraceutical, in critically ill children would be required to test this hypothesis further.

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The results from this work highlight the need to explore the relationships of glutamine, HSP70 and inflammatory mediators further. In order to achieve this stored plasma from a cohort of children with meningococcal disease during the acute phase of illness was analysed to investigate protein levels of HSP70, glutamine and inflammatory mediators.

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Chapter 4: Heat shock protein 70, glutamine and inflammatory markers during the course of meningococcal disease and the relationship to clinical indices

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Chapter 4: Heat shock protein 70, glutamine and inflammatory markers during the course of meningococcal disease and the relationship to clinical indices

4.1 Introduction Following infection, signalling pathways associated with inflammation and immunity become upregulated, influencing cell to cell signalling and promoting a gene regulated response to stress (338, 426). Activation of these pathways stimulates the release of numerous chemokines, cytokines and other mediators including catabolic hormones (glucagon, growth hormone, cortisol), prostaglandins and catecholamines promoting catabolism (breakdown and release of substrate for energy).

4.2 Glutamine and critical illness During critical illness glutamine synthesis is not diminished when compared to normal healthy volunteers, however as a result of the net efflux of glutamine from skeletal muscle, depletion occurs (427). Although plasma concentrations do not accurately reflect the intracellular glutamine concentration (428) which varies dependent on the type of cell (429, 430), it is currently the best proxy for glutamine depletion (431).

Glutamine deficiency in adult ICU patients is well documented and has been shown to be an independent risk factor for mortality (209, 240, 243, 432). Plasma glutamine levels of 0.3 – 0.5mmol/l and of 15mmol/l of glutamine in muscle are known to occur post surgery, trauma and in sepsis (433), which are lower than in healthy individuals with a usual range of 0.6 – 0.7mmol/l in plasma (216, 217) and 20 - 25mmol/l (216, 219, 220) in muscle.

Administration of glutamine has been shown to be safe and is not associated with a rise of toxic ammonia or glutamate levels (206, 209, 424). Orally administered glutamine has less of an effect on plasma levels, as it is rapidly metabolised by enterocytes within the gastrointestinal tract (434) and as such during critical illness IV glutamine has greater efficacy (282, 435). Pharmacokinetic studies investigating clearance and tolerance of glutamine have indicated no adverse effects and plasma levels of ≥2mmol/l are well tolerated (274). Glutamine dipeptides are metabolised almost immediately into their individual amino acids e.g. L-glutamine and L-alanyl (436, 437). In order to increase plasma levels to 0.6mmol/l an

113 infusion of 40 – 60g/24 hours was required in adults. A dose of 0.57g/kg/24 hours resulted in a plateauing of plasma glutamine approximately 25% above controls, which did increase ammonia or glutamate levels (438). Intravenous glutamine given at a dose of ≥0.3g/kg has been shown to restore plasma glutamine levels in critically ill adults, but larger doses of up to 0.86g/kg of glutamine are also shown to be are well tolerated (275). In post operative adult patients, a daily IV dose of glutamine prevented further skeletal muscle loss (439, 440), although it is not clear whether supplementation influences endogenous skeletal losses during critical illness (275). In stable critically ill adults with MODS net glutamine loss from muscles continued for 2 weeks (441), suggesting that losses continue despite a resolution of the immediate response to infection. In addition, if glutamine supplementation is discontinued prematurely then glutamine levels fall again (209).

Glutamine upregulates HSP70 release in vitro (203) and in vivo models of injury and adult trauma (204, 311, 442, 443), and has been correlated with decreased organ damage and mortality (310, 443), it could potentially be of benefit to critically ill children.

As children with meningococcal disease are often severely ill, they are a good clinical model for studying the effect of critical illness on glutamine, HSP70 and inflammatory mediators and the relationship to clinical indices.

4.3 Aims 1. To examine levels of HSP70, glutamine and markers of inflammation in children with meningococcal disease during the acute phase of illness compared to convalescence.

2. To examine relationships between clinical variables (including nutrition support), HSP70, markers of inflammation and glutamine in children with meningococcal disease during the acute phase of illness compared to convalescence.

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4.4 Results 4.4.1 Patient demographics and sample collection Acute plasma samples were obtained from 143 Caucasian children and 78 convalescent samples. Eighty-three (58%) were males and 60 (42%) were females. The median age of was 2 years (min 0.02 months – maximum 17.2 years) and the mortality rate was 4.7% (n=7). Blood samples were also obtained from 35 healthy aged matched paediatric controls, of which 13 (27%) were female and 22 (63%) were males, median age 3.8 years (min 0.38 – max 16 years). Table 4.1 outlines the patient demographics.

Samples were analysed for HSP70, inflammatory markers (IL-1β, IL-6, IL-8, IL-10, TNF-α) in addition to glutamine using methods described in chapter 2. In addition to this a folder review was completed. Patients with acute meningococcal disease were severely ill and required multi-organ support, including ventilation and renal support. Patient demographics and PICU support data is shown in Table 4.1, laboratory data in Table 4.2 and nutrition data in Table 4.3.

Table 4.1: Patient demographics and PICU support of children with acute meningococcal disease Number Median Range Mean Percentage (min – max) (±SD) (%) Decimal age (years) 2.0 0.02 -17.2 3.1 (±3.1) Weight (kg) 12 3 - 70 15.3 (±10.4) Weight for age Z 0.1 -4.1 - 4.8 0.2 (±1.2) scores * PRISM 26 10 - 89.8 31.9 (±20.3) PIM2 9.1 3.6 - 65.5 13.7 (±18.4) Days on PICU 7 3 – 63 8.8 (±7.6) ICU free days 21 0 – 25 18.7 (±6.7) Mechanic ventilation 143 (100%) Duration of 6 2 – 45 7.4 (±5.9) ventilation Deaths 7 (4.7%) Renal support 124 (81%) Urea (mmol/l)** 6.40 1 – 42.7 8.9 (±8.5)

Creatinine (μmol/L) 60 24 – 535 88 (±92.5) Inotropes 123 (83%)

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Steroids 108 (73%) Glucose (mmol/l) 8.8 3.8 – 29.5 10.4 (±5.7) Noradrenaline 0.25 0.4 – 2.4 0.5 (±0.7) (mcg/kg/min) Adrenaline 0.6 0.01 - 6.3 1.0 (±0.6) (mcg/kg/min) Dobutamine 10 5 – 40 12.6 (±7.7) (mcg/kg/min) Dopamine 10 2.5 – 28 12.2 (±6.9) (mcg/kg/min) *(normal range -1 to 1), **(normal range 2.5 – 7.8)

Patients with acute meningococcal disease had deranged clinical parameters. Although median levels of clinical variables including white cell count (WCC), internationalized normal ratio (INR), potassium, sodium, calcium, phosphate and magnesium were all found to be within normal range, 81% of the cohort were receiving renal support for raised levels of renal function markers. Up to 83% of children required inotropic support, which were administered in high doses. The majority of patients received steroids 73% which may have contributed to the high glucose levels seen within the cohort of 10.4mmol/l; ±SD 5.68 (Table 4.2).

Table 4.2: Patient demographics and clinical values of children with acute meningococcal disease Laboratory Value Paediatric Median Range Mean reference (min-max) (±SD) range WCC (x 109/l) 4.0 - 11.0 5.7 1.1 – 46 8.4 (±9.1) Platelets (x 109/l) 150 – 400 162 15 – 513 180 (±88.1) INR 0.9-1.2 1.5 0.9 - 5.9 2.4 (±1.3) CRP (mg/l) < 10 78.5 4.5 – 483 93.7 (±82.4) Potassium (mmol/l) 3.5 – 5.0 3.55 2.2 – 6 3.6 (±0.7) Base Excess (mEq/L) −2 - +2 -8.8 -24.4 – 20 -9.5 (±6.0) Lactate (mmol/l) 0.6 – 2.5 3.8 0.5 - 18.5 3.3 (±3.9) Sodium (mmol/l) 133 – 146 140 127 – 164 138 (±5.7) Albumin (g/L) 35 – 50 36 23 – 46 34 (±7.2) Calcium corrected 2.2 – 2.6 2.2 0.9 - 3.1 2.1 (±0.4) (mmol/l) Phosphate (mmol/l) 0.9 – 1.8 0.9 0.5 - 3.2 0.94 (±0.4) Magnesium (mmol/l) 0.7 – 1.0 0.7 0.4 - 1.5 0.8 (±0.1)

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4.4.2 Nutrition Support Although enteral feeding was initiated early post admission, it took a number of days before the prescribed goal rate was achieved which was likely to be related to the high prevalence of gastrointestinal disturbances and inotropic support. Thirty nine percent of feeds were delivered via NGT, with the remainder receiving NJT feeds and combination enteral/ parenteral (Table 4.4). The glutamine content of all the standard feeds (except Nutrini Energy, Infatrini) contained less than 0.5g per 100ml of glutamine. Even at full feeds the amount of glutamine delivered enterally was suboptimal at 0.14g/kg day providing only 28% of the recommended intake based on 0.5g/kg/day (Table 4.3).

Table 4.3: Feeding characteristics with glutamine intake and plasma levels Reference Median Range Mean (±SD) range Hours nil per mouth 4.5 1 – 96 13.3 (±15.8) Days to goal rate 3 0.3 – 10 3.2 (±2.0) Goal (ml/hr) 40 5 – 86 39.4 (±14.1) Glutamine (g/kg) at ≥ 0.3 – 0.5 0.14 0 - 0.7 0.14 (±0.1) goal rate of feed Glutamine (mmol/l) 0.6 – 0.7 0.29 0.05 – 0.6 0.3 (±0.1) HSP70 (ng/ml) 0.4 - 0.5 5.7 0.09 - 600.5 26.7 (±79.9) Gastric aspirates <5 9.3 0.5 – 63 10.4 (ml/kg) (±10.8)

Table 4.4: Nutrition Support, Route & Complications Percentage % Age 0 - 1 yr 15 > 1 - 5 yrs 68 > 5 yrs 16 Feeding Complications NPM - too unstable 4 Tolerated 24 Gastrointestinal Disturbances* 72 Gastric Aspirates 0 - 5ml/kg 19.6 > 5 - 10ml/kg 33.7 >10ml/kg 46.7 Route of Feeding NGT 39 NJT 31.5 117

Enteral & PN 28.5 PN 1 Type of feed % (glutamine content per 100ml) EBM 2 Infant formula 48.4 Infatrini (glutamine 0.542mg/l00ml) 1 Pediasure (glutamine 0.3mg/l00ml) 36.5 Nutrini Energy (glutamine 0.9 mg/100ml) 1 Jevity (glutamine 0.39mg/100ml 4.5 PN & Enteral 5.6 PN (no glutamine) 1 Glutamine g/kg at full feeds Glutamine g/kg 0.17 *vomiting/ abdominal distention/ large gastric aspirate/ diarrhoea; **recommendations ≥0.3 – 0.5g/kg

In summary these patient demographics reflect a cohort of severely ill children, requiring invasive monitoring and multi-organ system support to maintain haemodynamic stability, who received suboptimal amounts of glutamine.

4.5 The effect of freeze thaw cycles on glutamine stability As the paediatric plasma samples had been stored for more than 5 years in the department freezer, it was necessary to evaluate if repeated freeze/thaw cycles would have an effect on glutamine levels in this samples. Therefore, a series of freeze thaw (FT) cycles (B – H) on whole blood from healthy adult volunteers were completed prior to measuring plasma glutamine levels in the paediatric samples (Figure 4.1).

9000 A - baseline (- Gln+) B - baseline 8000 (+2mM Gln) C - FT1 7000 D - FT2 E -FT3 6000 F -FT4 G -FT5 5000 H -FT6

4000

3000 Glutamine (mmol/l)

2000

1000

0 A B C D E F G H Figure 4.1: Effect of freeze-thawing on glutamine plasma levels in healthy adult volunteers (n=3). Results are expressed as mean; ± SEM. FT = freeze-thaw

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Using Kruskal-Wallis one way ANOVA of variance there were no significant difference in glutamine concentration between samples B - H (p>0.05), suggesting that the paediatric samples may not be significantly affected by the freeze-thaw cycle. Plasma glutamine levels in the healthy volunteers (A) were within normal range mean (±SD) 0.64 mmol/l ± 43.1.

4.6.1 Plasma glutamine levels in children with acute meningococcal disease compared to convalescence Children with acute meningococcal disease were found to have significantly lower levels of glutamine 0.31mmol/l; ±SD 0.13 (median 0.31mmol/l; range 0.05 – 0.64) (p<0.001) compared to convalescence 0.40mmol/l; ±SD 0.14 (median 0.40mmol/l; range 0.07 – 0.64) (Figure 4.2). During the acute phase of illness, glutamine levels were 52% below the normal range (using 0.6mmol/l as the lower reference range) and levels in the convalescent samples, taken on average 55 days later (35 – 135 days) were also found to be 26% below normal range.

600

*** 500

400

300

200 Glutamine (mmol/l)

100

0 Acute Convalescence

Figure 4.2: Glutamine levels in children with acute (n=132) meningococcal disease and during convalescence (n=65). Results are expressed as mean; ± SEM ***p<0.0001

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4.6.2 HSP70 levels in critically ill children with meningococcal disease during the acute phase of their illness compared to convalescence Mean HSP70 level in children during the acute phase of illness was 26.7ng/ml; ±SD 79.95 (median 5.7ng/ml; range 0.09 – 600.5ng/ml) which was significantly higher compared to the levels in healthy controls (mean 1.26ng/ml; ±SD 1.38 (median 0.703ng/ml range 0.001 – 5.45ng/ml) (p<0.0001) and to convalescence mean levels, 3.16ng/ml; ±SD 5.67 (median 1.045ng/ml range 0.001 – 37.31ng/ml) (p<0.0001). HSP70 levels were also significantly higher in convalescent samples compared to healthy controls (p<0.05) (Figure 4.3).

40 ***

*** ***

20 HSP70 (ng/ml) HSP70

0 Controls Acute Convalescent

Figure 4.3: HSP70 levels on admission in children with acute meningococcal disease compared to convalescence and healthy paediatric controls. Controls n=35, acute samples n=143, convalescent samples n=78. Results are expressed as mean; ± SEM *p<0.05, ***p<0.0001

4.6.3 Inflammatory mediators in children with acute meningococcal disease compared to convalescence During the acute phase of illness patients with meningococcal disease had significantly higher levels of inflammatory mediators (IL-6, IL-10, TNF-α, IL-8, IL-1β) compared to convalescence and levels in healthy controls. There was a significantly higher IL-10: IL-6 ratio in children with acute meningococcal disease (mean 12.62pg/ml; ±SD 111.6) compared to convalescence (mean 1.47pg/ml; ±SD 3.61) (p= 0.0093) (Figure 4.4).

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1000000

*** *** IL6 100000 TNF- *** *** *** *** 1L 10 10000 IL - 8 pg/ml) ** *** 10 IL 1 1000 *** *** Ct = control A = acute 100

C = convalescence Cytokine (log Cytokine

1 Ct A C Ct A C Ct A C Ct A C Ct A C

0.1

Figure 4.4: Log10 of inflammatory mediators (IL-6, TNF-α, IL-10, IL-8 and IL-1β) in children with acute meningococcal disease compared to convalescence. Acute n=132, convalescent n=78, controls = 35. Results are expressed as mean; ± SEM **p<0.005, ***p<0.0001

4.6.4 Relationships between HSP70, glutamine and inflammatory mediators

As the data was skewed with a non-Gaussian distribution it was normalised by a log10 transformation and a Pearson’s correlation (parametric test) was used. HSP70 was positively correlated with IL-10 (n=88, r=0.425; p<0.0001), IL-6 (n=141, r=0.318; p<0.0001) and TNF-α (n=145, r=0.206; p=0.013). There were positive correlations of IL-6 with IL-10 (n=87, r=0.916; p<0.0001), TNF-α (n=88, r=0.911; p<0.0001) and for IL-8 with IL-1β (n=146, r=0.898; p<0.0001). However, glutamine levels did not correlate with levels of HSP70 or the inflammatory mediators.

4.6.5 Relationships with HSP70 levels and clinical indices in meningococcal disease cases during the acute phase of illness There were significant positive correlations with HSP70 levels and i) length for PICU stay (n=99, r=0.30, p<0.001), ii) duration of mechanical ventilation (n=99, r =0.31, p<0.001), iii) PRISM score (n=99, r = 0.26, p=<0.01), iv) lactate (n=99, r =0.26, p<0.001) and v) CRP (n=99, r =0.29, p<0.001). However, there were no correlations with any feeding or nutrition support indices.

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4.6.6 Relationships with glutamine and clinical measures in meningococcal disease cases during the acute phase of illness There were significant inverse correlations between the amount of glutamine (g/kg) administered at the goal rate of feeding and PRISM score (n=98, r= -0.27, p=0.007) and between glucose (mmol/l) (n=75, r= -0.26, p=0.015). The number of days to reach the goal rate of feeding was positively correlated with the amount of glutamine delivered (n=63, r= 0.242 p=0.026). Interestingly, no significant correlation was found between glucose and plasma glutamine levels, although there was a trend towards a significant inverse correlation (n=75, r=-0.20, NS p=0.069). Plasma glutamine and lactate were also inversely correlated (n=98, r=-0.41, p<0.001) as was plasma glutamine and CRP (n=98, r=-0.51, p<0.001).

There were other inverse correlations with plasma glutamine levels and i) number of days spent in PICU (n=98, r=-0.520, p<0.001) and ii) number of days spent on mechanical ventilation (n=98, r=-0.513, p<0.001).

4.6.7 Relationships within clinical variables of children with acute meningococcal disease The administration of adrenaline was significantly positively correlated with the number of hours a child was nil by mouth (n=62, r=0.229, p=0.046). Positive correlations were also found between lactate and PRISM score (n=98, r = 0.451, p<0.001).

In summary, HSP70 was positively correlated with cytokines IL-6, IL-10 and TNF-α, in addition to CRP, length of ICU stay and mechanical ventilation, suggesting that HSP70 is closely associated with the acute phase of critical illness. Glutamine levels and the amount of glutamine administered were inversely correlated with mechanical ventilation and the number of days spent in ICU, suggesting that low levels of glutamine are associated with an increase in morbidity. The amount of glutamine delivered at goal rate was also found to be positively correlated with the number of days to achieve goal rate of nutrition support. The amount of glutamine delivered via nutrition support was also associated with hyperglycaemia and PRISM score. However, there was a lack of association between glutamine and HSP70 levels and inflammatory mediators.

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4.6.8 Relationships between categorical and clinical variables in children with acute meningococcal disease To explore the strength of relationships between clinical variables further, direct logistic regression was performed on a number of categorical and continuous variables. A number of different models were constructed considering, for example the effect of insulin administration on a range of variables including glucose, glutamine, IL-6, HSP70 and IL-10. No significant impact was found and therefore these models were not included. The only model showing significance was the likelihood of certain variables in predicting steroid administration during the acute phase of illness. The model contained 7 independent variables (glutamine, HSP70, IL-10, IL-6, TNF-α, glucose, CRP). The model was able to predict which children received steroid or not in the acute phase with statistical precision of χ2 (7, n=49 = 17.1 p=0.014). The model as a whole explained between 30.2% (Cox and Snell R2) and 46.1% (Naglekerke R2) of the variance in steroid administration, and correctly classified 81.6% of cases. As shown in Table 4.5 four independent variables made a unique statistically significant contribution to the model (glutamine, IL-10, IL-6, TNF-α) although there was a trend towards significance with (glucose and CRP). The strongest predictor of steroid administration was IL-10, recording an odds ratio of 18.3. This indicated that children with acute meningococcal disease were 18.3 times more likely to require steroids if they had elevated IL-10 levels compared to those who did not. The odds ratio for glutamine was 1 indicating a weak relationship with steroid administration and glutamine levels.

Table 4.5: Factors predicting the use of steroid administration in children with acute meningococcal disease B Standard Wald p Odds 95% Confidence interval

values error Test value ratio for odds ratio Upper Lower Glutamine (mmol/l) 0.009 0.004 4.12 0.04 1.00 1.000 1.0 HSP70 (ng/ml) 0.95 0.62 2.34 0.12 2.59 0.76 8.78 IL-10 (pg/ml) 2.90 1.51 3.69 0.05 18.32 0.94 355.79 IL-6 (pg/ml) -0.51 0.72 0.50 0.47 0.60 0.14 2.45 TNF-α (pg/ml) -3.35 1.37 6.01 0.01 0.03 0.002 0.51 Glucose (mmol/l) 0.18 0.09 3.51 0.06 1.19 0.99 1.44 CRP (mg/l) -0.04 0.02 3.23 0.07 0.96 0.92 1.00 Constant -6.59 2.49 6.98 0.008 0.001

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4.7 Discussion Children with severe infection and trauma admitted to a PICU usually require multi-organ support, including mechanical ventilation and multiple drugs to sedate, paralyse and support cardiac output. Nutrition support during the acute phase of illness remains challenging, as infants and young children have limited metabolic stores (444), increasing the risk of malnutrition (214). The negative effects of malnutrition are not restricted to the PICU stay and can last for up to 6 months post discharge, especially in those children with prolonged illness (445).

Although early enteral nutrition support in PICU should be the norm, it is usually balanced against potential risk of feeding in clinically unstable patients (446). High severity of injury scores and cardiovascular support (inotropes) are associated with delayed commencement of enteral feeding (447, 448), and under these circumstance parenteral nutrition is often established in its place (426). However, the amino acid profile of parenteral and most enteral feeds lack sufficient amounts of some conditionally essential amino acids, thus increasing the risk of morbidity and mortality (206, 241). This is reflected in the above results, which show that a high PRISM score is correlated with lower glutamine levels and high IL-6 levels and lactate levels, reflective of the severity of inflammation, cellular dysfunction and possibly poor nutrition intake and or rapid use of endogenous glutamine stores. Inotrope administration, associated with delayed enteral feeding and feeding intolerance (448), was also predictive of low glutamine levels.

There is increasing recognition that nutrients (nutraceuticals) given in pharmacological doses could have a positive impact on outcome in critical illness through the better regulation inflammatory and immune response (207), of which glutamine is probably one of the most well studied (237). In critical illness there is a precipitous drop in plasma glutamine levels with the onset of catabolism increasing the risk of mortality (206, 241). During the acute phase of meningococcal disease, glutamine levels were found to be significantly lower (p<0.001) compared to convalescence and were 52% below the normal range. Convalescent samples taken on average 55 days later (35 – 135 days) were also found to be 26% below normal range. Griffiths et al showed that glutamine levels were depleted in critically ill adults by 58% up to 30 days post injury (209), which concurs with the findings in this cohort.

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Glutamine depletion occurred early on during the course of meningococcal disease and appeared to be related to the severity of disease. In this cohort of meningococcal patients, it took > 4 days to achieve goal rates of feeding in 33%, and 72% of the patients had gastrointestinal disturbances, which is similar to findings in other studies (214, 449).

In this cohort the total amount of glutamine delivered was only 0.14g/kg glutamine, which is insufficient to restore levels to within normal range. ASPEN/ ESPEN/ SCCM have given Level A recommendation for glutamine supplementation in doses of 0.3 – 0.5g/kg (271-273), which is been shown to replete plasma glutamine levels (274, 275) could be postulated to optimise HSP70 expression. These levels of supplementation are associated with decreased infectious morbidity (206, 283, 284), mortality (209, 285, 286), improved organ function and decreased length of hospital stay (238, 287, 288).

Given the low levels of glutamine derived from enteral sources in this cohort of children, it is not surprising that plasma glutamine levels were significantly below that of the normal range. Whilst paediatric enteral feeds such as Pediasure and adult enteral feeds such as Jevity have added L-glutamine (0.3 – 0.39g/100ml) respectively, standard infant feeds contain very little glutamine and this is in the form of glutamic acid. However, as most of the research has suggested that oral glutamine is not an effective route to restore glutamine levels (282, 435), glutamine supplementation should be administered parenterally. It has been suggested in order to ameliorate the inflammatory response during severe infections, a higher dose of glutamine is required (281) and in the most severely ill children, glutamine supplementation should be administered intravenously aiming for 0.5g/kg day (207, 438), especially as standard PN solutions also do not have any L-glutamine or glutamine dipeptides added.

The effects of glutamine are presumed to be through increased release of HSP70, which may help to first promote and then effectively attenuate, the inflammatory and immune response to critical illness (207, 239). In children with acute meningococcal disease, significantly higher levels of HSP70 were found during the acute phase of illness which declined during convalescence, in parallel with other inflammatory mediators. Acute HSP70 levels (median 5.7ng/ml; range 0.09 – 600.5ng/ml), were higher than those described in

125 adult critical illness (n=15, median 1.75ng/ml; 0.71 – 4.32ng/ml) (143) but lower than those found in paediatric septic shock (n=94, median 51.6ng/ml (0.75–294.9ng/ml) (141). However, the mortality rate in the study by Wheeler et al (141) was 13.4% compared to 4.7% in this cohort, and the septic shock children who died had significantly higher HSP70 levels 78.1ng/ml (range 1.43–319.1 ng/ml) compared to survivors 51.6ng/mL (range 0.75– 294.9ng/ml), which the authors suggest could be related to necrotic release of HSP70 into the extracellular milieu (141), rather than any cytotoxic effects of HSP70. In this cohort, HSP70 levels were high during the acute phase of illness and declined during convalescence, in parallel with inflammatory mediators.

There were significant differences between inflammatory mediators (1L-6, TNF-α, IL-10, IL-8 and IL-1β) release in children with acute meningococcal disease compared to convalescent samples and those from healthy controls. Inflammatory cytokines were high on admission and declined in convalescence, showing a pattern as described in other critically ill cohorts (61, 62). In addition to this, inflammatory mediators (IL-6, IL-10 and TNF-α) were positively correlated with HSP70 levels, although there was no relationship with glutamine. Although levels of HSP70 (136, 137), inflammatory mediators (72, 90, 450) and glutamine (209, 240, 243, 432) during critical illness have previously been described in adults, there are no reports of relationships between HSP70, glutamine and inflammatory mediators in critically ill children.

Inverse correlations were found between glutamine (g/kg) administered at the goal rate of feeding and the PRISM score and plasma glucose concentration, suggesting that low plasma glutamine levels were associated with disease severity and insulin resistance. Several investigators have found similar relationships between glutamine supplementation, glycemic control and outcome (284, 451, 452), although further work is required to understand the mechanisms. Although the correlation was not significant, there was a trend suggesting a relationship between glucose and plasma glutamine levels. It is also important to note that 83% of this cohort received renal support in the form of continuous renal replacement therapy. Amino acid losses in children receiving renal replacement therapy through the dialysate is documented as being 0.2g/kg per day with glutamine contributing 34.5µg/kg to those losses (453), further contributing to glutamine depletion.

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Other inverse correlations with plasma glutamine levels were found, such as the number of days spent in PICU (n=98, r=-0.520, p<0.001), number of days spent on mechanical ventilation (n=98, r=-0.513, p<0.001), which have been well described elsewhere with respect to increased risk of morbidity and mortality (209). The number of days to reach the goal rate of feeding was also positively correlated with the amount of glutamine delivered (n=63, r= 0.242 p=0.026).

Positive correlations between HSP70 levels were found with length of PICU stay, duration of mechanical ventilation, PRISM, lactate and CRP, suggesting that elevated levels of HSP70 may be associated with disease severity, inflammation and cell dysfunction, which is seen in adults with chronic inflammatory disease (diabetes) who have higher levels of HSP70, CRP and other inflammatory markers compared to healthy controls (454).

During critical illness CRP levels are used as a marker of inflammation (455, 456) and are associated with oxidative stress and increased production of reactive oxygen species (457) and levels decline as the inflammatory response resolves (456). Glutamine administration (0.5g/kg) is associated with significantly lower CRP levels post surgery in children (455) and in adults with acute pancreatitis (458), where it effectively mediates the inflammatory response (224). Children with acute meningococcal disease had elevated CRP and lactate levels, suggesting marked metabolic dysregulation. Plasma glutamine and CRP levels were inversely correlated.

High lactate levels in critical illness are correlated with disease severity and are a marker of tissue hypoxia, mitochondrial dysfunction (459, 460) with derangements in levels of ATP/ ADP (461) and NAD+/ NADH(462). In this meningococcal disease cohort, lactate levels were elevated above the reference range at 3mmol/l; ±SD 4.08, indicating cellular dysfunction. In rat models of sepsis glutamine supplementation preserved ATP and NAD+ levels, mediated by an increase in HSP70 levels (310, 311). Inhibition with quercitin abrogated the beneficial effect of glutamine supplementation by preventing HSP70 release (311). Other work has shown that when glutamine levels are replete to within normal range cellular homeostasis is restored with a resolution of lactic acidosis (270).

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Using regression analysis to predict factors involved in shocked children requiring steroids, the strongest predictor of steroid administration was the measured level of IL-10. The likelihood of steroid administration was also influenced by glutamine levels, albeit weakly. The majority of children received steroids (73%) which are known to exacerbate insulin resistance in critical illness (463), evident in this cohort with elevated plasma glucose levels (10.4mmol/l; ± SD 5.7). IL-6 has been shown to promote a net efflux of amino acids notably glutamine from muscles, attenuating protein synthesis in favour of protein breakdown with a net release of amino acids from skeletal muscle (94). Experimental models of sepsis work show that the concomitant administration of steroids and cytokines (IL-6 and TNF-α) stimulated alanine and glutamine transport in human hepatocytes (464) and rats (465, 466), providing substrate for the gluconeogenic pathway.

Glutamine can be used as a vehicle to increase the release of HSP70 (203-205). In adults glutamine administration significantly increased HSP70 levels, which was associated with a decreased length of ICU stay and fewer ventilator days (143). As glutamine is safe, non toxic and easy to administer it makes it an attractive modulator of the heat shock response (206- 210). In order for paediatric glutamine trials to be of success it is important to carefully select the patient population group as the lack of success in glutamine trials could be due to the heterogeneity of the study population, in addition to the use of supplementation in glutamine replete children. Routine measures of inflammation, such as CRP may be a useful proxy of low glutamine and high HSP70 levels, as CRP was positively correlated with HSP70 and negatively correlated with glutamine levels, suggesting that in this cohort of critically ill children, they may be useful in predicting who would benefit from glutamine supplementation with the aim of mediating HSP70 release.

There were several limitations of this work which included the use of stored plasma samples. There was also a lack of associated between glutamine and HSP70 levels which may be related to small sample size. Future work would aim to investigate the relationship between glutamine, HSP70 and inflammatory mediators in a larger samples size in addition to stratifying children into age based categories to investigate potential age related differences relative to HSP70 release.

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4.8 Conclusions The results from this work show that during the acute phase of illness both HSP70 and inflammatory mediators are significantly increased and glutamine levels are depleted. Glutamine supplementation may decrease morbidity and mortality associated with sepsis/septic shock by maintaining an appropriate heat shock response mediating the immune and inflammatory response to critical illness.

To explore the immunological/inflammatory benefit of glutamine, through HS70 release in critical illness further, the aim a future randomised controlled trial would be to supplement glutamine at a dose of 0.5g/kg in addition to enteral/PN support.

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Chapter 5: Transcriptomic profiling during the acute phase of meningococcal disease and the associations with HSP70,

glutamine and inflammatory mediators

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Chapter 5: Transcriptomic profiling during the acute phase of meningococcal disease and the associations with HSP70, glutamine and inflammatory mediators

5.1 Introduction During sepsis innate and adaptive immune functions are dysregulated (467). However, due to the heterogeneity amongst critically ill individuals there have been conflicting results with respect to the pathophysiological process of critical illness, especially related to the immune and inflammatory response (468, 469) .

Target based studies of genes and proteins have limitations arising from the timing of the measurement, the supervised nature of targets chosen and the challenges surrounding the interpretation of the host response based on the measurement of plasma inflammatory mediators alone (467). The use of genome-wide transcriptomic analysis has provided a global insight into changes and inter-relationships of gene transcripts during sepsis and critical illness providing greater insight into the host response to infection (470, 471). Transcriptomic studies of experimental and clinical sepsis in paediatric and adult patients have revealed, activation of genes relating to inflammatory mediator, with the use of IL-8 as a stratification biomarker for septic shock (71). Enrichment of pathways relating to signal transduction and inflammatory signalling pathways, such as TLR4, NF-κB, MAPK’s, janus kinase (JAK) and signal transducer and activator of transcription protein (STAT) is well described (467). In contrast there is significant downregulation of genes associated with immune function, notably the antigen presentation pathway (360, 472). The aim of this phase of the study was to examine how HSP70 gene expression is affected by meningococcal disease, and its association with inflammation and metabolism regulating genes and with clinical disease profile.

5.2 Endotoxin induced activation of inflammatory signalling pathways In order to investigate the inflammatory effects of MD it is important to understand the intracellular mediators associated with downstream endotoxin signalling. As discussed in Chapter 1, endotoxin activates genes associated with the IKK/ NF-κB signalling pathway, essential for the transcription of immune and inflammatory response genes leading to the release of inflammatory mediators such as TNF-α (45, 46). Endotoxin also stimulates the

131 activation of various MAPK’s. The MAPK’s family comprises of ERK, p38 kinase and JNK’s. Following activation the MAPK’s mediate the cell response to stress via the phosphorlyation of transcriptional factors and initiation of other kinases (473) promoting the production of inflammatory mediators IL-1, IL-8 and TNF-α (49).

Following the activation of inflammatory pathways such as CD14-MYD88-IKK/NF-κB and p38MAPK, genes associated with downstream inflammatory mediators become activated, particularly those associated with the p38MAPK pathway driving the release of inflammatory mediators and the pathogenesis of septic shock (360, 474). Inhibition of the p38MAPK pathway prevents septic lung damage in an endotoxin mouse model ameliorating the negative consequences of an over exuberant inflammatory response (475). In a genome- wide transcriptome analysis of children with acute meningococcal disease, the p38MAPK pathway was shown to be a feature during IL-6 mediated myocardial dysfunction. The authors hypothesise, there may be a clinical benefit in modulating the activation of up- or downstream effector molecules, in the p38MAPK pathway, attenuating the release of deleterious amounts of inflammatory mediators such as IL-6 (476).

5.3 HSP70 release and the regulation of MAPK and NF-κB signalling pathways The release of HSP70 in response to endotoxin has been shown to mediate the activation of genes associated with inflammatory mediators (IL-1, IL-10 and TNF-α) (352, 477), thus reducing mortality in experimental animal models of sepsis, endotoxaemia and adult respiratory distress syndrome (128). Following in vitro endotoxin stimulation of macrophages, HSP70 effectively mediates the inflammatory response via the inhibition of IKK and the NF-κB pathway (194). Other in vitro work using monocytes, has shown that preconditioning with heat shock (e.g. heating the cells up to 42oC) before endotoxin stimulation, is associated with the inhibition of mitogen activated protein kinase phosphatase-1 (MKP-1) phosphorylation and downstream activation of ERK, p38MAPK pathways inhibiting the release of TNF-α release (478).

There is growing evidence to suggest that phosphatases have a central role in the modulation of inflammatory signal transduction during times of stress in which MPK-1 has a pivotal role (478, 479). The p38MAPK pathway serves to regulate mRNA stability of genes

132 associated with inflammation (480). MPK-1 has been shown to inactivate p38MAPK pathway via dephosphorylation, suggesting cross talk between MPK-1 and p38MAPK and a possible mechanism for control over the release of inflammatory mediators during times of stress (199). During times of stress, MKP-1 and HSP70 appear to have protein: protein cross talk and HSP70 release increases MKP-1 phosphorylation, modulating p38MAPK activity and kinase activation (481) mediating an inflammatory response (199).

5.4 Glutamine supplementation and the regulation of MAPK and NF-κB signalling pathways Glutamine is known to regulate the expression of numerous genes associated with metabolism, signal transduction, cell defence and repair (235). Glutamine exerts a complex regulatory activity with respect to the activation of intracellular signalling pathways associated with inflammation including those related to MAPK (ERK and JNK’s) (482, 483). Glutamine has been shown to specifically stimulate MEK-1, an upstream kinase responsible for the activation of ERK-1 and ERK-2, which promotes the phosphorylation of ELK-1 involved in cellular differentiation. Glutamine also activates JNK signalling pathway switching on factor AP-1 involved in cellular proliferation (484).

The cellular response to infection is a complex process co-ordinated by a wide range of intra- and extracellular mediators. Within this dynamic, glutamine and HSP70 influence, and are influenced by the mix of inflammatory mediators, activating protective cellular defence mechanisms one of which includes the increased production of glutathione stimulated by HSP70 release (485). Glutathione (GSH) serves as a regulator of the cellular redox potential determining the antioxidant capacity of the cell (486) and is also dependent (in part), on the availability of sufficient precursor glutamine (487). During times of stress, glutamine and glutathione levels are correlated (488) and as glutamine levels are proportional to the severity of disease (243), glutathione levels become depleted (489). GSH exists in two forms, GSH (reduced) and GSSH (oxidised). Increased levels of GSSH alter the redox potential favouring oxidation with subsequent activation of inflammatory signalling pathways JNK, MAPK and NK-κB (486, 487). Glutamine supplementation has been shown to restore the GSH:GSSH redox balance by increasing glutathione levels, attenuating redox sensitive kinases (ERK 1-2, p38) inhibiting NF-KB activation and the inflammatory response (486).

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5.5 Glutamine and HLA expression during critical illness Secondary infectious complications following severe illness are a considerable problem in critically ill patients, occurring as a result of immune paresis (490, 491), which is characterized by diminished expression of MHC Class II antigens on monocytes. MHC Class II antigens are essential to the induction of the adaptive immune response to foreign antigen (492), as T lymphocytes will only recognize bacterial antigen when HLA-DR molecules are expressed on monocytes and macrophages (493). The maintenance of antigen presentation by MHC Class II receptors is necessary for an appropriate response to infection and preventing infectious complications, as diminished antigen presentation response is correlated with poorer clinical outcome in trauma patients (490, 494) and critically ill children (495). Glutamine depletion has been associated with reduced HLA-DR expression and it may be that be maintaining glutamine levels during critical illness forms part of an effective strategy to maintaining immune competence (347).

Genome-wide transcriptomic studies have been used successfully to profile inflammatory response pathways in sepsis that are significantly dysregulated , including innate immune responses and adaptive immunity (472, 496). Glutamine is known to influence the expression of numerous genes (235), however to date, there are no studies, considering the effect of glutamine supplementation on gene enrichment of HSP70, inflammatory signalling pathways and those of adaptive immune function during critical illness in children. This chapter will use gene expression profiling to investigate the network of relationships of genes related to HSP70 and the host response in children with meningococcal disease.

5.6 Aims 1. To examine the gene expression associated with HSP70 and inflammatory pathways in children with meningococcal disease compared to convalescence.

2. To examine whether the differential gene expression had any biological relevance with respect to plasma protein levels of HSP70, inflammatory mediators and glutamine.

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5.7 Results As discussed in Chapter 2, RNA extraction, amplification, hybridisation and microarray data analysis were previously completed by Dr Victoria Wright, Department of Paediatrics, Imperial College London. The data arising from these experiments were prospectively analysed using GeneSpring (Chapter 2.3.4.4) and Ingenuity Pathway analysis (IPA) (Chapter 2.3.4.5).

This group is a subset from the larger cohort (n=143) that was used to determine levels of HSP70, glutamine and inflammatory markers. The patient demographics include 23 males and 16 females, with a mean age 4.54 years; SD ± 4.53 (median 2.844 years; range 1.75 – 4.78 years). There was no significant difference p>0.05 between protein levels in this sub- cohort of acute and convalescent HSP70, glutamine, IL-1β and IL-8 samples (Table 5.1) and the sub-cohort was therefore a representative sample. Demographic, clinical and nutrition support data for the entire cohort have been summarized earlier in Chapter 4.4.1.

Table 5.1 Protein levels of HSP70, glutamine and inflammatory mediators in a sub-cohort of children with acute meningococcal disease and during convalescence Acute (mean; ±SD) Convalescence (mean; ±SD) HSP70 (ng/ml) 24.6; ±69.3 1.19; ± 4.961 Glutamine (mmol/l) 299; ± 148.4 486.5; ± 158.5 IL-1β (pg/ml) 291.6; ± 773.8 56.1; ± 14.18 IL-8 (pg/ml) 1238; ± 2777 132.4; ± 278

In order to understand whether there were any significantly differentially genes (SDE) genes in children with acute meningococcal disease compared to convalescence, a list of SDE genes was generated using GeneSpring, to examine HSP70 and its relationship to changes with the wider network of inflammation and metabolic pathways in meningococcal disease compared to convalescence.

The complete dataset is detailed in Appendix 8.1. There were 1,780 SDE gene transcripts with 1,614 unique genes (p<0.05) with a log2 fold change ≥ 2 between the acute compared to convalescent samples. Median values were used for duplicate genes were used due to similarities in fold changes. The dataset was explored using IPA. The SDE genes during the

135 acute phase were represented in 3 main biological categories as defined by IPA; infectious disease (p=1.89E-33), respiratory disease (p=1.89E-33) and inflammatory response (p=3.27E- 31), as expected given that meningococcal disease is associated with the release of inflammatory mediators.

5.8.1 Immune pathways were dysregulated during the acute phase of meningococcal disease adaptive compared to convalescence To understand how genes from within the dataset were related a pathway analysis was completed. Pathway analysis provides a two dimensional representation of the data by linking interconnected genes. Amongst genes most affected by meningococcal disease were those controlling T cell function and the adaptive immune response, and pathways controlling these functions were amongst the most dysregulated in acute disease compared to convalescence (Figure 5.1a and b). The notable exception for this was the glycolysis/gluconeogenic pathway (p=2.53E-06), which contained mainly upregulated genes, with up to 16% of the pathway represented by genes from the dataset. The most highly dysregulated pathway was “CD28 signalling in T helper cells” (p=3.83E-10), which was significantly downregulated with up to 25% of the pathway represented by genes from the dataset. Other signalling pathways to be affected included “Calcium induced T lymphocyte apoptosis” (p=1.4E-07), which was significantly downregulated with up to 30% of the pathway represented by genes from the dataset, and “role of NFAT in regulation if the immune response” (p=7.62-10) with 17% of the pathway represented by genes from the dataset, representing pathways associated with adaptive immunity.

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Figure 5.1 a and b: Top signalling pathways represented by the significantly differentially expressed genes between patients with meningococcal disease compared to convalescence (a) and a stacked representation of the same data in (b) indicating the proportion of genes upregulated (red) and downregulated (green). The y-axis is the –log of the Benjamini-Hochberg adjusted p-value, providing an indication of how likely a pathway is enriched in the dataset. The –log (p-value) of 1.3 as indicated by the horizontal line, represents a P value of 0.05. 137

5.8.2 Comparison of signalling pathways of SDE upregulated and downregulated genes To provide an overview of the signalling pathways within the dataset the 1,780 SDE gene transcripts was analysed using IPA, in an unsupervised manner. Following on from this to compare differences between significantly differentially expressed up- and downregulated genes within the dataset, separate datasets were created using IPA. The upregulated dataset contained 933 SDE gene transcripts compared to the downregulated dataset which contained 492 SDE gene transcripts, the nature of pathways within the separate datasets were examined. Appendices 2 show molecules within the signalling pathways for all, up- and downregulated datasets.

5.8.3 Upregulated genes, during the acute phase of meningococcal disease, were associated with pathways associated with cell to cell signalling and activation of the inflammatory response Amongst the upregulated dataset, genes most affected by meningococcal disease were those controlling cell to cell signaling, signal transduction via the TLR pathway (p=2.57E-08) (Figure 5.4) and inflammatory signaling pathways of IL-6 (p=1.7E-05) and IL-10 (p=1.37E-05) amongst others. The glycolysis and gluconeogenesis signalling pathway was the most significantly enriched (p=1E-09), with up to 17% of the pathway represented by the genes from the dataset. The TLR pathway had 27% of the pathway and IL-6 and IL-10 had between 12 – 18% of the pathways represented by the genes from the dataset.

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Figure 5.2: Top signalling pathways represented by the 933 SDE upregulated genes between patients with meningococcal disease compared to convalescence. The y-axis is the –log of the Benjamini-Hochberg adjusted p-value, providing an indication of how likely a pathway is enriched in the dataset. The –log (p-value) of 1.3 as indicated by the horizontal line, represents a P value of 0.05.

5.8.4 Downregulated genes, during the acute phase of meningococcal disease, were associated with apoptosis In contrast in the downregulated dataset, genes most affected by meningococcal disease were those controlling associated with adaptive immune functions specifically T cell signaling, cell growth, proliferation, development and apoptosis (Figure 5.3). Other downregulated pathways included calcium-induced T lymphocyte apoptosis (p=4.98E-15) with 30% of the pathway represented within the dataset. Adaptive immune cell signalling such as CD28 signalling in T helper cells was also significant enriched (p=1.56E-12) with 17% of the pathway represented within the dataset.

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Figure 5.3: Top signalling pathways represented by the 492 SDE differentially downregulated genes between patients with meningococcal disease compared to convalescence. The y-axis is the –log of the Benjamini-Hochberg adjusted p-value, providing an indication of how likely a pathway is enriched in the dataset. The –log (p-value) of 1.3 as indicated by the horizontal orange line, represents a P value of 0.05.

5.8.5 Summary of the results from signalling pathway analysis During the acute phase of meningococcal disease signalling pathways representing T cell function and adaptive immune response, particularly antigen presentation pathways were significantly downregulated compared to convalescence. Significantly upregulated signalling pathways were associated with glycolysis and gluconeogenic pathway and cell to cell signalling.

5.9.1 Gene network analysis of dataset with 1,780 SDE transcripts in acute meningococcal disease compared to convalescence and the role of HSP70 Network analysis allows for the multi-dimensional representation of the dataset based on statistical probability, showing direct and indirect physical interactions. A network analysis creates a list of possible networks, ranked according to how many genes in the IPA knowledge base correspond to the genes in the dataset. Every gene interaction within the

140 network is supported by published information. Network generation allows for the exploration of interconnections between genes, highlighting functional relationships.

To gain further understanding of the biological meaning of the genes relating to HSP70 expression within the dataset, a network analysis was focused on HSP70 related gene expression. The subsequent figures provide an overview of HSP70 gene interaction on a global level in addition to those within the up- and downregulated datasets. The dataset of 1,780 SDE transcripts yielded a highly significant gene network associated with HSP70. The network contained 35 genes of which 25 were represented in the dataset, and had a P value of 1.0E-28 (Figure 5.4).

The main functions represented by the network were protein folding, cell death and cell compromise. Within this network HSP70 gene expression was significantly differentially upregulated and had a number of direct relationships with genes within the network, (solid light blue lines), suggesting that HSP70 has a role in the acute phase of meningococcal disease. Several of the connected genes relate to apoptosis (APAF-1, PGLYRP1), a process which HSP70 is known to regulate. Genes are represented as nodes and the biological relationship between 2 molecules is represented as a line. The intensity of the node colour indicates the degree of upregulation (red) or downregulation (green). Corrected p-value and relative differential fold change are represented below each molecule. All direct relationships (solid line) are supported by at least 1 reference from the literature, textbook or from signalling pathway analysis information generated by the Ingenuity Knowledge Base.

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Figure 5.4: Representative gene network within the 1,780 SDE transcripts (upregulated and downregulated) in acute meningococcal disease compared to convalescence and the role of HSP70

5.9.2 Gene network analysis focused on 933 upregulated SDE transcripts in acute meningococcal disease compared to convalescence and the role of HSP70 The main functions represented by this network were cellular assembly and organisation, cellular function and maintenance and cell-cycle (Figure 5.5). This network has a score of 32 is equivalent to a P value of 1.0E-32. It contains 28 focus molecules, of which genes associated with HSP70 (HSPA1A/HSPA1B) are one. Several of the connected genes within this network relate to inflammatory signalling pathways including an indirect relationship with MAKP14 a member of the p38MAPK signalling pathway, which required further investigation. Other relationships were with apoptotic genes for example CARD16 (caspase recruitment domain family, member 16).

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Figure 5.5: Representative gene network within the 933 upregulated SDE transcripts in acute meningococcal disease compared to convalescence and the role of HSP70

5.9.3 Gene network analysis focused on 492 downregulated SDE transcripts in acute meningococcal disease compared to convalescence and the role of HSP70 The main functions represented by this network were DNA replication, recombination and repair, cell death and protein degradation (Figure 5.6). This network has a score of 23, which is equivalent to a P value of 1.0E-23. It contains 18 focus molecules, of which HSP70 is one. Considering the downregulated dataset only the network analysis revealed the genes associated with HSP70 is upregulated. Other connected genes within relate to other HSP families, which are downregulated, such as DNAJA3. HSP70 is known to have a link with ATR, which is protein kinase which promotes the assembly of DNA repair complexes at damaged sites with the chromosome. DNAJA3 is part of the HSP40 family and is an essential co- chaperone with HSP70 to enhance ATPase and substrate release. 143

Figure 5.6: Representative gene network within the 492 downregulated SDE transcripts in acute meningococcal disease compared to convalescence and the role of HSP70

5.9.4 Summary of network analysis considering the differential expression of HSP70 in acute meningococcal disease compared to convalescence To understand the biological relevance of HSP70 within the acute phase of meningococcal disease a network analysis was completed examining the entire dataset and then genes differentially up- or downregulated separately. Considering the network analysis from the 1,780 SDE gene transcripts (up- and downregulated), where HSP70 is SDE, the main functions of the gene network were; protein folding, cell death and cell compromise. HSP70 is known to regulate cell homeostasis, through protein folding of damaged self and non-self proteins.

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With respect to the 933 upregulated SDE genes transcripts the main functions of the gene network were; cellular assembly and organisation, cellular function and m maintenance and cell-cycle. Several of the connected genes within this network relate to inflammatory signalling pathways including an indirect relationship with MAKP14. HSP70 is known to mediate the inflammatory response to infection and is able to signal via the p38MAPK pathway.

With respect to the 492 downregulated SDE genes transcripts the main functions of the gene network were DNA replication, recombination and repair, cell death and protein degradation. HSP70 is known to co-chaperone with other HSP’s increasing its affinity for cell repair and substrate release in an ATP dependent manner.

5.10.1 Relationships between plasma protein levels of HSP70, glutamine, inflammatory mediators (IL-8, IL-6, IL-1β and TNF-α) and SDE transcripts associated with inflammatory and immune functions Pearson correlation was used to investigate whether there was a relationship between individual SDE genes of interest (> 2log2 fold change) and corresponding plasma protein levels of HSP70, glutamine, inflammatory mediators (IL-8, IL-6, IL-1β, TNF-α) during acute disease and in convalescence, as described by Sonna et al (497). Genes selected were those associated with HSP70, HLA, inflammatory mediators IL-1β, IL-8 and inflammatory signalling pathways p38MAPK. There are 164 genes within the p38MAPK signalling pathway (476) and 25% (n=40) were SDE within the dataset (Table 5.2), which are ordered by fold change. SDE gene transcripts for HSP70 IL-1β and IL-8 were upergulated and those associated with HLA were downregulated.

Genes associated with IL-6, TNF-α were not significantly differentially expressed (at a log2 FC ≥ 2) in the acute phase of meningococcal disease when compared to convalescence and were therefore not included in the analysis. For each patient, protein levels were linked to the expression values for each gene of interest. Where there was more than 1 transcript for the gene the median P value was taken, as there were very little differences between values.

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Table 5.2: SDE gene transcripts associated with p38MAPK signalling pathway in children with acute meningococcal disease compared to convalescence

Symbol Entrez Official full name Direction Corrected Absolute Gene of p-value Fold ID regulation Change IL1R2 7850 interleukin 1 receptor, type II 5.13E-06 43 GADD45A 1647 growth arrest and DNA-damage-inducible, alpha 5.27E-06 15.74 IL1RAP 3556 interleukin 1 receptor accessory protein 5.35E-06 9.2 MKNK1 8569 MAP kinase interacting serine/threonine kinase 1 5.13E-06 6.28 ZAP70 7535 zeta-chain (TCR) associated protein kinase 70kDa 8.66E-06 4.57 STK3 6788 serine/threonine kinase 3 (STE20 homolog, yeast) 1.09E-05 4.47 DUSP1 1843 dual specificity phosphatase 1 (MPK-1) 5.35E-06 4.08 CEBPB 1051 CCAAT/enhancer binding protein (C/EBP), beta 5.13E-06 3.89 CDC25B 994 cell division cycle 25 homolog B (S. pombe) 5.47E-06 3.7 MAPK14 1432 mitogen-activated protein kinase 14 3.55E-04 3.54 CDC42 998 cell division cycle 42 (GTP binding protein, 25kDa) 3.09E-05 3.41 RPS6KA5 9252 ribosomal protein S6 kinase, 90kDa, polypeptide 5 4.18E-05 3.29 LCK 3932 lymphocyte-specific protein tyrosine kinase 2.18E-05 3.15 STAT1 6772 signal transducer and activator of transcription 1, 9.15E-05 3.13 91kDa MAP3K8 1326 mitogen-activated protein kinase kinase kinase 8 5.27E-06 2.97 NCF2 4688 neutrophil cytosolic factor 1, (chronic 9.15E-05 2.94 granulomatous disease, autosomal 2) CASP5 838 caspase 5, apoptosis-related cysteine peptidase 0.007 2.86 CD14 929 CD14 molecule 7.68E-06 2.8 JAK2 3717 Janus kinase 2 (a protein tyrosine kinase) 2.33E-05 2.77 CREB5 9586 cAMP responsive element binding protein 5 0.002 2.7 NCF4 4689 neutrophil cytosolic factor 1, (chronic 5.17E-05 2.69 granulomatous disease, autosomal 4) FOSL2 2355 FOS-like antigen 1 0.015 2.66 ATF6 22926 activating transcription factor 6 3.50E-05 2.56 MYD88 4615 myeloid differentiation primary response gene (88) 5.27E-06 2.53 STK39 27347 serine threonine kinase 39 (STE20/SPS1 homolog, 1.67E-05 2.5 yeast) MEF2C 4208 myocyte enhancer factor 2C 4.26E-05 2.49 PRKCD 5580 protein kinase C, delta 6.74E-06 2.49 MAPK 9261 mitogen-activated protein kinase-activated protein 7.25E-06 2.45 APK2 kinase 2 SOCS2 8835 suppressor of cytokine signalling 2 5.70E-04 2.41 IL1B 3553 interleukin 1, beta 1.34E-04 2.4 JUNB 3726 jun B proto-oncogene 1.17E-05 2.4 ZAK 51776 sterile alpha motif and leucine zipper containing 1.67E-04 2.36 kinase AZK DDIT3 1649 DNA-damage-inducible transcript 3 8.28E-05 2.31 TNFRSF1A 7132 tumour necrosis factor receptor superfamily, 6.88E-05 2.22 member 1A TOLLIP 54472 toll interacting protein 5.13E-06 2.22 MEF2A 4205 myocyte enhancer factor 2A 0.002 2.09 PPM1B 5495 protein phosphatase 1B (formerly 2C), magnesium- 2.63E-05 2.08 dependent, beta isoform ZFP36 7538 zinc finger protein 36, C3H type, homolog (mouse) 1.28E-05 2.07 MAPK 7867 mitogen-activated protein kinase-activated protein 5.13E-06 2.05 APK3 kinase 3 MAP2K1 5604 mitogen-activated protein kinase kinase 1 7.68E-06 2.02 146

5.10.2 Plasma protein levels of HSP70, glutamine are positively correlated with genes associated with HSP70, p38MAPK and HLA during meningococcal disease

Plasma levels of HSP70 are positively correlated with genes (> 2log2 fold change) during the acute phase of meningococcal disease and in convalescence implying that during infection there is significant upregulation of genes associated with HSP70 resulting in increased plasma levels HSP70 (Table 5.3).

During the acute phase of meningococcal disease plasma levels of glutamine and HSP70 were correlated with genes from the MAPK family suggesting a relationship between glutamine, HSP70 and genes associated with the inflammatory response. During convalescence there were a number of negative correlations with respect to plasma levels of glutamine and gene transcripts from the MAPK family, suggesting that glutamine levels may impact on the MAPK signalling pathway. Negative relationships were also seen relative to immune function with multiple gene transcripts associated with HLA antigen presentation being negatively correlated with convalescent glutamine levels, suggesting that plasma glutamine levels may be an important determinant for effective antigen presentation.

Plasma protein levels of IL-1β were correlated with genes associated with IL-1β and IL-8. There was no relationship was found between glutamine and genes associated with HSP70 or plasma levels of inflammatory mediators; IL-8, IL-6, IL-1β, TNF-α, suggesting in this cohort there is no relationship between glutamine and genes associated with HSP70 and inflammatory mediators. However, the sample size was small and this could have impacted on the likelihood of achieving significance.

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Table 5.3: Correlations between protein levels and gene transcripts related to inflammation and immune function during the acute phase of meningococcal disease and convalescence Plasma Gene Enterez Direction Absolute Correlation protein association Gene of Fold (r)/significance (p) (number) symbol regulation Change Acute HSP70 HSP70 HSPA1A 2.45 r= 0.53; p=0.004 (n=27) HSPA1B 2.89 r= 0.72; p=<0.0001 p38MAPK JUNB 2.40 r= 0.44; p=0.01 Glutamine p38MAPK IL1RAP 9.20 r= 0.41; p=0.041 (n=25) MAP3K8 2.97 r= 0.41; p=0.04 CREB5 2.70 r= 0.51; p=0.009 JUNB 2.40 r= 0.4; p=0.04 MAP2K1 2.02 r= 0.4; p=0.04 TOLLIP 2.22 r= -0.47; p=0.01 CD14 2.80 r= -0.47; p=0.01 JAK2 2.77 r= -0.54; p=0.005 ZAP70 4.57 r= -0.47; p=0.01 IL-1β Inflammatory IL-1β 2.40 r= 0.38; p=0.048 (n=27) response IL-8 5.65 r= 0.38; p=0.048 Convalescence HSP70 HSP70 HSPA1A 2.45 r= -0.54; p=0.04 (n=15) HSPA1B 2.89 r= -0.45; p=0.09 p38MAPK CD14 2.80 r= -0.58; p=0.02 GADD45A 15.74 r= -0.54; p=0.03 SOCS2 2.41 r= -0.53; p=0.03 CASP5 2.80 r= 0.52; p=0.04 MEF2C 2.49 r= -0.53; p=0.03 RPS6KA5 3.29 r= -0.52; p=0.04

STAT1 3.13 r= -0.56; p=0.02 Glutamine p38MAPK LCK 3.15 r= 0.58; r=0.04 (n=12) MAP2K1 2.45 r= -0.40; p=0.04 CDC258 3.70 r= -0.65; p=0.02 CREB5 2.70 r= 0.67; p=0.01 DUSP-1 4.08 r= 0.62; p=0.03 JUNB 2.40 r= 0.64; p=0.02 JAK2 2.77 r= -0.65; p=0.02 MAPK14 3.54 r= -0.74; p=0.005 MAPKAPK3 2.05 r= -0.66; p=0.018 HLA – HLA-DMA 3.18 r=- 0.69; p=0.01 antigen HLA-DPA1 3.92 r= -0.84; p=0.001 presentation HLA-DQA1 3.73 r= -0.65; p=0.02 HLA-DRB3 3.68 r= -0.58; p=0.05 HLA-DRB6 3.09 r= -0.76; p=0.004

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5.11.1 Validation of SDE gene transcripts using ToppGene confirms dysregulated adaptive immune function and inflammatory signalling pathways in meningococcal disease Validation of 1,780 SDE gene transcripts, using gene ontology from ToppGene, confirms in acute meningococcal disease compared to convalescence, there was significant enrichment of signalling pathways representing adaptive immune function, the antigen presentation pathway and inflammatory signalling pathways as seen using IPA (Figure 5.7).

Figure 5.7: Validation of SDE gene transcripts using ToppGene molecular functions. Signalling pathways represent adaptive and innate immunity and the antigen presentation pathway in acute meningococcal disease compared to convalescence

5.11.2 Validation of SDE genes using an independent cohort of children with acute meningococcal disease compared to controls

The SDE genes (log2 fold change ≥ 2) in a comparable dataset of children with meningococcal sepsis were compared with the 1,780 SDE dataset using IPA (Figure 5.8). Signalling pathways in the independent cohort of children with acute meningococcal disease compared to controls were similarly enriched, representing adaptive immunity, inflammatory signalling pathways, the antigen presentation pathway and apoptotic pathways. These findings confirm the validity of the results generated from the transcriptomic analysis of children with acute meningococcal disease compared to convalescence.

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Figure 5.8: Significant terms for signalling pathways comparing an independent validation dataset with SDE gene transcripts for acute meningococcal disease. Dark blue bars (acute meningococcal disease); light blue bars (validation dataset)

5.12 Discussion Monocyte and macrophage stimulation by endotoxin leads to the upregulation of genes and transcription factors that promote the expression and release of inflammatory mediators, including cytokines (49).The pathophysiology of severe meningococcal disease is associated with high levels of endotoxaemia leading to a significant release of these inflammatory mediators provoking shock and multi-organ failure (498). Within the dataset, children with acute meningococcal disease had 1,780 SDE gene transcripts with 1614 unique genes (≥ 2 log2 fold change) compared to convalescence. The data was validated using protein levels from the cases during the acute and convalescent disease stages as well as assessing biological functions and pathways represented in the dataset by using a second meningococcal disease dataset and a different biological database. As expected the biological processes that the genes represented in the dataset fell into three main categories: respiratory disease, infectious disease and inflammatory response, of which the latter two are associated with the pathophysiology of meningococcal disease (498).

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The glycolysis and gluconeogenesis signalling pathway was the most significantly enriched, with up to 25% of the pathway in the dataset. Increased gluconeogenic and glycolytic pathways are hallmarks of critical illness and are associated with protein efflux from muscle delivering glutamine and alanine for participation in the citrate cycle for de novo glucose synthesis (499). Most of the genes were significantly upregulated in the gluconeogenic pathway in our children with meningococcal disease which may have contributed to the significantly decreased levels of plasma glutamine seen in this cohort.

Severe infection is associated with increased expression of PRR such as TLRs (500). In this database signal transduction pathways associated with TLR (p=2.63E-03) and NF-κB (p=5.74- 03) were significantly enriched with 12% and 20% of the pathways respectively represented within the dataset. The TLR pathway was significant in children with acute meningococcal disease highlighting the role of signal transduction via TLR2/4-CD14-MD2 activating the inflammatory pathways of NK-κB and p38MAPK, as has been described in other cohorts of septic patients (467) and in children with acute meningococcal disease (476).

Acute plasma glutamine was positively correlated with TLR 2 expression (n=25, r=0.45, p=0.02) and TLR4 expression (n=25, r=0.59; p=0.002). Interestingly, in a septic rat model, enteral glutamine administration attenuated TLR4, MYD88 and TRAF mRNA accumulation in the intestinal mucosa, downregulating the release of TNF-α, IL-6 and IL-1 (501), although in critically ill adults glutamine supplementation had no effect on TLR4 expression (502). In the context of this cohort it is known that malnutrition affects bacterial defence mechanism, including the release of DAMPs in response to PAMP’s and activation of PRRs. Therefore, glutamine depletion could affect bacterial recognition and phagocytosis resulting in the accumulation of bacterial wall fragments, which may intensifying the inflammatory response and release of inflammatory mediators (503), which is supported by data from the current research.

The network analysis of the SDE (up- and downregulated) transcripts revealed that genes associated with HSP70 are differentially upregulated during the acute phase of illness suggesting they have a role in meningococcal disease. Although not previously described in meningococcal disease, genes associated with HSP70 family have been shown to be

151 upregulated in adult patients, both with sepsis and non-infectious SIRS (497). In this cohort, plasma levels of HSP70 were positively correlated with HSPA1A and HSPA1B during the acute phase of illness, suggesting that upregulation of genes associated with HSP70 may be linked to the accumulation of HSP70 within the plasma. Although it would seem likely that an increase in HSPA1A and HSPA1B gene expression would result in changes to plasma levels of the protein, is that it is not possible to establish causality based on correlation alone (497).

The gene network associated with HSP70, included annotations for cell death, protein degradation and cell compromise. Several of the connected genes relate to apoptosis; apoptotic peptidase activating factor 1 (APAF-1) is involved in apoptosis, binding to cytochrome C, activating caspase-3 and caspase-7. HSP70 is known to mediate apoptosis by regulating APAF-1/ cytochrome C aggregation (504). Within the cohort, signalling pathways associated with apoptosis were significantly downregulated during acute phase of meningococcal disease, suggesting the influence of HSP70 on inhibiting apoptosis is dysregulated.

Another apoptotic protein, which is directly linked in this network, was peptidoglycan recognition protein 1 (PGLYRP-1), which functions as an innate immune regulator during times of stress forming a cytotoxic complex and provoking apoptotic cell death (505). CARD16 was also highly expressed within the network of only upregulated genes and directly connected to HSPA1A and HSPA1B (506). CARD16 is a member of the caspase family representing apoptotic genes, this in combination with upregulation of APAF-1 is often suggestive of an immunosuppressive effect mediated by meningococcal disease (507). HSP70 have been shown to prevent mitochondrial-mediated apoptosis by inhibiting APAF-1 and apoptosis inducing factor (AIF), leading to the suppression of caspase-dependent and independent apoptosis (508, 509). Thus in this cohort, where APAF-1 is upregulated, the anti-apoptotic effect of HSP70 may be reduced, increasing the risk of cell death, consistent with results of HSP70 inhibition studies.

Amongst genes that were represented in meningococcal disease the representative downregulated gene network associated with HSP70 included annotations for DNA replication, recombination and repair, cell death and protein degradation. Genes associated

152 with HSP70 were upregulated within the dataset, however, several of the connected genes within the network related to other HSP families such as DNAJA3, were differentially downregulated. HSP70 is known to have a link with ATR, a protein kinase, which promotes cell repair through the assembly of DNA repair complexes at damaged sites within the chromosome. Depletion of HSP70 is associated with impaired phosphorylation of ATR, increasing the cell related damage (510). DNAJA3 is part of the HSP40 or DnaJ family of chaperones DNAJA3 is part of the HSP40 family and is an essential co-chaperone with HSP70 to enhance ATPase and substrate release (33), by transiently binding to ATP promoting HSP70 function (511) and facilitating the folding of newly synthesized proteins in an ATP dependent manner (512). HSP70 is vital to the maintenance of protein folding and protein- protein interactions within the cell, preventing protein aggregation (512), however depletion of HSP70 can lead to cell death (513). Where there is mitochondrial dysfunction with reduced ability to produce ATP, necrotic cell death ensues (514). Within this network, housekeeping gene transcripts associated with HSP70 were downregulated (DNAJA3), which has been described in transcriptomic analysis of septic adults (497) and there may therefore by an increased risk of necrotic cell death in this cohort of children.

Within the dataset of upregulated genes only, signalling pathways for IL-6 (p=5.64E-04), IL- 10 (p=6.03E-04) were enriched, with 17% of the pathway represented within the dataset for both, during the acute phase of meningococcal disease. The upregulation of genes associated with signalling pathways for IL-8, IL-6 and IL-10 has been described in children with septic shock and meningococcal disease (338, 476). Genes associated with IL-1β and IL- 8, in children during the acute phase of their illness, was strongly correlated with plasma levels of inflammatory mediators (TNF-α, IL-1β, IL-8 and IL-6) during the acute and convalescent stages of the disease, although there were no correlations with either HSP70 or glutamine. As seen in Chapter 4, during the acute phase of meningococcal disease levels of inflammatory mediators were significantly higher when compared to convalescence. Other genome-wide trascriptomic studies of sepsis, including children, describe significant gene enrichment of signal transduction and inflammatory pathways early in the course of sepsis (467). It is increasingly recognised that the classic pattern of early an pro-inflammatory response with the upregulation of genes associated with TNF-α, IL-1β followed by a second phase of anti-inflammatory mediators (IL-10) may be too simplistic and that within a clinical

153 setting this classic response to infection is rarely seen (515). This is reflected in transcriptomic studies as there appears to be no consistent progression from an early pro- inflammatory state to a subsequent anti-inflammatory phase (467). HSP70 plays a central role in mediating the inflammatory response to stress, activating the NF-κB pathway (108), and then countering the inflammatory response by inhibiting the transcription of IL-1β (414) and TNF-α (516) preventing the further release of IL-8 and IL-6 (415), but this was not seen in this dataset as HSPA1A and HSPA1B were not correlated to cytokine levels or expression levels.

During the acute phase of meningococcal disease glutamine was positively correlated with JAK2 (n=27, r=0.41; p=0.04) and during convalescence HSP70 was negatively correlated with STAT (n=27, r-=0.56; p=0.02) and SOCS2 (n=27, r=-0.56; p=0.02). JAK2 is part of the JAK-STAT cascade, which is reported as being upregulated during sepsis. JAK-STAT inflammatory cascade has anti-inflammatory properties, which include negative feedback mechanisms involving suppressor of cytokine signalling (SOCS) induction, involved in the early inhibition of TLRs and inflammatory mediator signalling. Sepsis is associated with dysregulation of this pathway as a result of significant downregulation of genes associated with mitochondrial activity, decreasing cell metabolism including protein synthesis machinery (517). Glutamine has been shown to mediate anti-inflammatory effects through STAT pathways (483), it could be that glutamine depletion diminishes the efficacy of JAK2 expression during acute meningococcal disease compared to convalescence.

Genome-wide transcriptional studies of children with septic shock reveal that a large number of genes are differentially downregulated particularly those associated with adaptive immunity, such as those linked to a T-cell mediated immune response (518). The top signalling pathways, within the dataset of studied children with meningococcal disease were mainly associated with downregulated activity of genes associated with adaptive immunity. The antigen presentation pathway was also found to be affected since most of the genes in these pathways were significantly downregulated in the acute phase of the disease compared to convalescence.

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Genes associated with antigen presentation (particularly those related to MHC Class II) have also been found to be as being downregulated during adult trauma (496) and paediatric septic shock (360, 472). During critical illness there is depressed macrophage and monocytic expression of MHC class II particularly HLA-DR (519) and HLA-DQ (520). Ex vivo studies show monocytes from these patients have a decreased ability to mount an appropriate inflammatory response when stimulated with LPS (347). Reduced expression of HLA-DR is seen as a marker of the degree and duration of immune paralysis (521, 522) and within a clinical context is a good predictor of septic complications (490, 523) and mortality (347, 524, 525).

Maintenance of HLA-DR expression may be an important strategy to prevent infectious complications (347). It may be that maintenance of effective HLA-DR expression is dependent on glutamine availability (526) as there is evidence to suggest that APC cells including macrophages and monocytes are dependent on glutamine for optimal function (527). In vitro work has shown a 40% reduction in HLA-DR expression over the course of a week as glutamine levels declined (526) and that levels of < 0.4 mMol/L were associated with a 30% reduction in HLA-DR expression (346, 526). However, glutamine supplementation is not able to completely restore HDL-DR functionality (347). It could be that maintenance of plasma glutamine levels during critical illness could in some way promote an effective immune and inflammatory response to infection, through maintenance of MHC Class II expression on APC cells allowing optimal HSP70:antigen presentation. Further work would be required to corroborate such a hypothesis.

There were significant inverse correlations with plasma glutamine levels and SDE genes associated with MHC Class II during convalescence. In this subset of children, glutamine levels during convalescence were found to be 58% below the normal range. No correlation could be found with of HLA gene expression and low glutamine during the acute phase of the disease, suggesting that glutamine levels may contribute to an increased secondary infection risk during convalescence. The pattern of differentially downregulated genes associated with MHC Class II (HLA-DMA, HLA-DPA1, HLA-DQA1, HLA-DRB3 and HLA-DRB6) has been described in genome-wide analysis of trauma patients and was strongly correlate with multi-organ failure and 28 day mortality (496). Depressed MHC Class II expression on

155 monocytes/ macrophages is associated with increased risk of late infectious complications in septic adults (490) and critically ill children (495). Both parenteral and enterally administered glutamine in critically ill adults has been shown to be effective in promoting increased HLA-DR expression, in part due to the maintenance of normal plasma glutamine levels (346, 347).

HSP70 forms complexes with pathogenic bacteria (and endogenous proteins), which are capable of activating CD4+ (528, 529) and CD8+ complexes on T cells (530) facilitating MHC class I and II peptide assembly in addition to receptor-facilitated endocytosis (528). In experimental in vitro and in vivo models MHC Class II activation following HSP70 interaction facilitates the induction of innate immunity including the LPS-CD14+ signal transduction of NF-κB pathway leading to the release of inflammatory mediators (531). Although HSP70 antigenic protein presentation has not been described in paediatric critical illness, it could be hypothesised that if antigen presentation is downregulated, HSP70 processing of pathogenic proteins (including endotoxin), via MHC Class II receptors, may be impaired. However, there was no correlation between HLA and HSP70 protein levels.

Genes associated with p38MAPK and JNK play a pivotal role in the transcriptional and posttranscriptional regulation of inflammatory mediators (532). Within this cohort, there were multiple positive and negative correlations with plasma levels of glutamine, HSP70 and genes associated with the p38MAPK signalling pathway during the acute phase of illness and convalescence. This suggests that these proteins could influence the inflammatory response to meningococcal disease. In vitro mouse models of sepsis have shown that glutamine supplementation inhibits the activation of p38MAPK and JNK through de-phosphorylation of MAPK’s. This is affected by the activation of MKP-1 (also known as DUSP-1), an important regulator of the MAP kinases. The activation of MKP-1 following glutamine supplementation is rapid and protects against shock and mortality in animal models of sepsis (533). An in vivo model involving septic rats has shown that glutamine supplementation attenuates the activation of p38MAPK and ERK, through HSP70 upreguation, downregulating the release of TNF-α, IL-6 and IL-8 (534). In this study cohort MKP-1 was only correlated to plasma glutamine levels during the convalescent phase. HSP70 has also been shown to phosphorylate MKP-1 preventing the activation of p38MAPK (199, 535).

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In the context of this study cohort, glutamine levels were depleted for up to 55 days post admission of meningococcal disease. This could have a negative impact of the host’s ability to attenuate the inflammatory response, especially as there were numerous positive and negative correlations with both HSP70 and glutamine and p38MAPK signalling pathway. Further work will be required to understand the impact of glutamine supplementation on the transcriptional control of p38MAPK and the role of glutamine on HSP70 upregulation may play.

There are a number of limitations to this work. Due to the small sample size transcriptomic analysis based on age related stratification was not possible. Of note, other transcriptomic analysis of paediatric sepsis have shown that neonates with septic shock have more significantly downregulated expression of genes associated with innate and adaptive immunity compared to school aged children (338). Future work would aim to collect blood samples for proteomic and gene analysis at standardized time points throughout the study, in addition to stratifying patients into relevant age based categories matched to appropriate age matched controls. Although the transcriptomic findings reported in this cohort are well described in other related studies (360, 472, 536), further work is required to validate the functional importance of the transcriptomic results using functional assays (such as phytohemagglutinin (PHA) stimulation of whole blood) and flow cytometry, to describe cell surface expression of markers associated with T lymphocyte function e.g. CD11b and adaptive immunity e.g. HLA, as described in other critically ill cohorts (495, 537-540).

5.13 Conclusion The results from this transcriptomic analysis showed inflammatory signalling pathways were significantly upregulated whereas those relating to adaptive immunity and antigen presentation were downregulated. Genes associated with HSP70 were correlated with plasma levels of HSP70 suggesting that HSP70 does play a role in the pathophysiology of meningococcal disease. During the acute phase of the disease, plasma levels of glutamine and HSP70 were correlated with numerous genes associated with p38MAPK signalling pathways. In addition, convalescent plasma glutamine levels were correlated with genes associated with HLA suggesting a role for glutamine in antigen presentation. As glutamine levels were significantly depleted in the acute and convalescent phase it could be that the

157 positive results of glutamine supplementation is mediated by an appropriate resolution of inflammation in addition to promoting adaptive immunity. Future work would aim to understand the effect of glutamine supplementation on gene expression during critical illness, especially relative to genes associated with HSP70, inflammatory signalling pathways and those relating to HLA.

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Chapter 6: General Discussion and Conclusion

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Chapter 6: General Discussion and Conclusion 6.1 General Discussion Paediatric sepsis affects a significant proportion of children annually with a mortality rate of 10% (3-5). The inflammatory response to infection forms part of the host response to sepsis and is vital to survival. However, the dysregulated release of inflammatory mediators can result in organ damage increasing the risk of early and late mortality (17). There is renewed interest in the concept that nutraceuticals, such as glutamine may present as therapeutic strategies, particularly in promoting HSP70 release, thus mediating the immune and inflammatory response thereby improving outcome (207).

Glutamine supplementation in critically ill adults is associated with decreased morbidity and mortality (271-273), although the same benefits with respect to glutamine supplementation have not been reported in critically ill children (541). The beneficial effects of glutamine supplementation during critical illness may relate to the upregulation of HSP70 release. During times of stress HSP70 release is associated with improved outcome in experimental models of sepsis (310, 311, 345) and in critically ill adults (143).

The aim of this study was to investigate the effects of sepsis on the release of HSP70 and the relationship with glutamine and inflammatory mediators. The study was conducted in two parts, firstly using whole blood in an experimental in vitro endotoxin stimulation model and secondly in vivo analysis of HSP70 release and the relationship to glutamine and inflammatory mediators in children with acute meningococcal disease.

The aim of the in vitro study was to investigate the effect of glutamine supplementation on HSP70 release, using an in vitro endotoxin model. The results showed that 2mM glutamine in endotoxin stimulated blood significantly increased HSP70 release in whole blood from adults and children, in agreement with previous studies (93, 203, 407). HSP70 release was significantly higher in children when compared to adults with a similar pattern of release as described in critically ill adults (143) and children with septic shock (141).

The different effects of glutamine supplementation on the release of inflammatory mediators, in adults compared to children were surprising. In adults, glutamine

160 supplementation appeared to attenuate the release of inflammatory mediators, whereas in children glutamine supplementation was associated with pro-inflammatory response. In vitro work by others has shown that HSP70 can mediate both a pro- (399, 407) and anti- inflammatory (414-416) response. Glutamine supplementation has also been shown to be associated with a pro-inflammatory response in vitro (399) with the converse effect in vivo during sepsis (86, 399, 417, 418). Since in vitro studies cannot completely replicate conditions within a host, future work should focus on the relationship between glutamine and HSP70 release over time in critically ill children, investigating the role that HSP70 plays in the formation of complexes with pathogens and the subsequent immune and inflammatory responses with respect to gene transcription and protein levels within the cells/ tissue. Recent theories have focused on the concept that HSP70 acts as a DAMP in the presence of large amounts of pathogens; as infection resolves HSP70 helps to restore homeostasis by acting as a “DAMPER” downregulating the immune and inflammatory response (191).

The use of a HSP70 inhibitor KNK437 significantly decreased the intracellular and extracellular release of HSP70 and inflammatory mediators (IL-6, IL-10, TNF-α). These results suggest that HSP70, in part, mediates the endotoxin stimulated release of inflammatory mediators (IL-6, IL-10, TNF-α), as the addition of KNK437, diminishes HSP70 release with a concomitant downregulated release of IL-6, IL-10 and TNF-α release as described by other groups (385, 420, 421).

Results from the in vivo aspect of the study revealed HSP70 levels were significantly higher in children with meningococcal disease during the acute phase of illness, which has been reported in children with septic shock (141). Glutamine depletion was apparent early on during the disease, and in convalescent samples, taken up to 2 months later, an effect also reported in critically ill adults (209). Although feeding was initiated early, it took up to 4 days before goal rate was achieved. Even at goal rate the level of glutamine delivered was only 0.14g/kg glutamine, which is likely to be insufficient to restore levels to within normal range. A dose of 0.3 – 0.5g/kg has been shown to restore plasma glutamine levels to within normal range (274, 275) and benefits are possibly associated with increased HSP70 release during critical illness (207, 239).

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Inflammatory mediators were positively correlated with HSP70 levels, but not with glutamine. Positive correlations between HSP70 levels were found with length of PICU stay, duration of mechanical ventilation, severity of illness score PRISM, lactate and CRP. These relationships suggest that elevated levels of HSP70 may be associated with disease severity, inflammation and cell dysfunction, which is seen in adults with chronic inflammatory disease who have been found to have higher levels of HSP70, CRP and other inflammatory markers compared to healthy controls (454). In this cohort, plasma glutamine and CRP were inversely correlated consistent with the findings by others that administration of glutamine was associated with significantly lower CRP levels post surgery in children (455) and adults (458).

In adult patients, glutamine supplementation of PN significantly increased HSP70 levels which was associated with a decreased length of ICU stay and fewer ventilator days (143). As glutamine is safe, non toxic and easy to administer it is a potentially attractive modulator of the heat shock response (206-210). Glutamine administration is recommended by numerous nutrition associations including ESPEN as being of benefit (273). However, the results in children have been less convincing, the studies fraught with problems relating to design, dose of glutamine and heterogeneity of the study population (541). It has been suggested that plasma glutamine levels should be routinely measured, aiming to target treatment for maximal efficacy (246). However, as amino acids are not routinely measured in critically ill children other proxy measures could be used. C-reactive protein may be a more useful proxy of low glutamine and high HSP70 levels as it was positively correlated with HSP70 and negatively correlated with glutamine levels, suggesting that in this cohort of critically ill children, it may well be useful in predicting who would benefit from glutamine supplementation. Steroid administration was also predictive of low glutamine levels and could potentially also serve as a indicator for when glutamine supplementation should be used.

As glutamine levels remained low into convalescence, future work should consider the efficacy of post discharge glutamine supplementation and the impact this may have on rehabilitation and long term outcome. Follow up studies of survivors of N. meningitidis report that 5 – 20% of patients continue to have problems with intellectual functioning and illness related physical or social consequences (12). Since glutamine depletion has an impact

162 on 6 month mortality (209) it could be that glutamine supplementation is required in some children post ICU discharge.

Transcriptomic analysis of differentially regulated genes in children with meningococcal disease during the acute phase compared to convalescence was undertaken. Genes associated with HSP70 (HSPA1A and HSPA1B) were differentially upregulated during the acute phase of illness, suggesting that HSP70 plays a role in meningococcal disease. Although not previously described in meningococcal disease, genes associated with HSP70 family have been shown to be upregulated in adult patients, both with sepsis and non-infectious SIRS (497).

The top signalling pathways within the entire dataset of children with meningococcal disease during the acute phase of illness were mainly associated with downregulated activity of genes associated with adaptive immunity such as CD28 signalling in T helper cells, NUR77 signalling in T lymphocytes, which represented between 24 - 27% of genes within the entire dataset. The antigen presentation pathway was found to be significantly differentially downregulated in the entire dataset of children with meningococcal disease with up to 20% of genes from the entire dataset represented in the pathway, a finding described in other genomic work considering paediatric septic shock (360, 472).

When comparing plasma glutamine levels with differentially expressed genes associated with MHC Class II, there were significantly inverse correlations with glutamine levels (mmol/l) during convalescence. No correlation was found with acute measures of HLA gene expression and glutamine, suggesting that glutamine levels may contribute to an increased secondary infection risk during convalescence. The pattern of differentially downregulated genes associated with MHC Class II presentations has been described in genome-wide analysis of trauma patients and was found to strongly correlate with multi-organ failure and 28 day mortality (496). Depressed MHC Class II expression on monocytes/macrophages is associated with increased risk of late infectious complications in septic adults (490) and critically ill children (495). Both parenteral and enterally administered glutamine in critically ill adults has been shown to be effective in promoting increased HLA-DR expression, in part due to the maintenance of normal plasma glutamine levels (346, 347).

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The glycolysis and gluconoegenesis signalling pathway was the most significantly enriched and differentially upregulated signalling pathway, with up to 25% of the entire gene set represented in this pathway. Increased gluconeogenic and glycolytic pathways are hallmarks of critical illness and are associated with protein efflux from muscle delivering glutamine and alanine for participation in the citrate cycle for de novo glucose synthesis (499). It could be hypothesised that glutamine levels are depleted as a result of the upregulation of the gluconeogenic pathway in children with meningococcal disease.

During the acute phase of meningococcal disease plasma levels of HSP70 and glutamine were positively and negatively correlated with the expression of several up- and downstream kinases within the p38MAPK pathway including JAK2, STAT-1 and DUSP-1 (MKP- 1). p38 and JNK plays a pivotal role in the transcriptional and post-transcriptional regulation of inflammatory mediators (532). Other in vivo septic rat models have shown glutamine supplementation inactivates p38MAKP and ERK activation through HSP70 release, attenuating the release of TNF-α, IL-6 and IL-8 (534).

As glutamine is able to inactivate p38 and JNK through phosphorylation of MKP-1 (533) the administration of glutamine during critical illness may be a useful therapeutic adjunct. The beneficial effect of glutamine relative to the transcriptional activation of p38MAPK and JNK may be lost when glutamine levels are depleted, which has been described for up to 30 days post ICU admission (209). In the context of this meningococcal disease cohort glutamine levels were depleted for up to 55 days post admission which may have a negative impact over the host’s ability to limit the intensity of the inflammatory response, especially as there were numerous positive and negative correlations with both HSP70 and glutamine and p38MAPK. Further work will be required in the future to understand the impact of glutamine supplementation on transcriptional control of p38MAPK and the role HSP70 upregulation by glutamine may play.

As glutamine depletion is common in critical illness (307), this could lead to an insufficient ongoing HSP70 response in severe sepsis, which may contribute to cell injury, organ damage (388) and increased risk of secondary infections (189). Perhaps the role of glutamine supplementation, in severe sepsis is to facilitate an appropriate HSP70 response reducing

164 organ damage and enhancing survival (128, 143, 196). By promoting glutamine homeostasis, cells which rapidly consume glutamine (macrophages, lymphocytes, neutrophils and monocytes) (399) are able to continue producing appropriate amounts of HSP70 in addition to inflammatory mediators, in response to stress. As stress resolves, HSP70 is then able in inhibit the transcription of inflammatory mediators (414, 516).

The results from this transcriptomic analysis show that genes associated with HSP70 and inflammatory signalling pathways within the dataset are differentially upregulated and those relating to adaptive immunity are downregulated. In a glutamine deficient state, optimal upregulation of genes (237) and post transcriptional inactivation of genes associated with the inflammatory response does not occur (533). The transcriptomic findings reported in this cohort are well described in other related studies (360, 472, 536). However, it will also be important to validate the functional importance of these results data using multiplex assays and flow cytometry to measure T lymphocyte function, PRR function and adaptive immunity such as MHC II expression, as described in other critically ill cohorts (2, 495, 537-539, 542).

6.2 Conclusion This study showed in an experimental model of sepsis, that glutamine supplementation significantly increased HSP70 release. Children with meningococcal disease were glutamine depleted early in the course of the disease and well into convalescence. SDE gene transcripts associated with inflammatory signalling pathways and adaptive immunity were correlated with protein levels of HSP70 and glutamine, suggesting temporal relationships between plasma protein levels and gene expression. Future work should aim to understand the effect of glutamine supplementation on gene expression during critical illness, especially relative to genes associated with HSP70, inflammatory signalling pathways and those relating to HLA.

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8: Appendices

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Appendix 8.1: Differential fold changes of gene transcripts in children with acute meningococcal disease compared to convalescence

Entrez Symbol Official Name Corrected p- Fold Up or Gene ID value Change down Absolute regulation 51257 March-2 Homo sapiens membrane-associated ring finger 7.32E-04 2.1228623 up (C3HC4) 2 (MARCH2), mRNA. 23157 SEPT6-1 Homo sapiens septin 6 (SEPT6), transcript variant I, 1.14E-05 2.9239087 down mRNA. 23157 SEPT6-111 Homo sapiens septin 6 (SEPT6), transcript variant 1.96E-04 2.3934422 down III, mRNA. 23157 SEPT6-V Homo sapiens septin 6 (SEPT6), transcript variant 5.78E-05 2.2580981 down V, mRNA. 10801 SEPT9 Homo sapiens septin 9 (SEPT9), mRNA. 5.13E-06 2.1564415 down 55752 SEPT11 Homo sapiens septin 11 (SEPT11), mRNA. 6.49E-05 2.0454085 down 9403 SEPT15 Homo sapiens 15 kDa selenoprotein (SEP15), 6.31E-06 2.0176182 up transcript variant 1, mRNA. 19 ABCA1 Homo sapiens ATP-binding cassette, sub-family A 2.63E-05 2.9242759 up (ABC1), member 1 (ABCA1), mRNA. 25864 ABHD14A Homo sapiens abhydrolase domain containing 14A 1.34E-04 2.427685 down (ABHD14A), mRNA. 84836 ABHD14B Homo sapiens abhydrolase domain containing 14B 0.0025 2.05323 down (ABHD14B), mRNA. 11057 ABHD2 Homo sapiens abhydrolase domain containing 2 6.99E-04 2.9495397 up (ABHD2), transcript variant 2, mRNA. 51099 ABHD5 Homo sapiens abhydrolase domain containing 5 7.68E-06 3.468925 up (ABHD5), mRNA. 3983 ABLIM1 Homo sapiens actin binding LIM protein 1 6.31E-06 4.8815417 down (ABLIM1), transcript variant 4, mRNA. 30 ACAA1 Homo sapiens acetyl-Coenzyme A acyltransferase 1 5.47E-06 2.552882 up (peroxisomal 3-oxoacyl-Coenzyme A thiolase) (ACAA1), nuclear gene encoding mitochondrial protein, mRNA. 84129 ACAD11 Homo sapiens acyl-Coenzyme A dehydrogenase 4.78E-05 2.0209713 down family, member 11 (ACAD11), mRNA. 57001 ACN9 Homo sapiens ACN9 homolog (S. cerevisiae) 1.82E-05 2.6151311 up (ACN9), mRNA. 23597 ACOT9 Homo sapiens acyl-CoA thioesterase 9 (ACOT9), 4.53E-05 2.3838916 up mRNA. 51 ACOX1 Homo sapiens acyl-Coenzyme A oxidase 1, 3.18E-05 2.8341515 up palmitoyl (ACOX1), transcript variant 1, mRNA. 55 ACPP Homo sapiens acid phosphatase, prostate (ACPP), 5.22E-04 3.3458211 up mRNA. 2180 ACSL1 Homo sapiens acyl-CoA synthetase long-chain 5.13E-06 4.1557355 up family member 1 (ACSL1), mRNA. 2181 ACSL3 Homo sapiens acyl-CoA synthetase long-chain 4.6E-03 2.2107027 up family member 3 (ACSL3), transcript variant 1, mRNA. 2181 ACSL3 Homo sapiens acyl-CoA synthetase long-chain 1.00E-05 2.1797078 up family member 3 (ACSL3), transcript variant 2, mRNA. 2182 ACSL4 Homo sapiens acyl-CoA synthetase long-chain 5.35E-06 4.180476 up family member 4 (ACSL4), transcript variant 1, mRNA. 201

84532 ACSS1 Homo sapiens acyl-CoA synthetase short-chain 9.48E-06 2.3971343 down family member 1 (ACSS1), nuclear gene encoding mitochondrial protein, mRNA. 55902 ACSS2 Homo sapiens acyl-CoA synthetase short-chain 5.27E-06 2.471739 up family member 2 (ACSS2), transcript variant 2, mRNA. 55902 ACSS2 Homo sapiens acyl-CoA synthetase short-chain 5.13E-06 2.4205906 up family member 2 (ACSS2), transcript variant 2, mRNA. 55860 ACTR10 Homo sapiens actin-related protein 10 homolog (S. 1.52E-05 2.4555352 up cerevisiae) (ACTR10), mRNA. 10097 ACTR2 Homo sapiens ARP2 actin-related protein 2 1.39E-05 2.0837154 up homolog (yeast) (ACTR2), transcript variant 1, mRNA. 10096 ACTR3 Homo sapiens ARP3 actin-related protein 3 5.13E-06 2.145069 up homolog (yeast) (ACTR3), mRNA. 91 ACVR1B Homo sapiens activin A receptor, type IB (ACVR1B), 5.13E-06 4.154476 up transcript variant 1, mRNA. 100 ADA Homo sapiens adenosine deaminase (ADA), mRNA. 2.42E-05 2.4412327 down

8751 ADAM15 Homo sapiens ADAM metallopeptidase domain 15 2.1E-03 2.0619636 up (ADAM15), transcript variant 2, mRNA. 104 ADARB1 Homo sapiens adenosine deaminase, RNA-specific, 5.13E-06 3.298978 down B1 (RED1 homolog rat) (ADARB1), transcript variant 4, mRNA. 90956 ADCK2 Homo sapiens aarF domain containing kinase 2 5.27E-06 2.2083964 down (ADCK2), mRNA. 79934 ADCK4 Homo sapiens aarF domain containing kinase 4 6.22E-03 2.2813466 up (ADCK4), mRNA. 109 ADCY3 Homo sapiens adenylate cyclase 3 (ADCY3), mRNA. 6.53E-04 3.1877859 up 123 ADFP Homo sapiens adipose differentiation-related 5.13E-06 2.709949 up protein (ADFP), mRNA. 133 ADM Homo sapiens adrenomedullin (ADM), mRNA. 5.13E-06 4.2016716 up 135 ADORA2A Homo sapiens adenosine A2a receptor (ADORA2A), 4.15E-04 2.085995 up mRNA. 166 AES Homo sapiens amino-terminal enhancer of split 5.27E-06 2.3638194 down (AES), transcript variant 1, mRNA. 166 AES Homo sapiens amino-terminal enhancer of split 5.13E-06 3.0469098 down (AES), transcript variant 3, mRNA. 10555 AGPAT2 Homo sapiens 1-acylglycerol-3-phosphate O- 5.27E-06 2.6327765 up acyltransferase 2 (lysophosphatidic acid acyltransferase, beta) (AGPAT2), transcript variant 2, mRNA. 10555 AGPAT2 Homo sapiens 1-acylglycerol-3-phosphate O- 7.25E-06 3.3010685 up acyltransferase 2 (lysophosphatidic acid acyltransferase, beta) (AGPAT2), transcript variant 1, mRNA. 10555 AGPAT2 Homo sapiens 1-acylglycerol-3-phosphate O- 4.0E-03 3.4558003 up acyltransferase 2 (lysophosphatidic acid acyltransferase, beta) (AGPAT2), transcript variant 1, mRNA. 23287 AGTPBP1 Homo sapiens ATP/GTP binding protein 1 3.27E-05 3.9650426 up (AGTPBP1), mRNA. 57085 AGTRAP Homo sapiens angiotensin II receptor-associated 5.13E-06 3.645217 up protein (AGTRAP), mRNA. 57085 AGTRAP Homo sapiens angiotensin II receptor-associated 5.13E-06 3.9348745 up protein (AGTRAP), transcript variant 2, mRNA. 202

79026 AHNAK Homo sapiens AHNAK nucleoprotein (AHNAK), 3.50E-05 2.3069205 down transcript variant 1, mRNA. 199 AIF1 Homo sapiens allograft inflammatory factor 1 1.66E-05 2.925644 up (AIF1), transcript variant 3, mRNA. 199 AIF1 Homo sapiens allograft inflammatory factor 1 2.42E-05 2.4036589 up (AIF1), transcript variant 3, mRNA. 51390 AIG1 Homo sapiens androgen-induced 1 (AIG1), mRNA. 6.13E-05 2.7312293 up 231 AKR1B1 Homo sapiens aldo-keto reductase family 1, 5.27E-06 2.5981205 down member B1 (aldose reductase) (AKR1B1), mRNA. 214 ALCAM Homo sapiens activated leukocyte cell adhesion 1.33E-05 4.5643463 up molecule (ALCAM), mRNA. 216 ALDH1A1 Homo sapiens aldehyde dehydrogenase 1 family, 1.22E-04 3.0390568 down member A1 (ALDH1A1), mRNA. 221 ALDH3B1 Homo sapiens aldehyde dehydrogenase 3 family, 5.91E-06 3.0088317 up member B1 (ALDH3B1), transcript variant 2, mRNA. 8659 ALDH4A1 Homo sapiens aldehyde dehydrogenase 4 family, 6.53E-04 2.427308 up member A1 (ALDH4A1), nuclear gene encoding mitochondrial protein, transcript variant P5CDhS, mRNA. 226 ALDOA Homo sapiens aldolase A, fructose-bisphosphate 3.50E-05 2.1466763 up (ALDOA), transcript variant 2, mRNA. 226 ALDOA Homo sapiens aldolase A, fructose-bisphosphate 5.13E-06 2.3350735 up (ALDOA), transcript variant 2, mRNA. 91801 ALKBH8 Homo sapiens alkB, alkylation repair homolog 8 (E. 5.11E-04 2.0812833 down coli) (ALKBH8), mRNA. 240 ALOX5 Homo sapiens arachidonate 5-lipoxygenase 1.08E-05 2.9613776 up (ALOX5), mRNA. 241 ALOX5AP Homo sapiens arachidonate 5-lipoxygenase- 5.13E-06 3.6183558 up activating protein (ALOX5AP), mRNA. 80216 ALPK1 Homo sapiens alpha-kinase 1 (ALPK1), mRNA. 1.66E-05 3.0632231 up 249 ALPL Homo sapiens alkaline phosphatase, 3.18E-05 4.5773597 up liver/bone/kidney (ALPL), mRNA. 251 ALPPL2 Homo sapiens alkaline phosphatase, placental-like 7.68E-06 3.0623443 up 2 (ALPPL2), mRNA. 150864 ALS2CR13 Homo sapiens amyotrophic lateral sclerosis 2 1.52E-05 2.2309058 down (juvenile) chromosome region, candidate 13 (ALS2CR13), mRNA. 272 AMPD3 Homo sapiens adenosine monophosphate 4.67E-04 2.5294275 up deaminase (isoform E) (AMPD3), transcript variant 2, mRNA. 64682 ANAPC1 Homo sapiens anaphase promoting complex 8.34E-06 2.602417 down subunit 1 (ANAPC1), mRNA. 118932 ANKRD22 Homo sapiens ankyrin repeat domain 22 4.70E-04 10.628527 up (ANKRD22), mRNA. 341405 ANKRD33 Homo sapiens ankyrin repeat domain 33 6.31E-06 3.2745109 up (ANKRD33), mRNA. 57182 ANKRD50 Homo sapiens ankyrin repeat domain 50 2.51E-04 2.6015894 up (ANKRD50), mRNA. 79722 ANKRD55 Homo sapiens ankyrin repeat domain 55 4.18E-04 2.0538769 up (ANKRD55), transcript variant 2, mRNA. 23294 ANKS1A Homo sapiens ankyrin repeat and sterile alpha 5.13E-06 3.2355926 up motif domain containing 1A (ANKS1A), mRNA. 118429 ANTXR2 Homo sapiens anthrax toxin receptor 2 (ANTXR2), 2.86E-04 2.0358493 up mRNA. 301 ANXA1 Homo sapiens annexin A1 (ANXA1), mRNA. 5.13E-06 4.4185266 up

203

302 ANXA2 Homo sapiens annexin A2 (ANXA2), transcript 6.10E-05 2.2237422 up variant 2, mRNA. 306 ANXA3 Homo sapiens annexin A3 (ANXA3), mRNA. 6.38E-05 13.930081 up 308 ANXA5 Homo sapiens annexin A5 (ANXA5), mRNA. 5.27E-06 2.6713386 up 1176 AP3S1 Homo sapiens adaptor-related protein complex 3, 2.28E-05 2.7825558 up sigma 1 subunit (AP3S1), transcript variant 2, mRNA. 10239 AP3S2 Homo sapiens adaptor-related protein complex 3, 1.22E-04 2.235738 up sigma 2 subunit (AP3S2), mRNA. 317 APAF1 Homo sapiens apoptotic peptidase activating 5.47E-06 2.7191226 up factor 1 (APAF1), transcript variant 5, mRNA. 83464 APH1B Homo sapiens anterior pharynx defective 1 5.13E-06 5.455491 up homolog B (C. elegans) (APH1B), mRNA. 55911 APOB48R Homo sapiens apolipoprotein B48 receptor 8.78E-04 2.0926363 up (APOB48R), mRNA. 80833 APOL3 Homo sapiens apolipoprotein L, 3 (APOL3), 5.50E-05 3.018701 down transcript variant alpha/b, mRNA. 353 APRT Homo sapiens adenine phosphoribosyltransferase 2.20E-05 2.3390777 down (APRT), transcript variant 1, mRNA.

353 APRT Homo sapiens adenine phosphoribosyltransferase 2.24E-05 2.281276 down (APRT), transcript variant 2, mRNA.

375318 AQP12A Homo sapiens aquaporin 12A (AQP12A), mRNA. 1.96E-04 2.1761649 up 366 AQP9 Homo sapiens aquaporin 9 (AQP9), mRNA. 5.13E-06 3.3863466 up 375 ARF1 Homo sapiens ADP-ribosylation factor 1 (ARF1), 9.68E-04 2.0372055 up transcript variant 2, mRNA. 378 ARF4 Homo sapiens ADP-ribosylation factor 4 (ARF4), 4.16E-05 2.125652 up mRNA. 383 ARG1 Homo sapiens arginase, liver (ARG1), mRNA. 1.3E-02 39.776466 up 9824 ARHGAP11A Homo sapiens Rho GTPase activating protein 11A 9.97E-06 2.6994462 up (ARHGAP11A), transcript variant 2, mRNA. 93663 ARHGAP18 Homo sapiens Rho GTPase activating protein 18 4.90E-05 2.4310317 up (ARHGAP18), mRNA. 83478 ARHGAP24 Homo sapiens Rho GTPase activating protein 24 2.0E-03 2.272303 up (ARHGAP24), transcript variant 1, mRNA. 1820 ARID3A Homo sapiens AT rich interactive domain 3A 6.31E-06 2.3753872 up (BRIGHT- like) (ARID3A), mRNA. 10124 ARL4 Homo sapiens ADP-ribosylation factor-like 4 5.24E-04 3.6588619 up (ARL4), transcript variant 1, mRNA. 10124 ARL4 Homo sapiens ADP-ribosylation factor-like 4 5.41E-04 2.6687455 up (ARL4), transcript variant 1, mRNA. 10550 ARL6IP5 Homo sapiens ADP-ribosylation-like factor 6 5.27E-06 2.025481 up interacting protein 5 (ARL6IP5), mRNA. 10552 ARPC1A Homo sapiens actin related protein 2/3 complex, 5.35E-06 2.07487 up subunit 1A, 41kDa (ARPC1A), mRNA. 10094 ARPC3 Homo sapiens actin related protein 2/3 complex, 9.10E-06 2.4239552 up subunit 3, 21kDa (ARPC3), mRNA. 10092 ARPC5 Homo sapiens actin related protein 2/3 complex, 5.91E-06 2.273194 up subunit 5, 16kDa (ARPC5), mRNA. 409 ARRB2 Homo sapiens arrestin, beta 2 (ARRB2), transcript 1.17E-05 2.8576148 up variant 2, mRNA. 91947 ARRDC4 Homo sapiens arrestin domain containing 4 5.33E-05 4.0111256 up (ARRDC4), mRNA. 414 ARSD Homo sapiens arylsulfatase D (ARSD), transcript 3.59E-04 2.5105703 up variant 1, mRNA. 204

427 ASAH1 Homo sapiens N-acylsphingosine amidohydrolase 9.08E-05 2.5698562 up (acid ceramidase) 1 (ASAH1), transcript variant 1, mRNA. 430 ASCL2 Homo sapiens achaete-scute complex-like 2 6.31E-06 2.4324753 down (Drosophila) (ASCL2), mRNA. 444 ASPH Homo sapiens aspartate beta-hydroxylase (ASPH), 1.2E-02 2.8206544 up transcript variant 2, mRNA. 79058 ASPSCR1 Homo sapiens alveolar soft part sarcoma 7.25E-06 2.2351763 down chromosome region, candidate 1 (ASPSCR1), mRNA. 22926 ATF6 Homo sapiens activating transcription factor 6 3.50E-05 2.5570004 up (ATF6), mRNA. 10533 ATG7 Homo sapiens ATG7 autophagy related 7 homolog 2.42E-05 2.0359898 up (S. cerevisiae) (ATG7), mRNA. 471 ATIC Homo sapiens 5-aminoimidazole-4-carboxamide 7.68E-06 2.3492987 down ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), mRNA. 472 ATM Homo sapiens ataxia telangiectasia mutated 2.20E-05 2.2496457 down (ATM), transcript variant 1, mRNA. 475 ATOX1 Homo sapiens ATX1 antioxidant protein 1 homolog 5.47E-06 2.3077657 up (yeast) (ATOX1), mRNA. 23200 ATP11B Homo sapiens ATPase, Class VI, type 11B (ATP11B), 5.27E-06 3.285318 up mRNA. 493 ATP2B4 Homo sapiens ATPase, Ca++ transporting, plasma 1.1E-03 2.1196537 up membrane 4 (ATP2B4), transcript variant 1, mRNA. 509 ATP5C1 Homo sapiens ATP synthase, H+ transporting, 2.86E-04 2.117162 up mitochondrial F1 complex, gamma polypeptide 1 (ATP5C1), nuclear gene encoding mitochondrial protein, transcript variant 2, mRNA. 537 ATP6AP1 Homo sapiens ATPase, H+ transporting, lysosomal 5.27E-06 2.3823411 up accessory protein 1 (ATP6AP1), mRNA. 533 ATP6V0B Homo sapiens ATPase, H+ transporting, lysosomal 6.31E-06 2.6505432 up 21kDa, V0 subunit b (ATP6V0B), transcript variant 2, mRNA. 533 ATP6V0B Homo sapiens ATPase, H+ transporting, lysosomal 5.27E-06 2.245184 up 21kDa, V0 subunit c'' (ATP6V0B), mRNA. 9114 ATP6V0D1 Homo sapiens ATPase, H+ transporting, lysosomal 6.31E-06 2.246149 up 38kDa, V0 subunit d isoform 1 (ATP6V0D1), mRNA. 8992 ATP6V0E1 Homo sapiens ATPase, H+ transporting, lysosomal 5.13E-06 2.2897472 up 9kDa, V0 subunit e1 (ATP6V0E1), mRNA. 155066 ATP6V0E2L Homo sapiens ATPase, H+ transporting V0 subunit 5.13E-06 3.9404657 down E2-like (rat) (ATP6V0E2L), mRNA. 523 ATP6V1A Homo sapiens ATPase, H+ transporting, lysosomal 1.39E-05 2.476419 up 70kDa, V1 subunit A (ATP6V1A), mRNA. 526 ATP6V1B2 Homo sapiens ATPase, H+ transporting, lysosomal 8.44E-06 2.096081 up 56/58kDa, V1 subunit B, isoform 2 (ATP6V1B2), mRNA. 528 ATP6V1C1 Homo sapiens ATPase, H+ transporting, lysosomal 5.13E-06 4.5077534 up 42kDa, V1 subunit C1 (ATP6V1C1), transcript variant 1, mRNA. 51382 ATP6V1D Homo sapiens ATPase, H+ transporting, lysosomal 5.13E-06 2.702841 up 34kDa, V1 subunit D (ATP6V1D), mRNA. 529 ATP6V1E1 Homo sapiens ATPase, H+ transporting, lysosomal 5.13E-06 2.2574718 up 31kDa, V1 subunit E1 (ATP6V1E1), transcript variant 3, mRNA. 529 ATP6V1E1 Homo sapiens ATPase, H+ transporting, lysosomal 5.13E-06 2.375929 up 31kDa, V1 subunit E isoform 1 (ATP6V1E1), mRNA.

205

79895 ATP8B4 Homo sapiens ATPase, Class I, type 8B, member 4 5.13E-06 3.5032446 up (ATP8B4), mRNA. 10079 ATP9A Homo sapiens ATPase, Class II, type 9A (ATP9A), 5.27E-06 7.854341 up mRNA. 545 ATR Homo sapiens ataxia telangiectasia and Rad3 5.9E-03 2.2117977 down related (ATR), mRNA. 6310 ATXN1 Homo sapiens ataxin 1 (ATXN1), mRNA. 6.31E-06 3.1478345 up 389289 AXIIR Homo sapiens chromosome 5 open reading frame 5.13E-06 2.8115764 down 39 (C5orf39), mRNA. 8313 AXIN2 Homo sapiens axin 2 (conductin, axil) (AXIN2), 3.3E-03 2.4288762 down mRNA. 64651 AXUD1 Homo sapiens AXIN1 upregulated 1 (AXUD1), 1.22E-04 2.1785872 up mRNA. 54947 AYTL1 Homo sapiens acyltransferase like 1 (AYTL1), 3.14E-04 2.5456052 up mRNA. 64343 AZI2 Homo sapiens 5-azacytidine induced 2 (AZI2), 3.10E-05 2.7625904 up mRNA. 51582 AZIN1 Homo sapiens antizyme inhibitor 1 (AZIN1), 2.35E-03 2.0683444 up transcript variant 2, mRNA. 51582 AZIN1 Homo sapiens antizyme inhibitor 1 (AZIN1), 3.18E-05 2.1084754 up transcript variant 1, mRNA. 374907 B3GALT7 Homo sapiens UDP-GlcNAc:betaGal beta-1,3-N- 5.32E-04 3.2319646 up acetylglucosaminyltransferase 8 (B3GNT8), mRNA. 84002 B3GNT5 Homo sapiens UDP-GlcNAc:betaGal beta-1,3-N- 3.6E-03 6.9917035 up acetylglucosaminyltransferase 5 (B3GNT5), mRNA. 146712 B3GNTL1 Homo sapiens UDP-GlcNAc:betaGal beta-1,3-N- 1.74E-03 2.7826777 up acetylglucosaminyltransferase-like 1 (B3GNTL1), mRNA. 8702 B4GALT4 Homo sapiens UDP-Gal:betaGlcNAc beta 1,4- 1.81E-04 2.159077 up galactosyltransferase, polypeptide 4 (B4GALT4), transcript variant 2, mRNA. 9334 B4GALT5 Homo sapiens UDP-Gal:betaGlcNAc beta 1,4- 5.47E-06 4.0208426 up galactosyltransferase, polypeptide 5 (B4GALT5), mRNA. 60468 BACH2 Homo sapiens BTB and CNC homology 1, basic 1.19E-04 2.4713762 down leucine zipper transcription factor 2 (BACH2), mRNA. 9530 BAG4 Homo sapiens BCL2-associated athanogene 4 1.1E-03 2.0753407 up (BAG4), mRNA. 10409 BASP1 Homo sapiens brain abundant, membrane 2.42E-05 2.8821824 up attached signal protein 1 (BASP1), mRNA. 7920 BAT5 Homo sapiens HLA-B associated transcript 5 5.35E-06 2.311769 up (BAT5), mRNA. 10538 BATF Homo sapiens basic leucine zipper transcription 5.13E-06 4.0594397 up factor, ATF-like (BATF), mRNA. 11177 BAZ1A Homo sapiens bromodomain adjacent to zinc 1.17E-05 3.4623272 up finger domain, 1A (BAZ1A), transcript variant 2, mRNA. 11177 BAZ1A Homo sapiens bromodomain adjacent to zinc 6.88E-05 2.54265 up finger domain, 1A (BAZ1A), transcript variant 1, mRNA. 10134 BCAP31 Homo sapiens B-cell receptor-associated protein 5.47E-06 2.0151117 up 31 (BCAP31), mRNA. 55653 BCAS4 Homo sapiens breast carcinoma amplified 5.47E-06 3.270653 down sequence 4 (BCAS4), transcript variant 1, mRNA. 55653 BCAS4 Homo sapiens breast carcinoma amplified 5.47E-06 2.6149397 down sequence 4 (BCAS4), transcript variant 1, mRNA. 206

10295 BCKDK Homo sapiens branched chain ketoacid 1.28E-05 2.2914066 up dehydrogenase kinase (BCKDK), mRNA. 53335 BCL11A Homo sapiens B-cell CLL/lymphoma 11A (zinc 1.39E-05 2.4021606 down finger protein) (BCL11A), transcript variant 1, mRNA. 64919 BCL11B Homo sapiens B-cell CLL/lymphoma 11B (zinc 6.34E-06 5.764843 down finger protein) (BCL11B), transcript variant 2, mRNA. 596 BCL2 Homo sapiens B-cell CLL/lymphoma 2 (BCL2), 5.88E-06 3.32775 down nuclear gene encoding mitochondrial protein, transcript variant alpha, mRNA. 597 BCL2A1 Homo sapiens BCL2-related protein A1 (BCL2A1), 6.38E-05 9.320875 up mRNA. 602 BCL3 Homo sapiens B-cell CLL/lymphoma 3 (BCL3), 7.25E-06 2.5529904 up mRNA. 604 BCL6 Homo sapiens B-cell CLL/lymphoma 6 (zinc finger 1.39E-05 3.5367644 up protein 51) (BCL6), transcript variant 1, mRNA. 604 BCL6 Homo sapiens B-cell CLL/lymphoma 6 (zinc finger 2.28E-02 2.4570224 up protein 51) (BCL6), transcript variant 2, mRNA. 199786 BCNP1 Homo sapiens family with sequence similarity 129, 5.17E-05 2.9546525 down member C (FAM129C), mRNA. 274 BIN1 Homo sapiens bridging integrator 1 (BIN1), 5.13E-06 3.4572208 down transcript variant 1, mRNA. 274 BIN1 Homo sapiens bridging integrator 1 (BIN1), 5.13E-06 3.3422344 down transcript variant 6, mRNA. 329 BIRC2 Homo sapiens baculoviral IAP repeat-containing 2 1.39E-05 2.0821064 up (BIRC2), mRNA. 330 BIRC3 Homo sapiens baculoviral IAP repeat-containing 3 4.71E-04 2.2116282 down (BIRC3), transcript variant 1, mRNA. 330 BIRC3 Homo sapiens baculoviral IAP repeat-containing 3 3.48E-03 2.0205636 down (BIRC3), transcript variant 1, mRNA. 641 BLM Homo sapiens Bloom syndrome (BLM), mRNA. 6.26E-03 2.4511328 up 2647 BLOC1S1 Homo sapiens biogenesis of lysosome-related 5.13E-06 4.292033 up organelles complex-1, subunit 1 (BLOC1S1), mRNA. 643 BLR1 Homo sapiens Burkitt lymphoma receptor 1, GTP 1.67E-05 2.5610785 down binding protein (chemokine (C-X-C motif) receptor 5) (BLR1), transcript variant 2, mRNA. 9790 BMS1L Homo sapiens BMS1 homolog, ribosome assembly 5.27E-06 2.3050718 down protein (yeast) (BMS1), mRNA. 663 BNIP2 Homo sapiens BCL2/adenovirus E1B 19kDa 5.35E-06 3.4055154 up interacting protein 2 (BNIP2), mRNA. 666 BOK Homo sapiens BCL2-related ovarian killer (BOK), 2.20E-05 2.4652698 up mRNA. 669 BPGM Homo sapiens 2,3-bisphosphoglycerate mutase 3.67E-03 2.4872093 up (BPGM), transcript variant 1, mRNA. 671 BPI Homo sapiens bactericidal/permeability-increasing 3.18E-05 13.339196 up protein (BPI), mRNA. 25798 BRI3 Homo sapiens brain protein I3 (BRI3), mRNA. 5.13E-06 2.886382 up 25874 BRP44 Homo sapiens brain protein 44 (BRP44), mRNA. 5.13E-06 2.109027 up 254065 BRWD3 Homo sapiens bromodomain and WD repeat 1.2E03 2.0850143 up domain containing 3 (BRWD3), mRNA. 683 BST1 Homo sapiens bone marrow stromal cell antigen 1 5.13E-06 5.1550226 up (BST1), mRNA. 84280 BTBD10 Homo sapiens BTB (POZ) domain containing 10 5.13E-06 2.9469848 up (BTBD10), mRNA.

207

121551 BTBD11 Homo sapiens BTB (POZ) domain containing 11 1.55E-03 2.2038865 down (BTBD11), transcript variant 1, mRNA. 138151 BTBD14A Homo sapiens BTB (POZ) domain containing 14A 1.17E-05 2.4902163 up (BTBD14A), mRNA. 11119 BTN3A1 Homo sapiens butyrophilin, subfamily 3, member 5.47E-06 4.2925014 down A1 (BTN3A1), transcript variant 1, mRNA. 11118 BTN3A2 Homo sapiens butyrophilin, subfamily 3, member 1.52E-05 3.2264793 down A2 (BTN3A2), mRNA. 10384 BTN3A3 Homo sapiens butyrophilin, subfamily 3, member 1.29E-05 3.0910273 down A3 (BTN3A3), transcript variant 2, mRNA. 221061 C10orf38 Homo sapiens chromosome 10 open reading frame 1.31E-03 2.558776 down 38 (C10orf38), mRNA. 64115 C10orf54 Homo sapiens chromosome 10 open reading frame 5.27E-06 3.1372325 up 54 (C10orf54), mRNA. 8872 C10orf7 Homo sapiens cell division cycle 123 homolog (S. 6.31E-06 2.5030859 up cerevisiae) (CDC123), mRNA. 738 C11orf2 Homo sapiens chromosome 11 open reading 6.31E-06 2.131858 down frame2 (C11orf2), mRNA. 120534 C11orf46 Homo sapiens chromosome 11 open reading frame 7.68E-06 2.2513816 down 46 (C11orf46), mRNA. 84067 C11orf56 Homo sapiens chromosome 11 open reading frame 5.35E-06 2.6234212 up 56 (C11orf56), mRNA. 113246 C12orf57 Homo sapiens chromosome 12 open reading frame 7.57E-05 2.6421273 down 57 (C12orf57), mRNA. 51528 C14orf100 Homo sapiens chromosome 14 open reading frame 5.13E-06 2.8320215 up 100 (C14orf100), mRNA. 51527 C14orf129 Homo sapiens chromosome 14 open reading frame 2.24E-05 2.334114 up 129 (C14orf129), mRNA. 80017 C14orf159 Homo sapiens chromosome 14 open reading frame 1.82E-05 2.6340868 down 159 (C14orf159), mRNA. 64423 C14orf173 Homo sapiens chromosome 14 open reading frame 1.28E-05 2.4499786 down 173 (C14orf173), transcript variant 2, mRNA. 9556 C14orf2 Homo sapiens chromosome 14 open reading frame 5.17E-05 2.1398838 up 2 (C14orf2), mRNA. 55640 C14orf58 Homo sapiens feline leukemia virus subgroup C 5.27E-06 5.4932256 up cellular receptor family, member 2 (FLVCR2), mRNA. 54930 C14orf94 Homo sapiens chromosome 14 open reading frame 2.24E-03 2.8377788 up 94 (C14orf94), mRNA. 51187 C15orf15 Homo sapiens chromosome 15 open reading frame 1.65E-03 2.1335464 up 15 (C15orf15), mRNA. 79652 C16orf30 Homo sapiens chromosome 16 open reading frame 1.12E-03 2.762675 down 30 (C16orf30), mRNA. 79650 C16orf57 Homo sapiens chromosome 16 open reading frame 5.13E-06 3.4210134 up 57 (C16orf57), mRNA. 64755 C16orf58 Homo sapiens chromosome 16 open reading frame 5.27E-06 2.233512 down 58 (C16orf58), mRNA. 255919 C16orf69 Homo sapiens chromosome 16 open reading frame 1.19E-05 2.7329924 up 69 (C16orf69), mRNA. 9605 C16orf7 Homo sapiens chromosome 16 open reading frame 1.82E-05 2.6783915 up 7 (C16orf7), mRNA. 80233 C17orf70 Homo sapiens chromosome 17 open reading frame 1.82E-05 2.0779722 down 70 (C17orf70), mRNA. 125488 C18orf17 Homo sapiens chromosome 18 open reading frame 7.97E-06 2.7321863 down 17 (C18orf17), mRNA. 83636 C19orf12 Homo sapiens chromosome 19 open reading frame 5.28E-05 2.0819197 down 12 (C19orf12), transcript variant 1, mRNA.

208

8725 C19orf2 Homo sapiens chromosome 19 open reading frame 2.42E-05 2.0990937 down 2 (C19orf2), transcript variant 2, mRNA. 29071 C1GALT1C1 Homo sapiens C1GALT1-specific chaperone 1 9.97E-06 2.9396925 up (C1GALT1C1), transcript variant 2, mRNA. 128346 C1orf162 Homo sapiens chromosome 1 open reading frame 5.13E-06 2.8794515 up 162 (C1orf162), mRNA. 54680 C1orf181 Homo sapiens chromosome 1 open reading frame 5.35E-06 2.8106399 down 181 (C1orf181), mRNA. 116496 C1orf24 Homo sapiens chromosome 1 open reading frame 3.46E-04 2.4415965 up 24 (C1orf24), transcript variant 1, mRNA. 713 C1QB Homo sapiens complement component 1, q 7.57E-03 7.21154 up subcomponent, beta polypeptide (C1QB), mRNA. 22918 C1QR1 Homo sapiens complement component 1, q 1.39E-05 2.4950087 up subcomponent, receptor 1 (C1QR1), mRNA. 51279 C1RL Homo sapiens complement component 1, r 3.50E-05 2.6868606 up subcomponent-like (C1RL), mRNA. 84969 C20orf100 Homo sapiens chromosome 20 open reading frame 1.52E-05 2.8502917 down 100 (C20orf100), mRNA. 54675 C20orf155 Homo sapiens cardiolipin synthase 1 (CRLS1), 7.68E-06 2.053593 up mRNA. 128611 C20orf174 Homo sapiens chromosome 20 open reading frame 1.84E-04 2.2091079 down 174 (C20orf174), mRNA. 55969 C20orf24 Homo sapiens chromosome 20 open reading frame 5.13E-06 3.8125732 up 24 (C20orf24), transcript variant 1, mRNA. 55969 C20orf24 Homo sapiens chromosome 20 open reading frame 5.13E-06 3.026176 up 24 (C20orf24), transcript variant 1, mRNA. 55969 C20orf24 Homo sapiens chromosome 20 open reading frame 5.13E-06 3.0735211 up 24 (C20orf24), transcript variant 4, mRNA. 57136 C20orf3 Homo sapiens chromosome 20 open reading frame 1.01E-04 2.7450454 up 3 (C20orf3), mRNA. 140823 C20orf52 Homo sapiens chromosome 20 open reading frame 2.42E-05 2.0341792 up 52 (C20orf52), mRNA. 755 C21orf2 Homo sapiens chromosome 21 open reading frame 2.20E-05 2.4869087 down 2 (C21orf2), mRNA. 54059 C21orf57 Homo sapiens chromosome 21 open reading frame 5.13E-06 2.3074672 up 57 (C21orf57), transcript variant 1, mRNA. 192137 C2orf12 PREDICTED: Homo sapiens chromosome 2 open 9.97E-06 3.0588775 up reading frame 12, transcript variant 2 (C2orf12), mRNA. 27249 C2orf25 Homo sapiens chromosome 2 open reading frame 5.13E-06 2.964137 up 25 (C2orf25), mRNA. 719 C3AR1 Homo sapiens complement component 3a 1.42E-04 6.5555315 up receptor 1 (C3AR1), mRNA. 25871 C3orf17 Homo sapiens chromosome 3 open reading frame 5.13E-06 2.4203558 up 17 (C3orf17), transcript variant 1, mRNA. 84984 C3orf34 Homo sapiens chromosome 3 open reading frame 2.86E-04 2.2390769 up 34 (C3orf34), mRNA. 56941 C3orf37 Homo sapiens chromosome 3 open reading frame 5.91E-06 2.0405183 down 37 (C3orf37), transcript variant 2, mRNA. 131408 C3orf40 Homo sapiens family with sequence similarity 131, 6.13E-05 2.0521533 up member A (FAM131A), mRNA. 56983 C3orf9 Homo sapiens KTEL (Lys-Tyr-Glu-Leu) containing 1 5.27E-06 2.0646598 down (KTELC1), mRNA. 51313 C4orf18 Homo sapiens chromosome 4 open reading frame 6.25E-05 2.647916 up 18 (C4orf18), transcript variant 2, mRNA. 728 C5AR1 Homo sapiens complement component 5a 9.97E-06 3.1329455 up receptor 1 (C5AR1), mRNA.

209

10591 C6orf108 Homo sapiens chromosome 6 open reading frame 4.90E-05 2.2198157 down 108 (C6orf108), transcript variant 2, mRNA. 55122 C6orf166 Homo sapiens chromosome 6 open reading frame 5.13E-06 2.551163 up 166 (C6orf166), mRNA. 387357 C6orf190 Homo sapiens chromosome 6 open reading frame 5.72E-04 2.4721549 down 190 (C6orf190), mRNA. 79624 C6orf211 Homo sapiens chromosome 6 open reading frame 1.57E-05 2.9303398 up 211 (C6orf211), mRNA. 79020 C7orf25 Homo sapiens chromosome 7 open reading frame 6.11E-05 2.7131867 up 25 (C7orf25), mRNA. 51337 C8orf55 Homo sapiens chromosome 8 open reading frame 5.13E-06 2.7348912 down 55 (C8orf55), mRNA. 414328 C9orf103 Homo sapiens chromosome 9 open reading frame 1.22E-04 2.7826786 up 103 (C9orf103), mRNA. 399665 C9orf132 Homo sapiens chromosome 9 open reading frame 6.95E-03 2.0030742 down 132 (C9orf132), mRNA. 286257 C9orf142 Homo sapiens chromosome 9 open reading frame 5.13E-06 2.042369 down 142 (C9orf142), mRNA. 81571 C9orf45 Homo sapiens chromosome 9 open reading frame 9.97E-03 2.1675062 down 45 (C9orf45), mRNA. 157983 C9orf66 Homo sapiens chromosome 9 open reading frame 1.98E-05 2.251992 up 66 (C9orf66), mRNA. 84270 C9orf89 Homo sapiens chromosome 9 open reading frame 5.13E-06 2.610029 up 89 (C9orf89), mRNA. 203197 C9orf91 Homo sapiens chromosome 9 open reading frame 2.20E-04 2.0844665 down 91 (C9orf91), mRNA. 759 CA1 Homo sapiens carbonic anhydrase I (CA1), mRNA. 2.16E-03 3.3545828 up 762 CA4 Homo sapiens carbonic anhydrase IV (CA4), mRNA. 5.13E-06 14.921277 up 11238 CA5B Homo sapiens carbonic anhydrase VB, 3.82E-05 2.37991 down mitochondrial (CA5B), nuclear gene encoding mitochondrial protein, mRNA. 51719 CAB39 Homo sapiens calcium binding protein 39 (CAB39), 5.13E-06 2.9890585 up mRNA. 774 CACNA1B Homo sapiens calcium channel, voltage- 5.94E-05 2.4060564 up dependent, L type, alpha 1B subunit (CACNA1B), mRNA. 777 CACNA1E Homo sapiens calcium channel, voltage- 2.7E-02 2.810861 up dependent, R type, alpha 1E subunit (CACNA1E), mRNA. 91860 CALML4 Homo sapiens calmodulin-like 4 (CALML4), 7.25E-06 2.3930612 up transcript variant 1, mRNA. 820 CAMP Homo sapiens cathelicidin antimicrobial peptide 4.15E-04 2.447115 up (CAMP), mRNA. 10487 CAP1 Homo sapiens CAP, adenylate cyclase-associated 5.13E-06 2.2343907 up protein 1 (yeast) (CAP1), mRNA. 822 CAPG Homo sapiens capping protein (actin filament), 5.13E-06 3.7426062 up gelsolin-like (CAPG), mRNA. 825 CAPN3 Homo sapiens calpain 3, (p94) (CAPN3), transcript 1.46E-05 3.5455084 up variant 1, mRNA. 825 CAPN3 Homo sapiens calpain 3, (p94) (CAPN3), transcript 3.66E-02 3.1070983 up variant 3, mRNA. 830 CAPZA2 Homo sapiens capping protein (actin filament) 5.13E-06 3.4337802 up muscle Z-line, alpha 2 (CAPZA2), mRNA. 84433 CARD11 Homo sapiens caspase recruitment domain family, 5.13E-06 3.557567 down member 11 (CARD11), mRNA. 58484 CARD12 Homo sapiens NLR family, CARD domain containing 1.46E-05 9.0580435 up 4 (NLRC4), mRNA. 210

113201 CASC4 Homo sapiens cancer susceptibility candidate 4 1.04E-04 2.004669 up (CASC4), transcript variant 2, mRNA. 838 CASP5 Homo sapiens caspase 5, apoptosis-related 7.33E-03 2.8615973 up cysteine peptidase (CASP5), mRNA. 831 CAST Homo sapiens calpastatin (CAST), transcript variant 9.97E-06 2.176052 up 1, mRNA. 117144 CATSPER1 Homo sapiens cation channel, sperm associated 1 4.10E-03 2.1907773 up (CATSPER1), mRNA. 868 CBLB Homo sapiens Cas-Br-M (murine) ecotropic 5.91E-06 2.4897604 down retroviral transforming sequence b (CBLB), mRNA. 875 CBS Homo sapiens cystathionine-beta-synthase (CBS), 2.0E-02 2.0217743 up mRNA. 55246 CCDC25 Homo sapiens coiled-coil domain containing 25 6.14E-06 2.082612 down (CCDC25), transcript variant 2, mRNA. 6352 CCL5 Homo sapiens chemokine (C-C motif) ligand 5 5.13E-06 4.938709 down (CCL5), mRNA. 894 CCND2 Homo sapiens cyclin D2 (CCND2), mRNA. 1.08E-05 2.8811862 down 896 CCND3 Homo sapiens cyclin D3 (CCND3), mRNA. 5.27E-06 2.41005 up 23582 CCNDBP1 Homo sapiens cyclin D-type binding-protein 1 5.13E-06 2.2842042 up (CCNDBP1), transcript variant 2, mRNA. 23582 CCNDBP1 Homo sapiens cyclin D-type binding-protein 1 2.49E-04 2.1258645 up (CCNDBP1), transcript variant 1, mRNA. 9236 CCPG1 Homo sapiens cell cycle progression 1 (CCPG1), 1.22E-03 3.1285686 up transcript variant 2, mRNA. 9236 CCPG1 Homo sapiens cell cycle progression 1 (CCPG1), 5.13E-06 4.030477 up transcript variant 1, mRNA. 1230 CCR1 Homo sapiens chemokine (C-C motif) receptor 1 9.97E-06 3.8070688 up (CCR1), mRNA. 1232 CCR3 Homo sapiens chemokine (C-C motif) receptor 3 6.85E-04 4.8691397 down (CCR3), transcript variant 2, mRNA. 1236 CCR7 Homo sapiens chemokine (C-C motif) receptor 7 1.52E-05 3.168401 down (CCR7), mRNA. 929 CD14 Homo sapiens CD14 antigen (CD14), mRNA. 7.68E-06 2.7952507 up 929 CD14 Homo sapiens CD14 molecule (CD14), transcript 1.17E-05 2.1337862 up variant 2, mRNA. 9332 CD163 Homo sapiens CD163 molecule (CD163), transcript 5.13E-06 20.528105 up variant 2, mRNA. 9332 CD163 Homo sapiens CD163 molecule (CD163), transcript 2.20E-04 23.009604 up variant 1, mRNA. 8763 CD164 Homo sapiens CD164 molecule, sialomucin 4.26E-05 2.144466 up (CD164), mRNA. 930 CD19 Homo sapiens CD19 antigen (CD19), mRNA. 4.18E-04 2.2766738 down 914 CD2 Homo sapiens CD2 antigen (p50), sheep red blood 5.13E-06 4.5879564 down cell receptor (CD2), mRNA. 934 CD24 Homo sapiens CD24 molecule (CD24), mRNA. 5.17E-05 4.0911636 up 919 CD247 Homo sapiens CD247 molecule (CD247), transcript 5.13E-06 4.7183876 down variant 1, mRNA. 919 CD247 Homo sapiens CD247 molecule (CD247), transcript 5.13E-06 4.827429 down variant 1, mRNA. 57124 CD248 Homo sapiens CD248 molecule, endosialin 4.15E-04 2.6990235 down (CD248), mRNA. 146722 CD300LF Homo sapiens CD300 molecule-like family member 5.13E-06 3.3200364 up f (CD300LF), mRNA. 948 CD36 Homo sapiens CD36 molecule (thrombospondin 1.96E-04 2.13865 up receptor) (CD36), transcript variant 1, mRNA.

211

915 CD3D Homo sapiens CD3d molecule, delta (CD3-TCR 1.52E-05 3.9694395 down complex) (CD3D), transcript variant 1, mRNA. 915 CD3D Homo sapiens CD3d molecule, delta (CD3-TCR 7.68E-06 4.237124 down complex) (CD3D), transcript variant 2, mRNA. 916 CD3E Homo sapiens CD3e molecule, epsilon (CD3-TCR 1.14E-05 4.158275 down complex) (CD3E), mRNA. 917 CD3G Homo sapiens CD3g molecule, gamma (CD3-TCR 3.00E-05 4.8877897 down complex) (CD3G), mRNA. 920 CD4 Homo sapiens CD4 antigen (p55) (CD4), mRNA. 1.39E-05 2.7685466 down 960 CD44 Homo sapiens CD44 molecule (Indian blood group) 9.10E-06 2.3961015 up (CD44), transcript variant 3, mRNA. 960 CD44 Homo sapiens CD44 molecule (Indian blood group) 2.20E-05 2.699219 up (CD44), transcript variant 5, mRNA. 921 CD5 Homo sapiens CD5 molecule (CD5), mRNA. 2.20E-05 4.3049035 down 963 CD53 Homo sapiens CD53 antigen (CD53), mRNA. 5.13E-06 2.6352615 up 963 CD53 Homo sapiens CD53 antigen (CD53), mRNA. 5.13E-06 2.6252496 up 1604 CD55 Homo sapiens CD55 molecule, decay accelerating 5.13E-06 4.717547 up factor for complement (Cromer blood group) (CD55), mRNA. 965 CD58 Homo sapiens CD58 molecule (CD58), mRNA. 5.13E-06 6.3527145 up 923 CD6 Homo sapiens CD6 molecule (CD6), mRNA. 5.13E-06 5.6552644 down 967 CD63 Homo sapiens CD63 antigen (melanoma 1 antigen) 5.27E-06 5.858538 up (CD63), mRNA. 924 CD7 Homo sapiens CD7 molecule (CD7), mRNA. 5.13E-06 4.597035 down 972 CD74 Homo sapiens CD74 molecule, major 5.13E-06 3.0446384 down histocompatibility complex, class II invariant chain (CD74), transcript variant 2, mRNA. 972 CD74 Homo sapiens CD74 molecule, major 5.27E-06 2.9287467 down histocompatibility complex, class II invariant chain (CD74), transcript variant 3, mRNA. 973 CD79A Homo sapiens CD79A antigen (immunoglobulin- 4.69E-05 2.4491968 down associated alpha) (CD79A), transcript variant 1, mRNA. 973 CD79A Homo sapiens CD79A antigen (immunoglobulin- 4.85E-04 2.1259444 down associated alpha) (CD79A), transcript variant 1, mRNA. 974 CD79B Homo sapiens CD79b molecule, immunoglobulin- 7.57E-05 2.333049 down associated beta (CD79B), transcript variant 3, mRNA. 974 CD79B Homo sapiens CD79B antigen (immunoglobulin- 5.69E-05 2.680019 down associated beta) (CD79B), transcript variant 1, mRNA. 974 CD79B Homo sapiens CD79B antigen (immunoglobulin- 3.50E-05 2.7322147 down associated beta) (CD79B), transcript variant 2, mRNA. 3732 CD82 Homo sapiens CD82 antigen (CD82), transcript 5.88E-06 2.9834602 up variant 1, mRNA. 3732 CD82 Homo sapiens CD82 molecule (CD82), transcript 5.13E-06 4.1557465 up variant 2, mRNA. 942 CD86 Homo sapiens CD86 antigen (CD28 antigen ligand 6.74E-06 2.2853835 down 2, B7-2 antigen) (CD86), transcript variant 1, mRNA. 925 CD8A Homo sapiens CD8a molecule (CD8A), transcript 5.13E-06 4.806471 down variant 2, mRNA. 925 CD8A Homo sapiens CD8 antigen, alpha polypeptide 5.13E-06 5.052119 down (p32) (CD8A), transcript variant 1, mRNA. 212

10225 CD96 Homo sapiens CD96 molecule (CD96), transcript 5.13E-06 4.9419694 down variant 2, mRNA. 10225 CD96 Homo sapiens CD96 antigen (CD96), transcript 8.68E-05 3.9020596 down variant 1, mRNA. 978 CDA Homo sapiens cytidine deaminase (CDA), mRNA. 1.11E-04 2.7909765 up 994 CDC25B Homo sapiens cell division cycle 25 homolog B (S. 5.47E-06 3.699055 down pombe) (CDC25B), transcript variant 2, mRNA. 998 CDC42 Homo sapiens cell division cycle 42 (GTP binding 5.13E-06 2.2075868 up protein, 25kDa) (CDC42), transcript variant 1, mRNA. 998 CDC42 Homo sapiens cell division cycle 42 (GTP binding 3.09E-05 3.407237 up protein, 25kDa) (CDC42), transcript variant 2, mRNA. 10435 CDC42EP2 Homo sapiens CDC42 effector protein (Rho GTPase 9.15E-05 2.4797053 up binding) 2 (CDC42EP2), mRNA. 10602 CDC42EP3 Homo sapiens CDC42 effector protein (Rho GTPase 1.19E-02 2.862828 up binding) 3 (CDC42EP3), mRNA. 1019 CDK4 Homo sapiens cyclin-dependent kinase 4 (CDK4), 2.63E-05 2.1375792 down mRNA. 1020 CDK5 Homo sapiens cyclin-dependent kinase 5 (CDK5), 3.14E-04 2.0153193 up mRNA. 55755 CDK5RAP2 Homo sapiens CDK5 regulatory subunit associated 1.55E-03 2.992742 up protein 2 (CDK5RAP2), transcript variant 2, mRNA.

55755 CDK5RAP2 Homo sapiens CDK5 regulatory subunit associated 8.96E-04 7.327741 up protein 2 (CDK5RAP2), transcript variant 2, mRNA.

1021 CDK6 Homo sapiens cyclin-dependent kinase 6 (CDK6), 3.14E-05 2.1420696 down mRNA. 1030 CDKN2B Homo sapiens cyclin-dependent kinase inhibitor 2B 4.44E-03 2.1050932 up (p15, inhibits CDK4) (CDKN2B), transcript variant 2, mRNA. 1032 CDKN2D Homo sapiens cyclin-dependent kinase inhibitor 2D 5.13E-06 3.649528 up (p19, inhibits CDK4) (CDKN2D), transcript variant 1, mRNA. 1039 CDR2 Homo sapiens cerebellar degeneration-related 7.25E-06 2.7418623 down protein 2, 62kDa (CDR2), mRNA. 634 CEACAM1 Homo sapiens carcinoembryonic antigen-related 8.44E-06 14.709088 up cell adhesion molecule 1 (biliary glycoprotein) (CEACAM1), transcript variant 1, mRNA. 634 CEACAM1 Homo sapiens carcinoembryonic antigen-related 5.27E-06 11.678248 up cell adhesion molecule 1 (biliary glycoprotein) (CEACAM1), transcript variant 2, mRNA. 634 CEACAM1 Homo sapiens carcinoembryonic antigen-related 5.27E-06 11.331404 up cell adhesion molecule 1 (biliary glycoprotein) (CEACAM1), transcript variant 2, mRNA. 1089 CEACAM4 Homo sapiens carcinoembryonic antigen-related 7.88E-05 3.9512918 up cell adhesion molecule 4 (CEACAM4), mRNA. 1088 CEACAM8 Homo sapiens carcinoembryonic antigen-related 1.19E-04 9.751434 up cell adhesion molecule 8 (CEACAM8), mRNA. 1050 CEBPA Homo sapiens CCAAT/enhancer binding protein 9.10E-06 3.0340652 up (C/EBP), alpha (CEBPA), mRNA. 1051 CEBPB Homo sapiens CCAAT/enhancer binding protein 5.13E-06 3.8940663 up (C/EBP), beta (CEBPB), mRNA. 1052 CEBPD Homo sapiens CCAAT/enhancer binding protein 5.47E-06 3.6535134 up (C/EBP), delta (CEBPD), mRNA.

213

51816 CECR1 Homo sapiens cat eye syndrome chromosome 1.52E-05 3.6679435 down region, candidate 1 (CECR1), transcript variant 1, mRNA. 1675 CFD Homo sapiens complement factor D (adipsin) 5.61E-03 2.3166604 down (CFD), mRNA. 118487 CHCHD1 Homo sapiens coiled-coil-helix-coiled-coil-helix 6.17E-06 2.0627074 up domain containing 1 (CHCHD1), mRNA. 55790 ChGn Homo sapiens chondroitin beta1,4 N- 8.39E-05 3.3370051 up acetylgalactosaminyltransferase (ChGn), mRNA. 26511 CHIC2 Homo sapiens cysteine-rich hydrophobic domain 2 6.74E-06 2.2700129 up (CHIC2), mRNA. 1119 CHKA Homo sapiens choline kinase alpha (CHKA), 3.09E-05 2.2814727 up transcript variant 1, mRNA. 27243 CHMP2A Homo sapiens chromatin modifying protein 2A 1.03E-04 2.2441735 up (CHMP2A), transcript variant 1, mRNA. 27243 CHMP2A Homo sapiens chromatin modifying protein 2A 1.52E-05 2.016407 up (CHMP2A), transcript variant 1, mRNA. 11261 CHP Homo sapiens calcium binding protein P22 (CHP), 6.31E-06 2.4448466 up mRNA. 56994 CHPT1 Homo sapiens choline phosphotransferase 1 1.28E-05 2.6655567 up (CHPT1), mRNA. 166012 CHST13 Homo sapiens carbohydrate (chondroitin 4) 5.61E-03 2.0742118 up sulfotransferase 13 (CHST13), mRNA. 22856 CHSY1 Homo sapiens carbohydrate (chondroitin) synthase 1.82E-05 2.6951137 up 1 (CHSY1), mRNA. 1147 CHUK Homo sapiens conserved helix-loop-helix 5.27E-06 2.8588862 up ubiquitous kinase (CHUK), mRNA. 27141 CIDEB Homo sapiens cell death-inducing DFFA-like 3.50E-05 2.126732 up effector b (CIDEB), mRNA. 10970 CKAP4 Homo sapiens cytoskeleton-associated protein 4 5.13E-06 5.6540203 up (CKAP4), mRNA. 51192 CKLF Homo sapiens chemokine-like factor (CKLF), 5.13E-06 4.214922 up transcript variant 6, mRNA. 51192 CKLF Homo sapiens chemokine-like factor (CKLF), 5.13E-06 4.150689 up transcript variant 3, mRNA. 1178 CLC Homo sapiens Charcot-Leyden crystal protein 3.46E-04 3.859848 down (CLC), mRNA. 51266 CLEC1B Homo sapiens C-type lectin domain family 1, 1.01E-04 4.534882 up member B (CLEC1B), mRNA. 50856 CLEC4A Homo sapiens C-type lectin domain family 4, 5.35E-06 3.9043794 up member A (CLEC4A), transcript variant 4, mRNA. 338339 CLEC4D Homo sapiens C-type lectin domain family 4, 0.013338441 6.2865214 up member D (CLEC4D), mRNA. 1192 CLIC1 Homo sapiens chloride intracellular channel 1 5.27E-06 3.0021625 up (CLIC1), mRNA. 9022 CLIC3 Homo sapiens chloride intracellular channel 3 1.17E-05 4.232528 down (CLIC3), mRNA. 6249 CLIP1 Homo sapiens CAP-GLY domain containing linker 1.28E-05 2.2602222 up protein 1 (CLIP1), transcript variant 1, mRNA. 22883 CLSTN1 Homo sapiens calsyntenin 1 (CLSTN1), transcript 5.13E-06 2.889167 down variant 1, mRNA. 1213 CLTC Homo sapiens clathrin, heavy polypeptide (Hc) 1.09E-05 2.247254 up (CLTC), mRNA. 171425 CLYBL Homo sapiens citrate lyase beta like (CLYBL), 3.80E-04 2.326929 down mRNA. 29097 CNIH4 Homo sapiens cornichon homolog 4 (Drosophila) 5.13E-06 5.2493296 up (CNIH4), mRNA.

214

22796 COG2 Homo sapiens component of oligomeric golgi 5.13E-06 2.0754697 down complex 2 (COG2), mRNA. 10087 COL4A3BP Homo sapiens collagen, type IV, alpha 3 0.004497504 2.289607 up (Goodpasture antigen) binding protein (COL4A3BP), transcript variant 1, mRNA. 51122 COMMD2 Homo sapiens COMM domain containing 2 2.57E-05 2.039293 up (COMMD2), mRNA. 54951 COMMD8 Homo sapiens COMM domain containing 8 4.12E-04 2.0294437 up (COMMD8), mRNA. 114769 COP1 Homo sapiens caspase-1 dominant-negative 0.002195084 2.6545103 up inhibitor pseudo-ICE (COP1), transcript variant 2, mRNA. 114769 COP1 Homo sapiens caspase-1 dominant-negative 1.54E-04 2.2014716 up inhibitor pseudo-ICE (COP1), transcript variant 2, mRNA. 10987 COPS5 Homo sapiens COP9 constitutive 5.35E-06 2.1435437 up photomorphogenic homolog subunit 5 (Arabidopsis) (COPS5), mRNA. 27235 COQ2 Homo sapiens coenzyme Q2 homolog, 5.13E-06 2.334017 up prenyltransferase (yeast) (COQ2), mRNA. 23603 CORO1C Homo sapiens coronin, actin binding protein, 1C 5.91E-06 2.095292 up (CORO1C), mRNA. 7464 CORO2A Homo sapiens coronin, actin binding protein, 2A 5.13E-06 3.0214834 up (CORO2A), transcript variant 2, mRNA. 1352 COX10 Homo sapiens COX10 homolog, cytochrome c 5.13E-06 2.0721776 down oxidase assembly protein, heme A: farnesyltransferase (yeast) (COX10), nuclear gene encoding mitochondrial protein, mRNA. 10063 COX17 Homo sapiens COX17 cytochrome c oxidase 7.25E-06 2.1722808 up assembly homolog (S. cerevisiae) (COX17), nuclear gene encoding mitochondrial protein, mRNA. 1347 COX7A2 Homo sapiens cytochrome c oxidase subunit VIIa 3.87E-05 2.5286677 up polypeptide 2 (liver) (COX7A2), mRNA. 1349 COX7B Homo sapiens cytochrome c oxidase subunit VIIb 3.65E-04 2.8020906 up (COX7B), nuclear gene encoding mitochondrial protein, mRNA. 1362 CPD Homo sapiens carboxypeptidase D (CPD), mRNA. 5.13E-06 5.0072336 up 80315 CPEB4 Homo sapiens cytoplasmic polyadenylation 1.87E-04 3.4604418 up element binding protein 4 (CPEB4), mRNA. 8895 CPNE3 Homo sapiens copine III (CPNE3), mRNA. 1.17E-05 2.3390002 up 54504 CPVL Homo sapiens carboxypeptidase, vitellogenic-like 0.003804619 2.2566674 down (CPVL), transcript variant 1, mRNA. 54504 CPVL Homo sapiens carboxypeptidase, vitellogenic-like 0.002355844 2.2606637 down (CPVL), transcript variant 1, mRNA. 1378 CR1 PREDICTED: Homo sapiens complement 0.014260175 2.5448275 up component (3b/4b) receptor 1 (Knops blood group) (CR1), mRNA. 1378 CR1 Homo sapiens complement component (3b/4b) 1.47E-04 3.6902504 up receptor 1 (Knops blood group) (CR1), transcript variant F, mRNA. 9586 CREB5 Homo sapiens cAMP responsive element binding 0.001689941 2.7032115 up protein 5 (CREB5), transcript variant 1, mRNA. 8804 CREG1 Homo sapiens cellular repressor of E1A-stimulated 5.13E-06 2.3837616 up genes 1 (CREG1), mRNA. 1396 CRIP1 Homo sapiens cysteine-rich protein 1 (intestinal) 1.82E-05 2.199152 down (CRIP1), mRNA.

215

1438 CSF2RA Homo sapiens colony stimulating factor 2 receptor, 5.27E-06 3.4945817 up alpha, low-affinity (granulocyte-macrophage) (CSF2RA), transcript variant 6, mRNA. 1438 CSF2RA PREDICTED: Homo sapiens colony stimulating 5.13E-06 3.6573358 up factor 2 receptor, alpha, low-affinity (granulocyte- macrophage) (CSF2RA), mRNA. 1441 CSF3R Homo sapiens colony stimulating factor 3 receptor 1.82E-05 2.3448637 up (granulocyte) (CSF3R), transcript variant 4, mRNA.

1441 CSF3R Homo sapiens colony stimulating factor 3 receptor 1.28E-05 3.6351035 up (granulocyte) (CSF3R), transcript variant 1, mRNA.

8530 CST7 Homo sapiens cystatin F (leukocystatin) (CST7), 1.66E-05 4.5753226 up mRNA. 1475 CSTA Homo sapiens cystatin A (stefin A) (CSTA), mRNA. 1.17E-04 2.297074 up 1476 CSTB Homo sapiens cystatin B (stefin B) (CSTB), mRNA. 5.13E-06 2.0360916 up 414189 CTGLF3 Homo sapiens centaurin, gamma-like family, 1.52E-05 2.2590318 down member 3 (CTGLF3), mRNA. 1495 CTNNA1 Homo sapiens catenin (cadherin-associated 7.68E-06 2.4322417 up protein), alpha 1, 102kDa (CTNNA1), mRNA. 1075 CTSC Homo sapiens cathepsin C (CTSC), transcript 5.13E-06 3.614826 up variant 1, mRNA. 1075 CTSC Homo sapiens cathepsin C (CTSC), transcript 2.90E-05 2.2673752 up variant 1, mRNA. 1509 CTSD Homo sapiens cathepsin D (CTSD), mRNA. 6.74E-06 3.128691 up 1512 CTSH Homo sapiens cathepsin H (CTSH), transcript 1.78E-04 2.1109343 up variant 1, mRNA. 1512 CTSH Homo sapiens cathepsin H (CTSH), transcript 0.009319386 2.005697 up variant 1, mRNA. 1514 CTSL1 Homo sapiens cathepsin L1 (CTSL1), transcript 1.65E-04 4.7943954 up variant 2, mRNA. 1514 CTSL1 Homo sapiens cathepsin L1 (CTSL1), transcript 1.98E-05 5.7686768 up variant 2, mRNA. 1519 CTSO Homo sapiens cathepsin O (CTSO), mRNA. 6.31E-06 2.3710158 down 1521 CTSW Homo sapiens cathepsin W (lymphopain) (CTSW), 5.48E-06 4.979813 down mRNA. 1522 CTSZ Homo sapiens cathepsin Z (CTSZ), mRNA. 2.17E-04 2.0581768 up 51596 CUTA Homo sapiens cutA divalent cation tolerance 5.27E-06 2.4104688 down homolog (E. coli) (CUTA), transcript variant 5, mRNA. 51596 CUTA Homo sapiens cutA divalent cation tolerance 5.27E-06 2.0102203 down homolog (E. coli) (CUTA), transcript variant 1, mRNA. 51076 CUTC Homo sapiens cutC copper transporter homolog (E. 9.10E-06 2.1719136 up coli) (CUTC), mRNA. 1524 CX3CR1 Homo sapiens chemokine (C-X3-C motif) receptor 1 3.18E-05 2.7759042 down (CX3CR1), mRNA. 58191 CXCL16 Homo sapiens chemokine (C-X-C motif) ligand 16 5.13E-06 4.317199 up (CXCL16), mRNA. 139231 CXorf39 Homo sapiens chromosome X open reading frame 1.31E-05 2.0749292 up 39 (CXorf39), mRNA. 51523 CXXC5 Homo sapiens CXXC finger 5 (CXXC5), mRNA. 9.10E-06 2.487575 down 51706 CYB5R1 Homo sapiens cytochrome b5 reductase 1 8.44E-06 2.9186919 up (CYB5R1), mRNA. 51167 CYB5R4 Homo sapiens cytochrome b5 reductase 4 1.08E-05 3.213936 up (CYB5R4), mRNA. 216

1535 CYBA Homo sapiens cytochrome b-245, alpha 5.13E-06 2.2248816 up polypeptide (CYBA), mRNA. 220002 CYBASC3 Homo sapiens cytochrome b, ascorbate dependent 1.28E-05 2.310716 down 3 (CYBASC3), mRNA. 1536 CYBB Homo sapiens cytochrome b-245, beta polypeptide 5.27E-06 2.9885292 up (chronic granulomatous disease) (CYBB), mRNA.

246126 CYorf15A Homo sapiens chromosome Y open reading frame 0.0019786 2.0988035 down 15A (CYorf15A), mRNA. 1545 CYP1B1 Homo sapiens cytochrome P450, family 1, 6.17E-06 11.84795 up subfamily B, polypeptide 1 (CYP1B1), mRNA. 1595 CYP51A1 Homo sapiens cytochrome P450, family 51, 4.99E-04 2.0392642 up subfamily A, polypeptide 1 (CYP51A1), mRNA. 27065 D4S234E Homo sapiens DNA segment on chromosome 4 0.006639309 3.2519257 down (unique) 234 expressed sequence (D4S234E), mRNA. 1602 DACH1 Homo sapiens dachshund homolog 1 (Drosophila) 6.17E-06 10.448672 up (DACH1), transcript variant 1, mRNA. 1622 DBI Homo sapiens diazepam binding inhibitor (GABA 2.17E-04 2.1242292 up receptor modulator, acyl-Coenzyme A binding protein) (DBI), transcript variant 2, mRNA. 167227 DCP2 Homo sapiens DCP2 decapping enzyme homolog 5.47E-06 2.4441154 up (S. cerevisiae) (DCP2), mRNA. 51164 DCTN4 Homo sapiens dynactin 4 (p62) (DCTN4), mRNA. 8.44E-06 2.1044273 up 123879 DCUN1D3 Homo sapiens DCN1, defective in cullin 1.66E-05 2.5469155 up neddylation 1, domain containing 3 (S. cerevisiae) (DCUN1D3), mRNA. 23564 DDAH2 Homo sapiens dimethylarginine 1.08E-02 3.4107604 up dimethylaminohydrolase 2 (DDAH2), mRNA. 50807 DDEF1 Homo sapiens development and differentiation 3.18E-05 2.189919 up enhancing factor 1 (DDEF1), mRNA. 1649 DDIT3 Homo sapiens DNA-damage-inducible transcript 3 8.28E-05 2.3088243 up (DDIT3), mRNA. 57062 DDX24 Homo sapiens DEAD (Asp-Glu-Ala-Asp) box 5.13E-06 3.6519904 down polypeptide 24 (DDX24), mRNA. 51202 DDX47 Homo sapiens DEAD (Asp-Glu-Ala-Asp) box 1.08E-05 2.0098073 down polypeptide 47 (DDX47), transcript variant 2, mRNA. 83479 DDX59 Homo sapiens DEAD (Asp-Glu-Ala-Asp) box 1.00E-05 2.3997624 up polypeptide 59 (DDX59), transcript variant 1, mRNA. 83479 DDX59 Homo sapiens DEAD (Asp-Glu-Ala-Asp) box 1.22E-04 2.0845 up polypeptide 59 (DDX59), transcript variant 1, mRNA. 1667 DEFA1 Homo sapiens defensin, alpha 1 (DEFA1), mRNA. 2.45E-02 2.4638147 up 79961 DENND2D Homo sapiens DENN/MADD domain containing 2D 5.13E-06 2.1206117 down (DENND2D), mRNA. 22898 DENND3 Homo sapiens DENN/MADD domain containing 3 3.87E-05 2.1982214 up (DENND3), mRNA. 28955 DEXI Homo sapiens dexamethasone-induced transcript 8.44E-06 2.0605617 down (DEXI), mRNA. 84649 DGAT2 Homo sapiens diacylglycerol O-acyltransferase 4.54E-04 3.2562783 up homolog 2 (mouse) (DGAT2), mRNA. 1609 DGKQ Homo sapiens diacylglycerol kinase, theta 110kDa 2.61E-04 2.0521948 down (DGKQ), mRNA. 9249 DHRS3 Homo sapiens dehydrogenase/reductase (SDR 5.95E-04 2.559416 down family) member 3 (DHRS3), mRNA. 217

51635 DHRS7 Homo sapiens dehydrogenase/reductase (SDR 8.44E-06 2.4672217 up family) member 7 (DHRS7), mRNA. 10170 DHRS9 Homo sapiens dehydrogenase/reductase (SDR 5.35E-06 9.970834 up family) member 9 (DHRS9), transcript variant 1, mRNA. 10170 DHRS9 Homo sapiens dehydrogenase/reductase (SDR 6.31E-06 8.799593 up family) member 9 (DHRS9), transcript variant 1, mRNA. 23405 DICER1 Homo sapiens Dicer1, Dcr-1 homolog (Drosophila) 1.08E-05 3.2951527 up (DICER1), transcript variant 2, mRNA. 84925 DIRC2 Homo sapiens disrupted in renal carcinoma 2 5.13E-06 4.497308 up (DIRC2), mRNA. 25923 DKFZP564J0863 Homo sapiens DKFZP564J0863 protein 5.13E-06 3.3129492 up (DKFZP564J0863), mRNA. 91056 DKFZp761E198 Homo sapiens DKFZp761E198 protein 3.71E-03 2.0044436 up (DKFZp761E198), mRNA. 1738 DLD Homo sapiens dihydrolipoamide dehydrogenase 1.57E-05 2.079882 up (E3 component of pyruvate dehydrogenase complex, 2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex) (DLD), mRNA. 9093 DNAJA3 Homo sapiens DnaJ (Hsp40) homolog, subfamily A, 1.66E-05 2.1748948 down member 3 (DNAJA3), mRNA. 10049 DNAJB6 Homo sapiens DnaJ (Hsp40) homolog, subfamily B, 9.10E-06 2.0758972 up member 6 (DNAJB6), transcript variant 1, mRNA. 80331 DNAJC5 Homo sapiens DnaJ (Hsp40) homolog, subfamily C, 5.27E-06 2.5996194 up member 5 (DNAJC5), mRNA. 1774 DNASE1L1 Homo sapiens deoxyribonuclease I-like 1 5.27E-06 3.1884494 up (DNASE1L1), transcript variant 4, mRNA. 83658 DNCL2A Homo sapiens dynein, cytoplasmic, light 2.98E-05 2.0942302 up polypeptide 2A (DNCL2A), transcript variant 2, mRNA. 1786 DNMT1 Homo sapiens DNA (cytosine-5-)-methyltransferase 5.13E-06 2.9878216 down 1 (DNMT1), mRNA. 116092 DNTTIP1 Homo sapiens deoxynucleotidyltransferase, 5.27E-06 2.5209358 up terminal, interacting protein 1 (DNTTIP1), mRNA. 55619 DOCK10 Homo sapiens dedicator of cytokinesis 10 3.18E-05 2.9987986 down (DOCK10), mRNA. 80005 DOCK5 Homo sapiens dedicator of cytokinesis 5 (DOCK5), 1.47E-04 2.2335734 up mRNA. 1802 DPH2 Homo sapiens DPH2 homolog (S. cerevisiae) 5.35E-06 2.048709 down (DPH2), transcript variant 1, mRNA. 285381 DPH3 Homo sapiens DPH3, KTI11 homolog (S. cerevisiae) 5.13E-06 3.376012 up (DPH3), transcript variant 2, mRNA. 29952 DPP7 Homo sapiens dipeptidyl-peptidase 7 (DPP7), 5.19E-05 2.3446808 down mRNA. 1806 DPYD Homo sapiens dihydropyrimidine dehydrogenase 9.55E-04 2.2364478 up (DPYD), mRNA. 51108 DREV1 Homo sapiens DORA reverse strand protein 1 5.91E-06 4.0783753 up (DREV1), mRNA. 1824 DSC2 Homo sapiens desmocollin 2 (DSC2), transcript 3.18E-05 4.42125 up variant Dsc2b, mRNA. 1843 DUSP1 Homo sapiens dual specificity phosphatase 1 5.35E-06 4.0786195 up (DUSP1), mRNA. 1845 DUSP3 Homo sapiens dual specificity phosphatase 3 1.28E-05 2.2295356 up (vaccinia virus phosphatase VH1-related) (DUSP3), mRNA.

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1847 DUSP5 Homo sapiens dual specificity phosphatase 5 5.20E-04 2.0630016 down (DUSP5), mRNA. 6993 DYNLT1 Homo sapiens dynein, light chain, Tctex-type 1 1.39E-05 3.1938145 up (DYNLT1), mRNA. 8445 DYRK2 Homo sapiens dual-specificity tyrosine-(Y)- 2.42E-05 2.9621196 down phosphorylation regulated kinase 2 (DYRK2), transcript variant 1, mRNA. 8291 DYSF Homo sapiens dysferlin, limb girdle muscular 1.28E-05 4.5595326 up dystrophy 2B (autosomal recessive) (DYSF), mRNA. 1870 E2F2 Homo sapiens E2F transcription factor 2 (E2F2), 2.37E-04 2.3872674 up mRNA. 1871 E2F3 Homo sapiens E2F transcription factor 3 (E2F3), 1.52E-05 3.0421667 up mRNA. 1880 EBI2 Homo sapiens Epstein-Barr virus induced gene 2 3.07E-05 2.7170253 down (lymphocyte-specific G protein-coupled receptor) (EBI2), mRNA. 1889 ECE1 Homo sapiens endothelin converting enzyme 1 5.73E-03 2.105879 up (ECE1), mRNA. 55268 ECHDC2 Homo sapiens enoyl Coenzyme A hydratase 5.13E-06 3.6648486 down domain containing 2 (ECHDC2), mRNA. 79746 ECHDC3 Homo sapiens enoyl Coenzyme A hydratase 3.26E-05 8.847016 up domain containing 3 (ECHDC3), mRNA. 80267 EDEM3 Homo sapiens ER degradation enhancer, 5.35E-06 2.2210698 up mannosidase alpha-like 3 (EDEM3), mRNA. 53637 EDG8 Homo sapiens endothelial differentiation, 3.80E-04 5.379586 down sphingolipid G-protein-coupled receptor, 8 (EDG8), mRNA. 10289 EIF1B Homo sapiens eukaryotic translation initiation 6.74E-06 2.4711375 up factor 1B (EIF1B), mRNA. 192670 EIF2C4 Homo sapiens eukaryotic translation initiation 2.84E-04 2.6301434 up factor 2C, 4 (EIF2C4), mRNA. 317649 EIF4E3 Homo sapiens eukaryotic translation initiation 1.17E-05 2.9506123 up factor 4E family member 3 (EIF4E3), mRNA. 1998 ELF2 Homo sapiens E74-like factor 2 (ets domain 1.95E-04 2.1745768 down transcription factor) (ELF2), transcript variant 2, mRNA. 255520 ELMOD2 Homo sapiens ELMO/CED-12 domain containing 2 1.00E-04 3.9240546 up (ELMOD2), mRNA. 55140 ELP3 Homo sapiens elongation protein 3 homolog (S. 5.13E-06 2.1816473 down cerevisiae) (ELP3), mRNA. 84034 EMILIN2 Homo sapiens elastin microfibril interfacer 2 5.35E-06 4.6457095 up (EMILIN2), mRNA. 2015 EMR1 Homo sapiens egf-like module containing, mucin- 5.47E-06 4.5677238 up like, hormone receptor-like 1 (EMR1), mRNA. 30817 EMR2 Homo sapiens egf-like module containing, mucin- 2.42E-05 3.4380198 up like, hormone receptor-like 2 (EMR2), transcript variant 2, mRNA. 84658 EMR3 Homo sapiens egf-like module containing, mucin- 2.37E-04 3.4959276 down like, hormone receptor-like 3 (EMR3), transcript variant 1, mRNA. 55556 ENOSF1 Homo sapiens enolase superfamily member 1 2.51E-05 2.3088577 down (ENOSF1), mRNA. 22875 ENPP4 Homo sapiens ectonucleotide 4.41E-04 2.450817 up pyrophosphatase/phosphodiesterase 4 (putative function) (ENPP4), mRNA. 953 ENTPD1 Homo sapiens ectonucleoside triphosphate 2.30E-04 2.4557087 up diphosphohydrolase 1 (ENTPD1), mRNA.

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56943 ENY2 Homo sapiens enhancer of yellow 2 homolog 5.47E-06 3.3467193 up (Drosophila) (ENY2), mRNA. 8320 EOMES Homo sapiens eomesodermin homolog (Xenopus 1.31E-03 3.7983854 down laevis) (EOMES), mRNA. 57634 EP400 Homo sapiens E1A binding protein p400 (EP400), 6.14E-06 2.0384111 down mRNA. 54749 EPDR1 Homo sapiens ependymin related protein 1 1.96E-02 2.021042 up (zebrafish) (EPDR1), mRNA. 2041 EPHA1 Homo sapiens EPH receptor A1 (EPHA1), mRNA. 2.09E02 2.0412483 down 94240 EPSTI1 Homo sapiens epithelial stromal interaction 1 9.78E-05 4.230828 down (breast) (EPSTI1), transcript variant 2, mRNA. 51327 ERAF Homo sapiens erythroid associated factor (ERAF), 3.51E-03 2.3858838 up mRNA. 30001 ERO1L Homo sapiens ERO1-like (S. cerevisiae) (ERO1L), 5.13E-06 3.475923 up mRNA. 2107 ETF1 Homo sapiens eukaryotic translation termination 5.91E-06 2.065806 up factor 1 (ETF1), mRNA. 2110 ETFDH Homo sapiens electron-transferring-flavoprotein 5.27E-06 2.23076 up dehydrogenase (ETFDH), nuclear gene encoding mitochondrial protein, mRNA. 55500 ETNK1 Homo sapiens ethanolamine kinase 1 (ETNK1), 1.74E-04 2.2017987 up mRNA. 2113 ETS1 Homo sapiens v-ets erythroblastosis virus E26 9.97E-06 2.558997 down oncogene homolog 1 (avian) (ETS1), mRNA. 2114 ETS2 Homo sapiens v-ets erythroblastosis virus E26 5.13E-06 8.58237 up oncogene homolog 2 (avian) (ETS2), mRNA. 51466 EVL Homo sapiens Enah/Vasp-like (EVL), mRNA. 5.13E-06 4.689364 down 54536 EXOC6 Homo sapiens exocyst complex component 6 1.08E-05 4.4522514 up (EXOC6), transcript variant 2, mRNA. 54512 EXOSC4 Homo sapiens exosome component 4 (EXOSC4), 5.27E-06 7.319126 up mRNA. 56915 EXOSC5 Homo sapiens exosome component 5 (EXOSC5), 2.07E-05 2.0822344 down mRNA. 2161 F12 Homo sapiens coagulation factor XII (Hageman 6.99E-04 2.637752 up factor) (F12), mRNA. 9214 FAIM3 Homo sapiens Fas apoptotic inhibitory molecule 3 5.13E-06 3.2987125 down (FAIM3), mRNA. 359845 FAM101B Homo sapiens family with sequence similarity 101, 4.69E-05 2.82394 up member B (FAM101B), mRNA. 399665 FAM102A Homo sapiens family with sequence similarity 102, 5.13E-06 4.415383 down member A (FAM102A), transcript variant 1, mRNA.

54491 FAM105A Homo sapiens family with sequence similarity 105, 1.47E-03 2.868355 up member A (FAM105A), mRNA. 91523 FAM113B Homo sapiens family with sequence similarity 113, 5.13E-06 3.6542397 down member B (FAM113B), mRNA. 83982 FAM14A Homo sapiens family with sequence similarity 14, 2.17E-04 2.1707006 up member A (FAM14A), mRNA. 56975 FAM20C Homo sapiens family with sequence similarity 20, 2.70E-04 2.9187624 up member C (FAM20C), mRNA. 9780 FAM38A Homo sapiens family with sequence similarity 38, 5.13E-06 2.3264341 down member A (FAM38A), mRNA. 404636 FAM45A Homo sapiens family with sequence similarity 45, 7.25E-06 2.554875 up member A (FAM45A), mRNA. 81553 FAM49A Homo sapiens family with sequence similarity 49, 2.63E-05 2.0053241 up member A (FAM49A), mRNA.

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51571 FAM49B Homo sapiens family with sequence similarity 49, 5.27E-06 2.8720198 up member B (FAM49B), mRNA. 26240 FAM50B Homo sapiens family with sequence similarity 50, 5.90E-05 2.0443442 down member B (FAM50B), mRNA. 51307 FAM53C Homo sapiens family with sequence similarity 53, 1.11E-04 2.2430544 up member C (FAM53C), mRNA. 23344 FAM62A Homo sapiens family with sequence similarity 62 5.13E-06 3.2714174 down (C2 domain containing), member A (FAM62A), mRNA. 57488 FAM62B Homo sapiens family with sequence similarity 62 1.08E-05 2.0318565 down (C2 domain containing) member B (FAM62B), mRNA. 54629 FAM63B Homo sapiens family with sequence similarity 63, 1.08E-05 2.1487174 up member B (FAM63B), mRNA. 375061 FAM89A Homo sapiens family with sequence similarity 89, 4.38E-02 3.9909568 up member A (FAM89A), mRNA. 375061 FAM89A Homo sapiens family with sequence similarity 89, 5.13E-06 3.994492 up member A (FAM89A), mRNA. 157769 FAM91A1 Homo sapiens family with sequence similarity 91, 9.36E-04 2.0552425 up member A1 (FAM91A1), mRNA. 10667 FARS2 Homo sapiens phenylalanine-tRNA synthetase 2 5.62E-06 2.2093713 down (mitochondrial) (FARS2), nuclear gene encoding mitochondrial protein, mRNA. 2194 FASN Homo sapiens fatty acid synthase (FASN), mRNA. 2.08E-04 2.1970415 down 2091 FBL Homo sapiens fibrillarin (FBL), mRNA. 5.27E-06 2.6103055 down 2201 FBN2 Homo sapiens fibrillin 2 (congenital contractural 4.12E-04 2.5507998 up arachnodactyly) (FBN2), mRNA. 2203 FBP1 Homo sapiens fructose-1,6-bisphosphatase 1 7.57E-05 2.1626742 up (FBP1), mRNA. 84678 FBXL10 Homo sapiens F-box and leucine-rich repeat 5.13E-06 2.3947892 down protein 10 (FBXL10), transcript variant 1, mRNA. 26234 FBXL5 Homo sapiens F-box and leucine-rich repeat 6.74E-05 2.429683 up protein 5 (FBXL5), transcript variant 1, mRNA. 26234 FBXL5 Homo sapiens F-box and leucine-rich repeat 1.39E-05 2.7659962 up protein 5 (FBXL5), transcript variant 1, mRNA. 23014 FBXO21 Homo sapiens F-box protein 21 (FBXO21), 5.35E-06 2.3136113 down transcript variant 2, mRNA. 84085 FBXO30 Homo sapiens F-box protein 30 (FBXO30), mRNA. 5.13E-06 4.14862 up 79791 FBXO31 Homo sapiens F-box protein 31 (FBXO31), mRNA. 5.13E-06 2.6231027 down 81545 FBXO38 Homo sapiens F-box protein 38 (FBXO38), 9.10E-06 2.018637 up transcript variant 1, mRNA. 26190 FBXW2 Homo sapiens F-box and WD-40 domain protein 2 5.27E-06 2.899193 up (FBXW2), mRNA. 2204 FCAR Homo sapiens Fc fragment of IgA, receptor for 8.09E-05 5.352728 up (FCAR), transcript variant 10, mRNA. 2207 FCER1G Homo sapiens Fc fragment of IgE, high affinity I, 5.13E-06 6.26573 up receptor for; gamma polypeptide (FCER1G), mRNA. 2208 FCER2 Homo sapiens Fc fragment of IgE, low affinity II, 3.07E-05 2.8810222 down receptor for (CD23) (FCER2), mRNA. 8857 FCGBP Homo sapiens Fc fragment of IgG binding protein 8.18E-03 2.414376 down (FCGBP), mRNA. 2209 FCGR1A Homo sapiens Fc fragment of IgG, high affinity Ia, 8.55E-04 3.9725544 up receptor (CD64) (FCGR1A), mRNA. 2210 FCGR1B Homo sapiens Fc fragment of IgG, high affinity Ib, 1.00E-03 4.0869117 up receptor (CD64) (FCGR1B), transcript variant 2, mRNA.

221

2210 FCGR1B Homo sapiens Fc fragment of IgG, high affinity Ib, 4.99E-04 4.0404263 up receptor (CD64) (FCGR1B), transcript variant 1, mRNA. 2212 FCGR2A Homo sapiens Fc fragment of IgG, low affinity IIa, 1.89E-03 2.2866335 up receptor (CD32) (FCGR2A), mRNA. 2212 FCGR2A Homo sapiens Fc fragment of IgG, low affinity IIa, 1.55E-03 2.3266907 up receptor (CD32) (FCGR2A), mRNA. 115548 FCHO2 Homo sapiens FCH domain only 2 (FCHO2), mRNA. 1.47E-03 2.0208118 up 84824 FCRLM1 Homo sapiens Fc receptor-like A (FCRLA), mRNA. 1.94E-04 2.5381489 down 56929 FEM1C Homo sapiens fem-1 homolog c (C.elegans) 5.27E-06 2.2946901 up (FEM1C), mRNA. 26509 FER1L3 Homo sapiens fer-1-like 3, myoferlin (C. elegans) 1.11E-03 2.2462935 up (FER1L3), transcript variant 1, mRNA. 2242 FES Homo sapiens feline sarcoma oncogene (FES), 4.26E-05 2.876374 up mRNA. 2867 FFAR2 Homo sapiens free fatty acid receptor 2 (FFAR2), 7.79E-04 2.1559656 up mRNA. 26127 FGFR1OP2 Homo sapiens FGFR1 oncogene partner 2 1.79E-03 2.002944 up (FGFR1OP2), mRNA. 2268 FGR Homo sapiens Gardner-Rasheed feline sarcoma 5.13E-06 3.743808 up viral (v-fgr) oncogene homolog (FGR), transcript variant 3, mRNA. 2268 FGR Homo sapiens Gardner-Rasheed feline sarcoma 5.13E-06 4.515146 up viral (v-fgr) oncogene homolog (FGR), mRNA. 2274 FHL2 Homo sapiens four and a half LIM domains 2 2.23E-03 2.1835103 up (FHL2), transcript variant 1, mRNA. 29109 FHOD1 Homo sapiens formin homology 2 domain 5.13E-06 2.517802 up containing 1 (FHOD1), mRNA. 2280 FKBP1A Homo sapiens FK506 binding protein 1A, 12kDa 5.13E-06 2.599086 up (FKBP1A), transcript variant 12B, mRNA. 2280 FKBP1A Homo sapiens FK506 binding protein 1A, 12kDa 5.47E-06 4.229929 up (FKBP1A), transcript variant 12A, mRNA. 2280 FKBP1A Homo sapiens FK506 binding protein 1A, 12kDa 1.17E-05 3.3661363 up (FKBP1A), transcript variant 12A, mRNA. 2289 FKBP5 Homo sapiens FK506 binding protein 5 (FKBP5), 5.27E-06 7.846178 up mRNA. 55277 FLJ10986 Homo sapiens hypothetical protein FLJ10986 5.13E-06 2.177325 up (FLJ10986), mRNA. 55314 FLJ11155 Homo sapiens transmembrane protein 144 8.89E-03 2.1348088 up (TMEM144), mRNA. 55332 FLJ11259 Homo sapiens hypothetical protein FLJ11259 6.17E-06 5.74057 up (FLJ11259), mRNA. 79621 FLJ11712 Homo sapiens ribonuclease H2, subunit B 2.04E-05 2.285728 down (RNASEH2B), mRNA. 65095 FLJ12949 Homo sapiens hypothetical protein FLJ12949 7.68E-06 2.2615159 down (FLJ12949), transcript variant 1, mRNA. 55601 FLJ20035 Homo sapiens hypothetical protein FLJ20035 1.39E-02 2.1092248 down (FLJ20035), mRNA. 54863 FLJ20245 Homo sapiens hypothetical protein FLJ20245 5.13E-06 2.6427622 up (FLJ20245), mRNA. 55622 FLJ20272 Homo sapiens tetratricopeptide repeat domain 27 5.69E-05 2.1794796 down (TTC27), mRNA. 54502 FLJ20273 Homo sapiens RNA-binding protein (FLJ20273), 5.47E-06 3.967706 up mRNA. 80194 FLJ21749 Homo sapiens hypothetical protein FLJ21749 6.74E-06 2.1933904 down (FLJ21749), mRNA.

222

79641 FLJ22386 Homo sapiens rogdi homolog (Drosophila) (ROGDI), 6.74E-06 2.9610865 up mRNA. 80212 FLJ22471 Homo sapiens coiled-coil domain containing 92 5.13E-06 2.2204547 down (CCDC92), mRNA. 79887 FLJ22662 Homo sapiens hypothetical protein FLJ22662 1.17E-05 3.7287767 up (FLJ22662), mRNA. 220042 FLJ25416 Homo sapiens chromosome 11 open reading frame 4.90E-05 3.7852185 up 82 (C11orf82), mRNA. 122060 FLJ30046 Homo sapiens hypothetical protein FLJ30046 2.97E-05 2.1388574 down (FLJ30046), mRNA. 220388 FLJ38159 Homo sapiens coiled-coil domain containing 89 1.67E-04 2.0847433 up (CCDC89), mRNA. 353116 FLJ39378 Homo sapiens hypothetical protein FLJ39378 3.82E-04 2.1353474 up (FLJ39378), mRNA. 286006 FLJ39575 Homo sapiens hypothetical protein FLJ39575 9.06E-03 2.4017336 up (FLJ39575), mRNA. 10211 FLOT1 Homo sapiens flotillin 1 (FLOT1), mRNA. 5.13E-06 5.5313544 up 2319 FLOT2 Homo sapiens flotillin 2 (FLOT2), mRNA. 1.08E-05 2.4410415 up 2323 FLT3LG Homo sapiens fms-related tyrosine kinase 3 ligand 5.35E-06 4.096451 down (FLT3LG), mRNA. 79672 FN3KRP Homo sapiens fructosamine-3-kinase-related 5.27E-06 2.313113 down protein (FN3KRP), mRNA. 22862 FNDC3A Homo sapiens fibronectin type III domain 3.87E-05 2.7806582 up containing 3A (FNDC3A), mRNA. 22862 FNDC3A Homo sapiens fibronectin type III domain 4.22E-05 2.6544664 up containing 3A (FNDC3A), transcript variant 1, mRNA. 2352 FOLR3 Homo sapiens folate receptor 3 (gamma) (FOLR3), 6.74E-06 8.076672 up mRNA. 2353 FOS Homo sapiens v-fos FBJ murine osteosarcoma viral 6.25E-05 2.8882043 up oncogene homolog (FOS), mRNA. 2355 FOSL2 Homo sapiens FOS-like antigen 2 (FOSL2), mRNA. 1.47E-02 2.664296 up 2308 FOXO1A Homo sapiens forkhead box O1A 5.13E-06 2.1501672 down (rhabdomyosarcoma) (FOXO1A), mRNA. 2357 FPR1 Homo sapiens formyl peptide receptor 1 (FPR1), 5.47E-06 3.370015 up mRNA. 2358 FPRL1 Homo sapiens formyl peptide receptor-like 1 1.08E-05 4.9162574 up (FPRL1), transcript variant 1, mRNA. 2358 FPRL1 Homo sapiens formyl peptide receptor-like 1 7.68E-06 4.874851 up (FPRL1), transcript variant 1, mRNA. 10023 FRAT1 Homo sapiens frequently rearranged in advanced 5.10E-03 2.0954587 up T-cell lymphomas (FRAT1), transcript variant 2, mRNA. 2503 FTHL11 Homo sapiens ferritin, heavy polypeptide-like 11 1.01E-04 2.1077788 up (FTHL11) on chromosome 8. 2497 FTHL2 Homo sapiens ferritin, heavy polypeptide-like 2 8.33E-05 2.1774118 up (FTHL2) on chromosome 1. 2498 FTHL3 Homo sapiens ferritin, heavy polypeptide-like 3 8.33E-05 2.0295854 up (FTHL3) on chromosome 2. 5045 FURIN Homo sapiens furin (paired basic amino acid 5.47E-06 2.4903831 up cleaving enzyme) (FURIN), mRNA. 2529 FUT7 Homo sapiens fucosyltransferase 7 (alpha (1,3) 2.48E-03 3.273772 up fucosyltransferase) (FUT7), mRNA. 2534 FYN Homo sapiens FYN oncogene related to SRC, FGR, 5.13E-06 3.246567 down YES (FYN), transcript variant 1, mRNA.

223

2534 FYN Homo sapiens FYN oncogene related to SRC, FGR, 5.48E-06 4.2108383 down YES (FYN), transcript variant 1, mRNA. 2534 FYN Homo sapiens FYN oncogene related to SRC, FGR, 8.34E-06 2.8713717 down YES (FYN), transcript variant 3, mRNA. 2537 G1P3 Homo sapiens interferon, alpha-inducible protein 3.46E-04 2.0230227 down (clone IFI-6-16) (G1P3), transcript variant 1, mRNA.

2539 G6PD Homo sapiens glucose-6-phosphate 5.13E-06 3.3805568 up dehydrogenase (G6PD), nuclear gene encoding mitochondrial protein, mRNA. 2539 G6PD Homo sapiens glucose-6-phosphate 5.47E-06 2.9979424 up dehydrogenase (G6PD), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA. 11345 GABARAPL2 Homo sapiens GABA(A) receptor-associated 5.13E-06 2.5661192 up protein-like 2 (GABARAPL2), mRNA. 1647 GADD45A Homo sapiens growth arrest and DNA-damage- 5.27E-06 15.739641 up inducible, alpha (GADD45A), mRNA. 2581 GALC Homo sapiens galactosylceramidase (Krabbe 5.27E-06 2.274529 up disease) (GALC), mRNA. 55454 GALNACT-2 Homo sapiens chondroitin sulfate GalNAcT-2 4.51E-02 2.7099066 up (GALNACT-2), mRNA. 8693 GALNT4 Homo sapiens UDP-N-acetyl-alpha-D- 1.52E-05 3.0352993 up galactosamine:polypeptide N- acetylgalactosaminyltransferase 4 (GalNAc-T4) (GALNT4), mRNA. 2597 GAPDH Homo sapiens glyceraldehyde-3-phosphate 5.13E-06 2.756057 up dehydrogenase (GAPDH), mRNA. 2597 GAPDH Homo sapiens glyceraldehyde-3-phosphate 5.13E-06 3.2379358 up dehydrogenase (GAPDH), mRNA. 8522 GAS7 Homo sapiens growth arrest-specific 7 (GAS7), 1.17E-05 3.5803542 up transcript variant b, mRNA. 2629 GBA Homo sapiens glucosidase, beta; acid (includes 5.13E-06 4.539533 up glucosylceramidase) (GBA), transcript variant 3, mRNA. 2632 GBE1 Homo sapiens glucan (1,4-alpha-), branching 5.27E-06 3.4127076 up enzyme 1 (glycogen branching enzyme, Andersen disease, glycogen storage disease type IV) (GBE1), mRNA. 2633 GBP1 Homo sapiens guanylate binding protein 1, 2.37E-04 3.2470932 down interferon-inducible, 67kDa (GBP1), mRNA. 115361 GBP4 Homo sapiens guanylate binding protein 4 (GBP4), 2.75E-05 4.328669 down mRNA. 115362 GBP5 Homo sapiens guanylate binding protein 5 (GBP5), 3.87E-05 3.633565 down mRNA. 25801 GCA Homo sapiens grancalcin, EF-hand calcium binding 5.13E-06 4.173537 up protein (GCA), mRNA. 10985 GCN1L1 Homo sapiens GCN1 general control of amino-acid 1.52E-05 2.0828094 down synthesis 1-like 1 (yeast) (GCN1L1), mRNA. 2648 GCN5L2 Homo sapiens GCN5 general control of amino-acid 9.78E-05 2.1211884 down synthesis 5-like 2 (yeast) (GCN5L2), mRNA. 50628 GEMIN4 Homo sapiens gem (nuclear organelle) associated 5.88E-06 2.248674 down protein 4 (GEMIN4), mRNA. 23062 GGA2 Homo sapiens golgi associated, gamma adaptin ear 1.67E-05 2.2716928 down containing, ARF binding protein 2 (GGA2), mRNA.

224

8836 GGH Homo sapiens gamma-glutamyl hydrolase 2.25E-04 2.856294 up (conjugase, folylpolygammaglutamyl hydrolase) (GGH), mRNA. 55340 GIMAP5 Homo sapiens GTPase, IMAP family member 5 5.13E-06 5.042632 down (GIMAP5), mRNA. 474344 GIMAP6 Homo sapiens GTPase, IMAP family member 6 5.27E-06 2.0248408 down (GIMAP6), transcript variant 3, mRNA. 168537 GIMAP7 Homo sapiens GTPase, IMAP family member 7 4.02E-05 2.5305545 down (GIMAP7), mRNA. 10755 GIPC1 Homo sapiens GIPC PDZ domain containing family, 6.74E-06 2.4096584 down member 1 (GIPC1), transcript variant 3, mRNA.

2710 GK Homo sapiens glycerol kinase (GK), transcript 2.42E-05 4.174195 up variant 1, mRNA. 2717 GLA Homo sapiens galactosidase, alpha (GLA), mRNA. 5.13E-06 3.0528233 up 2745 GLRX Homo sapiens glutaredoxin (thioltransferase) 9.10E-06 3.4667118 up (GLRX), mRNA. 51022 GLRX2 Homo sapiens glutaredoxin 2 (GLRX2), transcript 5.35E-06 2.002984 up variant 1, mRNA. 79709 GLT25D1 Homo sapiens glycosyltransferase 25 domain 5.13E-06 3.0869725 up containing 1 (GLT25D1), mRNA. 51228 GLTP Homo sapiens glycolipid transfer protein (GLTP), 5.13E-06 2.156737 up mRNA. 29997 GLTSCR2 Homo sapiens glioma tumor suppressor candidate 5.13E-06 2.1925943 down region gene 2 (GLTSCR2), mRNA. 2760 GM2A Homo sapiens GM2 ganglioside activator (GM2A), 1.08E-02 2.3641477 up mRNA. 2764 GMFB Homo sapiens glia maturation factor, beta (GMFB), 5.13E-06 2.363177 up mRNA. 9535 GMFG Homo sapiens glia maturation factor, gamma 5.91E-06 2.7988567 up (GMFG), mRNA. 2769 GNA15 Homo sapiens guanine nucleotide binding protein 5.13E-06 4.735776 up (G protein), alpha 15 (Gq class) (GNA15), mRNA.

2773 GNAI3 Homo sapiens guanine nucleotide binding protein 6.31E-06 3.7357645 up (G protein), alpha inhibiting activity polypeptide 3 (GNAI3), mRNA. 2776 GNAQ Homo sapiens guanine nucleotide binding protein 6.13E-05 4.012052 up (G protein), q polypeptide (GNAQ), mRNA. 2783 GNB2 Homo sapiens guanine nucleotide binding protein 7.68E-06 2.3462515 up (G protein), beta polypeptide 2 (GNB2), mRNA.

2790 GNG10 Homo sapiens guanine nucleotide binding protein 1.22E-04 4.1735535 up (G protein), gamma 10 (GNG10), mRNA. 2787 GNG5 Homo sapiens guanine nucleotide binding protein 5.13E-06 3.2356536 up (G protein), gamma 5 (GNG5), mRNA. 10578 GNLY Homo sapiens granulysin (GNLY), transcript variant 6.49E-05 4.525536 down NKG5, mRNA. 10578 GNLY Homo sapiens granulysin (GNLY), transcript variant 3.50E-05 4.8709884 down 519, mRNA. 2799 GNS Homo sapiens glucosamine (N-acetyl)-6-sulfatase 1.28E-05 3.1057098 up (Sanfilippo disease IIID) (GNS), mRNA. 440270 GOLGA8B Homo sapiens golgi autoantigen, golgin subfamily 5.13E-06 3.4558465 down a, 8B (GOLGA8B), mRNA. 9570 GOSR2 Homo sapiens golgi SNAP receptor complex 1.17E-05 2.0457952 up member 2 (GOSR2), transcript variant B, mRNA.

225

151306 GPBAR1 Homo sapiens G protein-coupled bile acid receptor 2.32E-03 2.3214679 down 1 (GPBAR1), mRNA. 151306 GPBAR1 Homo sapiens G protein-coupled bile acid receptor 9.03E-05 3.1991289 down 1 (GPBAR1), transcript variant 1, mRNA. 2821 GPI Homo sapiens glucose phosphate isomerase (GPI), 1.34E-04 2.0323052 up mRNA. 221188 GPR114 Homo sapiens G protein-coupled receptor 114 1.66E-05 3.541269 down (GPR114), mRNA. 7107 GPR137B Homo sapiens G protein-coupled receptor 137B 7.19E-05 2.6216893 up (GPR137B), mRNA. 353345 GPR141 Homo sapiens G protein-coupled receptor 141 1.14E-02 3.8366795 up (GPR141), mRNA. 387129 GPR154 Homo sapiens neuropeptide S receptor 1 (NPSR1), 1.39E-03 2.4623287 down transcript variant 1, mRNA. 26996 GPR160 Homo sapiens G protein-coupled receptor 160 7.68E-06 4.6498666 up (GPR160), mRNA. 2841 GPR18 Homo sapiens G protein-coupled receptor 18 7.04E-05 3.0063965 down (GPR18), mRNA. 9289 GPR56 Homo sapiens G protein-coupled receptor 56 2.90E-05 7.2792954 down (GPR56), transcript variant 2, mRNA. 9289 GPR56 Homo sapiens G protein-coupled receptor 56 5.81E-05 6.536251 down (GPR56), transcript variant 3, mRNA. 8477 GPR65 Homo sapiens G protein-coupled receptor 65 3.87E-05 2.542324 up (GPR65), mRNA. 53831 GPR84 Homo sapiens G protein-coupled receptor 84 1.08E-02 21.183023 up (GPR84), mRNA. 57121 GPR92 Homo sapiens G protein-coupled receptor 92 5.27E-06 2.9020178 down (GPR92), mRNA. 222487 GPR97 Homo sapiens G protein-coupled receptor 97 5.91E-06 4.3022423 up (GPR97), mRNA. 222487 GPR97 Homo sapiens G protein-coupled receptor 97 3.60E-05 4.3218193 up (GPR97), mRNA. 57655 GRAMD1A Homo sapiens GRAM domain containing 1A 1.82E-05 2.3442535 up (GRAMD1A), mRNA. 10750 GRAP Homo sapiens GRB2-related adaptor protein 5.13E-06 3.9750528 down (GRAP), mRNA. 2887 GRB10 Homo sapiens growth factor receptor-bound 3.59E-02 5.036894 up protein 10 (GRB10), transcript variant 1, mRNA. 2907 GRINA Homo sapiens glutamate receptor, ionotropic, N- 5.13E-06 5.1376505 up methyl D-asparate-associated protein 1 (glutamate binding) (GRINA), transcript variant 1, mRNA.

2907 GRINA Homo sapiens glutamate receptor, ionotropic, N- 5.13E-06 4.68124 up methyl D-asparate-associated protein 1 (glutamate binding) (GRINA), transcript variant 2, mRNA.

2870 GRK6 Homo sapiens G protein-coupled receptor kinase 6 9.97E-06 2.1693056 up (GRK6), transcript variant 2, mRNA. 2870 GRK6 Homo sapiens G protein-coupled receptor kinase 6 2.63E-05 2.0858104 up (GRK6), transcript variant 2, mRNA. 2896 GRN Homo sapiens granulin (GRN), transcript variant 2, 6.88E-05 2.3178787 up mRNA. 2896 GRN Homo sapiens granulin (GRN), mRNA. 1.17E-05 2.5333452 up 55876 GSDML Homo sapiens gasdermin-like (GSDML), mRNA. 5.78E-05 2.4000099 down 2934 GSN Homo sapiens gelsolin (amyloidosis, Finnish type) 2.78E-04 2.350788 up (GSN), transcript variant 2, mRNA.

226

23708 GSPT2 Homo sapiens G1 to S phase transition 2 (GSPT2), 1.11E-04 2.0439358 down mRNA. 2944 GSTM1 Homo sapiens glutathione S-transferase M1 1.25E-02 2.3481777 down (GSTM1), transcript variant 1, mRNA. 2946 GSTM2 Homo sapiens glutathione S-transferase M2 2.56-02 2.2712932 down (muscle) (GSTM2), mRNA. 9446 GSTO1 Homo sapiens glutathione S-transferase omega 1 5.13E-06 3.0588915 up (GSTO1), mRNA. 79712 GTDC1 Homo sapiens glycosyltransferase-like domain 3.71E-03 3.36884 up containing 1 (GTDC1), transcript variant 1, mRNA. 84705 GTPBP3 Homo sapiens GTP binding protein 3 5.27E-06 2.6767316 down (mitochondrial) (GTPBP3), transcript variant V, mRNA. 29083 GTPBP8 Homo sapiens GTP-binding protein 8 (putative) 5.35E-06 2.015749 down (GTPBP8), transcript variant 1, mRNA. 375513 GUSBL2 Homo sapiens glucuronidase, beta-like 2 (GUSBL2), 2.65E-05 2.0234268 down transcript variant 3, mRNA. 2992 GYG1 Homo sapiens glycogenin 1 (GYG1), mRNA. 5.13E-06 9.672594 up 2994 GYPB Homo sapiens glycophorin B (MNS blood group) 2.68E-02 2.1582923 up (GYPB), mRNA. 3001 GZMA Homo sapiens granzyme A (granzyme 1, cytotoxic 2.09E-05 3.887896 down T-lymphocyte-associated serine esterase 3) (GZMA), mRNA. 3002 GZMB Homo sapiens granzyme B (granzyme 2, cytotoxic 9.29E-04 3.0920699 down T-lymphocyte-associated serine esterase 1) (GZMB), mRNA. 2999 GZMH Homo sapiens granzyme H (cathepsin G-like 2, 1.32E-05 6.2872043 down protein h-CCPX) (GZMH), mRNA. 3003 GZMK Homo sapiens granzyme K (granzyme 3; tryptase II) 1.67E-05 4.805156 down (GZMK), mRNA. 3004 GZMM Homo sapiens granzyme M (lymphocyte met-ase 1) 2.51E-05 4.4124956 down (GZMM), mRNA. 55766 H2AFJ Homo sapiens H2A histone family, member J 5.35E-06 3.563822 up (H2AFJ), transcript variant 2, mRNA. 55506 H2AFY2 Homo sapiens H2A histone family, member Y2 2.90E-05 2.1035583 down (H2AFY2), mRNA. 3015 H2AFZ Homo sapiens H2A histone family, member Z 5.13E-06 2.0381615 up (H2AFZ), mRNA. 54145 H2BFS Homo sapiens H2B histone family, member S 6.53E-04 2.321558 up (H2BFS), mRNA. 3033 HADHSC Homo sapiens L-3-hydroxyacyl-Coenzyme A 5.35E-06 2.2370574 down dehydrogenase, short chain (HADHSC), mRNA. 8520 HAT1 Homo sapiens histone acetyltransferase 1 (HAT1), 1.43E-05 3.015796 up transcript variant 1, mRNA. 10542 HBXIP Homo sapiens hepatitis B virus x interacting 5.13E-06 3.0590484 up protein (HBXIP), mRNA. 3055 HCK Homo sapiens hemopoietic cell kinase (HCK), 5.13E-06 3.530682 up mRNA. 10866 HCP5 Homo sapiens HLA complex P5 (HCP5), mRNA. 5.27E-06 2.039402 down 10870 HCST Homo sapiens hematopoietic cell signal transducer 1.66E-05 2.0092502 down (HCST), transcript variant 1, mRNA. 9759 HDAC4 Homo sapiens histone deacetylase 4 (HDAC4), 7.25E-06 2.7702913 up mRNA. 23593 HEBP2 Homo sapiens heme binding protein 2 (HEBP2), 5.13E-06 2.7443266 up mRNA. 51191 HERC5 Homo sapiens hect domain and RLD 5 (HERC5), 1.28E-05 3.366833 down mRNA. 227

55008 HERC6 Homo sapiens hect domain and RLD 6 (HERC6), 6.88E-05 2.8096554 down transcript variant 4, mRNA. 57801 HES4 Homo sapiens hairy and enhancer of split 4 1.58E-03 2.5995913 down (Drosophila) (HES4), mRNA. 3087 HHEX Homo sapiens hematopoietically expressed 2.20E-05 2.217241 up homeobox (HHEX), mRNA. 84641 HIATL1 Homo sapiens hippocampus abundant transcript- 7.59E-06 2.417627 up like 1 (HIATL1), mRNA. 3091 HIF1A Homo sapiens hypoxia-inducible factor 1, alpha 4.24E-05 2.4934282 up subunit (basic helix-loop-helix transcription factor) (HIF1A), transcript variant 1, mRNA. 3091 HIF1A Homo sapiens hypoxia-inducible factor 1, alpha 7.68E-06 2.9970105 up subunit (basic helix-loop-helix transcription factor) (HIF1A), transcript variant 2, mRNA. 25994 HIGD1A Homo sapiens HIG1 domain family, member 1A 7.25E-06 2.981488 up (HIGD1A), mRNA. 3092 HIP1 Homo sapiens huntingtin interacting protein 1 8.41E-04 2.9434607 up (HIP1), mRNA. 3006 HIST1H1C Homo sapiens histone cluster 1, H1c (HIST1H1C), 4.54E-04 2.0319352 up mRNA. 8334 HIST1H2AC Homo sapiens histone cluster 1, H2ac (HIST1H2AC), 3.14E-04 2.0233493 up mRNA. 3017 HIST1H2BD Homo sapiens histone cluster 1, H2bd 1.08E-05 3.1049051 up (HIST1H2BD), transcript variant 2, mRNA. 3017 HIST1H2BD Homo sapiens histone cluster 1, H2bd 8.44E-06 3.0353427 up (HIST1H2BD), transcript variant 2, mRNA. 8344 HIST1H2BE Homo sapiens histone cluster 1, H2be (HIST1H2BE), 1.47E-03 2.3153474 up mRNA. 85236 HIST1H2BK Homo sapiens histone cluster 1, H2bk (HIST1H2BK), 2.42E-05 2.2010486 up mRNA. 8351 HIST1H3D Homo sapiens histone cluster 1, H3d (HIST1H3D), 1.62E-05 2.476441 up mRNA. 8365 HIST1H4H Homo sapiens histone cluster 1, H4h (HIST1H4H), 1.11E-02 2.050118 up mRNA. 8337 HIST2H2AA3 Homo sapiens histone cluster 2, H2aa3 1.17E-05 3.6270607 up (HIST2H2AA3), mRNA. 8338 HIST2H2AC Homo sapiens histone cluster 2, H2ac (HIST2H2AC), 5.35E-06 3.8891788 up mRNA. 8349 HIST2H2BE Homo sapiens histone cluster 2, H2be (HIST2H2BE), 1.82E-05 2.9119258 up mRNA. 3099 HK2 Homo sapiens hexokinase 2 (HK2), mRNA. 9.10E-06 2.731334 up 3101 HK3 Homo sapiens hexokinase 3 (white cell) (HK3), 5.13E-06 9.296805 up nuclear gene encoding mitochondrial protein, mRNA. 3108 HLA-DMA Homo sapiens major histocompatibility complex, 7.68E-06 3.18049 down class II, DM alpha (HLA-DMA), mRNA. 3109 HLA-DMB Homo sapiens major histocompatibility complex, 5.47E-06 3.6686242 down class II, DM beta (HLA-DMB), mRNA. 3111 HLA-DOA Homo sapiens major histocompatibility complex, 1.39E-05 3.3640552 down class II, DO alpha (HLA-DOA), mRNA. 3112 HLA-DOB Homo sapiens major histocompatibility complex, 1.00E-04 2.6358018 down class II, DO beta (HLA-DOB), mRNA. 3113 HLA-DPA1 Homo sapiens major histocompatibility complex, 5.47E-06 3.927301 down class II, DP alpha 1 (HLA-DPA1), mRNA. 3115 HLA-DPB1 Homo sapiens major histocompatibility complex, 4.19E-03 2.0948849 down class II, DP beta 1 (HLA-DPB1), mRNA.

228

3117 HLA-DQA1 PREDICTED: Homo sapiens major 2.68E-05 3.730297 down histocompatibility complex, class II, DQ alpha 1, transcript variant 2 (HLA-DQA1), mRNA. 3119 HLA-DQB1 Homo sapiens major histocompatibility complex, 1.02E-04 3.3586028 down class II, DQ beta 1 (HLA-DQB1), mRNA. 3122 HLA-DRA Homo sapiens major histocompatibility complex, 1.66E-05 2.1284666 down class II, DR alpha (HLA-DRA), mRNA. 3125 HLA-DRB3 Homo sapiens major histocompatibility complex, 1.08E-05 3.6839347 down class II, DR beta 3 (HLA-DRB3), mRNA. 3128 HLA-DRB6 Homo sapiens major histocompatibility complex, 1.17E-05 3.099395 down class II, DR beta 6 (pseudogene) (HLA-DRB6) on chromosome 6. 3142 HLX1 Homo sapiens H2.0-like homeobox (HLX), mRNA. 1.76E-04 2.6222506 up 84803 HMFN0839 Homo sapiens lung cancer metastasis-associated 1.28E-05 3.180867 up protein (MAG1), mRNA. 10042 HMG2L1 Homo sapiens high-mobility group protein 2-like 1 1.05E-03 2.5495625 up (HMG2L1), transcript variant 1, mRNA. 3148 HMGB2 Homo sapiens high-mobility group box 2 (HMGB2), 5.13E-06 4.538895 up mRNA. 3156 HMGCR Homo sapiens 3-hydroxy-3-methylglutaryl- 5.27E-06 2.139759 up Coenzyme A reductase (HMGCR), mRNA. 3162 HMOX1 Homo sapiens heme oxygenase (decycling) 1 5.13E-06 3.8246636 up (HMOX1), mRNA. 664709 HNRPA1L-2 Homo sapiens heterogeneous nuclear 2.62E-05 2.2492115 down ribonucleoprotein A1 pseudogene (HNRPA1L-2) on chromosome 19. 9987 HNRPDL Homo sapiens heterogeneous nuclear 9.10E-06 2.0014539 down ribonucleoprotein D-like (HNRPDL), transcript variant 2, mRNA. 92906 HNRPLL Homo sapiens heterogeneous nuclear 9.18E-05 2.000882 up ribonucleoprotein L-like (HNRPLL), mRNA. 84376 HOOK3 Homo sapiens hook homolog 3 (Drosophila) 7.42E-03 2.0374558 up (HOOK3), mRNA. 3211 HOXB1 Homo sapiens homeo box B1 (HOXB1), mRNA. 5.13E-06 2.4493742 up 3240 HP Homo sapiens haptoglobin (HP), mRNA. 1.00E-04 56.395782 up 3248 HPGD Homo sapiens hydroxyprostaglandin 1.43E-03 9.55992 up dehydrogenase 15-(NAD) (HPGD), mRNA. 3251 HPRT1 Homo sapiens hypoxanthine 7.57E-05 2.0362668 up phosphoribosyltransferase 1 (Lesch-Nyhan syndrome) (HPRT1), mRNA. 10855 HPSE Homo sapiens heparanase (HPSE), mRNA. 3.09E-04 2.5663595 up 84263 HSDL2 Homo sapiens hydroxysteroid dehydrogenase like 1.28E-05 2.8731604 up 2 (HSDL2), mRNA. 84941 HSH2D Homo sapiens hematopoietic SH2 domain 2.86E-04 2.708776 down containing (HSH2D), mRNA. 3303 HSPA1A Homo sapiens heat shock 70kDa protein 1A 4.26E-05 2.4528234 up (HSPA1A), mRNA. 3304 HSPA1B Homo sapiens heat shock 70kDa protein 1B 4.26E-05 2.8954086 up (HSPA1B), mRNA. 29094 HSPC159 Homo sapiens galectin-related protein (HSPC159), 3.59E-02 4.072594 up mRNA. 10553 HTATIP2 Homo sapiens HIV-1 Tat interactive protein 2, 5.91E-06 3.2115207 up 30kDa (HTATIP2), mRNA. 3344 HTLF Homo sapiens human T-cell leukemia virus 5.35E-06 2.65232 up enhancer factor (HTLF), mRNA.

229

255488 IBRDC2 Homo sapiens IBR domain containing 2 (IBRDC2), 5.13E-06 3.5402398 up mRNA. 3384 ICAM2 Homo sapiens intercellular adhesion molecule 2 5.13E-06 2.3652453 down (ICAM2), mRNA. 29851 ICOS Homo sapiens inducible T-cell co-stimulator (ICOS), 1.20E-04 2.6306055 down mRNA. 3399 ID3 Homo sapiens inhibitor of DNA binding 3, 5.13E-06 3.3014498 down dominant negative helix-loop-helix protein (ID3), mRNA. 3417 IDH1 Homo sapiens isocitrate dehydrogenase 1 (NADP+), 5.13E-06 3.8925538 up soluble (IDH1), mRNA. 3422 IDI1 Homo sapiens isopentenyl-diphosphate delta 6.74E-06 7.6172905 up isomerase 1 (IDI1), mRNA. 8870 IER3 Homo sapiens immediate early response 3 (IER3), 4.90E-05 5.1915226 up transcript variant long, mRNA. 10437 IFI30 Homo sapiens interferon, gamma-inducible protein 5.91E-06 2.7071104 up 30 (IFI30), mRNA. 10561 IFI44 Homo sapiens interferon-induced protein 44 4.37E-03 2.9018476 down (IFI44), mRNA. 10964 IFI44L Homo sapiens interferon-induced protein 44-like 2.88E-02 2.9018915 down (IFI44L), mRNA. 2537 IFI6 Homo sapiens interferon, alpha-inducible protein 6 2.42E-03 2.4678319 down (IFI6), transcript variant 2, mRNA. 64135 IFIH1 Homo sapiens interferon induced with helicase C 1.86E-04 2.667858 down domain 1 (IFIH1), mRNA. 3433 IFIT2 Homo sapiens interferon-induced protein with 6.25E-05 5.4927115 down tetratricopeptide repeats 2 (IFIT2), mRNA. 3437 IFIT3 Homo sapiens interferon-induced protein with 1.13E-04 6.5571265 down tetratricopeptide repeats 3 (IFIT3), mRNA. 10581 IFITM2 Homo sapiens interferon induced transmembrane 3.50E-05 2.0075166 up protein 2 (1-8D) (IFITM2), mRNA. 10410 IFITM3 Homo sapiens interferon induced transmembrane 1.10E-03 3.0724144 up protein 3 (1-8U) (IFITM3), mRNA. 3454 IFNAR1 Homo sapiens interferon (alpha, beta and omega) 5.13E-06 2.283704 up receptor 1 (IFNAR1), mRNA. 3459 IFNGR1 Homo sapiens interferon gamma receptor 1 5.13E-06 4.2705493 up (IFNGR1), mRNA. 3460 IFNGR2 Homo sapiens interferon gamma receptor 2 5.13E-06 2.3572378 up (interferon gamma transducer 1) (IFNGR2), mRNA. 3475 IFRD1 Homo sapiens interferon-related developmental 3.14E-04 2.2098625 up regulator 1 (IFRD1), transcript variant 1, mRNA. 10643 IGF2BP3 Homo sapiens insulin-like growth factor 2 mRNA 1.29E-04 2.824558 up binding protein 3 (IGF2BP3), mRNA. 3490 IGFBP7 Homo sapiens insulin-like growth factor binding 2.85E-04 2.6079216 up protein 7 (IGFBP7), mRNA. 10261 IGSF6 Homo sapiens immunoglobulin superfamily, 2.63E-05 3.0399635 up member 6 (IGSF6), mRNA. 3588 IL10RB Homo sapiens interleukin 10 receptor, beta 5.13E-06 2.7541602 up (IL10RB), mRNA. 3590 IL11RA Homo sapiens interleukin 11 receptor, alpha 5.27E-06 2.9951108 down (IL11RA), transcript variant 1, mRNA. 3594 IL12RB1 Homo sapiens interleukin 12 receptor, beta 1 1.68E-05 2.462098 down (IL12RB1), transcript variant 2, mRNA. 23765 IL17R Homo sapiens interleukin 17 receptor (IL17R), 9.15E-05 2.2415168 up mRNA. 10068 IL18BP Homo sapiens interleukin 18 binding protein 2.09E-05 2.3088887 down (IL18BP), transcript variant A, mRNA.

230

8809 IL18R1 Homo sapiens interleukin 18 receptor 1 (IL18R1), 6.74E-06 15.37721 up mRNA. 8807 IL18RAP Homo sapiens interleukin 18 receptor accessory 5.35E-06 6.7150755 up protein (IL18RAP), mRNA. 3553 IL1B Homo sapiens interleukin 1, beta (IL1B), mRNA. 1.34E-04 2.4005222 up 7850 IL1R2 Homo sapiens interleukin 1 receptor, type II 3.71E-02 6.081163 up (IL1R2), transcript variant 1, mRNA. 7850 IL1R2 Homo sapiens interleukin 1 receptor, type II 5.13E-06 43.003864 up (IL1R2), transcript variant 2, mRNA. 3556 IL1RAP Homo sapiens interleukin 1 receptor accessory 5.35E-06 9.201493 up protein (IL1RAP), transcript variant 1, mRNA. 3556 IL1RAP Homo sapiens interleukin 1 receptor accessory 3.86E-02 6.361552 up protein (IL1RAP), transcript variant 2, mRNA. 3557 IL1RN Homo sapiens interleukin 1 receptor antagonist 6.25E-05 3.7269661 up (IL1RN), transcript variant 1, mRNA. 3557 IL1RN Homo sapiens interleukin 1 receptor antagonist 4.15E-04 3.0954857 up (IL1RN), transcript variant 1, mRNA. 50615 IL21R Homo sapiens interleukin 21 receptor (IL21R), 1.25E-05 2.160711 down transcript variant 2, mRNA. 9466 IL27RA Homo sapiens interleukin 27 receptor, alpha 5.13E-06 2.29831 down (IL27RA), mRNA. 3560 IL2RB Homo sapiens interleukin 2 receptor, beta (IL2RB), 3.40E-05 5.49469 down mRNA. 9235 IL32 Homo sapiens interleukin 32 (IL32), transcript 7.25E-06 3.5453367 down variant 3, mRNA. 9235 IL32 Homo sapiens interleukin 32 (IL32), transcript 5.13E-06 4.5582767 down variant 4, mRNA. 3566 IL4R Homo sapiens interleukin 4 receptor (IL4R), 6.25E-05 2.0086331 up transcript variant 1, mRNA. 3575 IL7R Homo sapiens interleukin 7 receptor (IL7R), mRNA. 3.87E-05 3.04446 down

3576 IL8 Homo sapiens interleukin 8 (IL8), mRNA. 1.37E-03 5.659095 up 3577 IL8RA Homo sapiens interleukin 8 receptor, alpha 2.11E-03 2.431496 up (IL8RA), mRNA. 3609 ILF3 Homo sapiens interleukin enhancer binding factor 6.14E-06 2.3132324 down 3, 90kDa (ILF3), transcript variant 1, mRNA. 3613 IMPA2 Homo sapiens inositol(myo)-1(or 4)- 5.47E-06 4.700455 up monophosphatase 2 (IMPA2), mRNA. 3614 IMPDH1 Homo sapiens IMP (inosine monophosphate) 9.10E-06 2.7492611 up dehydrogenase 1 (IMPDH1), transcript variant 1, mRNA. 3615 IMPDH2 Homo sapiens IMP (inosine monophosphate) 9.10E-06 2.2711034 down dehydrogenase 2 (IMPDH2), mRNA. 3632 INPP5A PREDICTED: Homo sapiens inositol polyphosphate- 1.34E-04 2.2702928 up 5-phosphatase, 40kDa (INPP5A), mRNA. 51141 INSIG2 Homo sapiens insulin induced gene 2 (INSIG2), 2.11E-05 2.3774726 up mRNA. 51194 IPO11 Homo sapiens importin 11 (IPO11), mRNA. 5.13E-06 4.0466456 up 8826 IQGAP1 Homo sapiens IQ motif containing GTPase 2.42E-05 2.3597813 up activating protein 1 (IQGAP1), mRNA. 3656 IRAK2 Homo sapiens interleukin-1 receptor-associated 3.75E-04 2.0473778 up kinase 2 (IRAK2), mRNA. 11213 IRAK3 Homo sapiens interleukin-1 receptor-associated 5.13E-06 14.552744 up kinase 3 (IRAK3), mRNA. 8660 IRS2 Homo sapiens insulin receptor substrate 2 (IRS2), 1.52E-05 3.6279347 up mRNA.

231

9636 ISG15 Homo sapiens ISG15 ubiquitin-like modifier 6.53E-04 2.964672 down (ISG15), mRNA. 64782 ISG20L1 Homo sapiens interferon stimulated exonuclease 3.68E-05 2.0694807 down gene 20kDa-like 1 (ISG20L1), mRNA. 51015 ISOC1 Homo sapiens isochorismatase domain containing 4.37E-04 2.0040698 down 1 (ISOC1), mRNA. 3684 ITGAM Homo sapiens integrin, alpha M (complement 5.13E-06 5.406939 up component receptor 3, alpha; also known as CD11b (p170), macrophage antigen alpha polypeptide) (ITGAM), mRNA. 3687 ITGAX Homo sapiens integrin, alpha X (complement 6.53E-04 2.201949 up component 3 receptor 4 subunit) (ITGAX), mRNA. 3695 ITGB7 Homo sapiens integrin, beta 7 (ITGB7), mRNA. 5.13E-06 4.3152237 down 3702 ITK Homo sapiens IL2-inducible T-cell kinase (ITK), 2.63E-05 2.8544672 down mRNA. 9452 ITM2A Homo sapiens integral membrane protein 2A 1.11E-04 2.8810825 down (ITM2A), mRNA. 81618 ITM2C Homo sapiens integral membrane protein 2C 1.39E-05 3.3436434 down (ITM2C), transcript variant 1, mRNA. 81618 ITM2C Homo sapiens integral membrane protein 2C 1.17E-05 3.1908162 down (ITM2C), transcript variant 2, mRNA. 3710 ITPR3 Homo sapiens inositol 1,4,5-triphosphate receptor, 5.13E-06 3.7986212 down type 3 (ITPR3), mRNA. 10625 IVNS1ABP Homo sapiens influenza virus NS1A binding protein 5.91E-06 2.5936842 up (IVNS1ABP), mRNA. 10625 IVNS1ABP Homo sapiens influenza virus NS1A binding protein 1.39E-05 2.729983 up (IVNS1ABP), mRNA. 3717 JAK2 Homo sapiens Janus kinase 2 (a protein tyrosine 2.33E-05 2.7726579 up kinase) (JAK2), mRNA. 126119 JOSD2 Homo sapiens Josephin domain containing 2 5.10E-04 2.247258 up (JOSD2), mRNA. 3726 JUNB Homo sapiens jun B proto-oncogene (JUNB), 1.17E-05 2.3996542 up mRNA. 9119 K6HF Homo sapiens keratin 75 (KRT75), mRNA. 7.57E-05 2.390776 up 84078 KBTBD7 Homo sapiens kelch repeat and BTB (POZ) domain 8.33E-05 3.0779214 up containing 7 (KBTBD7), mRNA. 56888 KCMF1 Homo sapiens potassium channel modulatory 3.93E-02 3.3099513 up factor 1 (KCMF1), mRNA. 3772 KCNJ15 Homo sapiens potassium inwardly-rectifying 7.79E-04 2.254441 up channel, subfamily J, member 15 (KCNJ15), transcript variant 2, mRNA. 3772 KCNJ15 Homo sapiens potassium inwardly-rectifying 6.50E-04 2.4360135 up channel, subfamily J, member 15 (KCNJ15), transcript variant 3, mRNA. 3759 KCNJ2 Homo sapiens potassium inwardly-rectifying 2.20E-05 3.027121 up channel, subfamily J, member 2 (KCNJ2), mRNA. 23199 KIAA0182 Homo sapiens KIAA0182 (KIAA0182), mRNA. 1.37E-03 2.0617087 down 23080 KIAA0241 Homo sapiens KIAA0241 (KIAA0241), mRNA. 1.83E-04 2.3476229 up 9710 KIAA0355 Homo sapiens KIAA0355 (KIAA0355), mRNA. 5.47E-06 2.5729055 down 23223 KIAA0690 Homo sapiens ribosomal RNA processing 12 1.01E-03 2.109059 up homolog (S. cerevisiae) (RRP12), mRNA. 22904 KIAA0963 Homo sapiens strawberry notch homolog 2 6.74E-06 2.5096743 up (Drosophila) (SBNO2), mRNA. 56261 KIAA1434 Homo sapiens hypothetical protein KIAA1434 5.13E-06 3.3259358 up (KIAA1434), mRNA.

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80256 KIAA1539 Homo sapiens KIAA1539 (KIAA1539), mRNA. 5.91E-06 2.7394087 up 57700 KIAA1600 Homo sapiens KIAA1600 (KIAA1600), mRNA. 2.63E-05 2.3203564 up 80856 KIAA1715 Homo sapiens KIAA1715 (KIAA1715), mRNA. 3.36E-05 2.4645565 up 57498 KIDINS220 Homo sapiens kinase D-interacting substrate of 5.69E-05 2.167831 up 220 kDa (KIDINS220), mRNA. 23095 KIF1B Homo sapiens kinesin family member 1B (KIF1B), 2.87E-05 3.790065 up transcript variant 2, mRNA. 23095 KIF1B Homo sapiens kinesin family member 1B (KIF1B), 7.25E-06 4.390986 up transcript variant 1, mRNA. 3799 KIF5B Homo sapiens kinesin family member 5B (KIF5B), 1.00E-05 2.1791384 up mRNA. 22920 KIFAP3 Homo sapiens kinesin-associated protein 3 4.51E-02 2.1541207 down (KIFAP3), mRNA. 54758 KLHDC4 Homo sapiens kelch domain containing 4 1.32E-05 2.488812 down (KLHDC4), mRNA. 11275 KLHL2 Homo sapiens kelch-like 2, Mayven (Drosophila) 5.27E-06 7.49346 up (KLHL2), mRNA. 84861 KLHL22 Homo sapiens kelch-like 22 (Drosophila) (KLHL22), 5.13E-06 2.5531943 down mRNA. 26249 KLHL3 Homo sapiens kelch-like 3 (Drosophila) (KLHL3), 6.15E-03 3.6103013 down mRNA. 57563 KLHL8 Homo sapiens kelch-like 8 (Drosophila) (KLHL8), 5.35E-06 3.3154922 up mRNA. 3820 KLRB1 Homo sapiens killer cell lectin-like receptor 9.97E-06 4.9018617 down subfamily B, member 1 (KLRB1), mRNA. 3824 KLRD1 Homo sapiens killer cell lectin-like receptor 4.04E-05 2.783256 down subfamily D, member 1 (KLRD1), transcript variant 1, mRNA. 51348 KLRF1 Homo sapiens killer cell lectin-like receptor 1.92E-05 4.0498667 down subfamily F, member 1 (KLRF1), mRNA. 3840 KPNA4 Homo sapiens karyopherin alpha 4 (importin alpha 9.97E-06 2.3995998 up 3) (KPNA4), mRNA. 3845 KRAS Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral 8.44E-06 2.2416167 up oncogene homolog (KRAS), transcript variant b, mRNA. 83999 KREMEN1 Homo sapiens kringle containing transmembrane 3.70E-02 2.5115707 up protein 1 (KREMEN1), transcript variant 1, mRNA. 337973 KRTAP19-6 Homo sapiens keratin associated protein 19-6 1.01E-04 2.9269497 up (KRTAP19-6), mRNA. 83888 KSP37 Homo sapiens fibroblast growth factor binding 1.66E-05 6.4447947 down protein 2 (FGFBP2), mRNA. 83746 L3MBTL2 Homo sapiens l(3)mbt-like 2 (Drosophila) 5.13E-06 2.0660145 down (L3MBTL2), mRNA. 114294 LACTB Homo sapiens lactamase, beta (LACTB), nuclear 4.04E-04 2.121644 up gene encoding mitochondrial protein, transcript variant 2, mRNA. 3903 LAIR1 Homo sapiens leukocyte-associated Ig-like receptor 1.52E-05 3.1167188 up 1 (LAIR1), transcript variant c, mRNA. 3920 LAMP2 Homo sapiens lysosomal-associated membrane 1.17E-05 2.3016438 up protein 2 (LAMP2), transcript variant LAMP2B, mRNA. 3920 LAMP2 Homo sapiens lysosomal-associated membrane 9.29E-04 2.1473026 up protein 2 (LAMP2), transcript variant LAMP2A, mRNA. 10314 LANCL1 Homo sapiens LanC lantibiotic synthetase 9.10E-06 2.3512146 down component C-like 1 (bacterial) (LANCL1), mRNA.

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9741 LAPTM4A Homo sapiens lysosomal-associated protein 6.88E-05 2.215206 up transmembrane 4 alpha (LAPTM4A), mRNA. 27040 LAT Homo sapiens linker for activation of T cells (LAT), 3.50E-05 2.6552627 down transcript variant 2, mRNA. 7462 LAT2 Homo sapiens linker for activation of T cells family, 5.47E-06 2.9489973 up member 2 (LAT2), transcript variant 1, mRNA.

7462 LAT2 Homo sapiens linker for activation of T cells family, 5.13E-06 2.945858 up member 2 (LAT2), transcript variant 1, mRNA.

54900 LAX1 Homo sapiens lymphocyte transmembrane 6.31E-06 3.5568678 down adaptor 1 (LAX1), mRNA. 81606 LBH Homo sapiens limb bud and heart development 5.27E-06 3.9515767 down homolog (mouse) (LBH), mRNA. 3930 LBR Homo sapiens lamin B receptor (LBR), transcript 2.37E-04 2.1497772 up variant 1, mRNA. 3932 LCK Homo sapiens lymphocyte-specific protein tyrosine 2.18E-05 3.151416 down kinase (LCK), transcript variant 1, mRNA. 3934 LCN2 Homo sapiens lipocalin 2 (oncogene 24p3) (LCN2), 1.17E-05 33.34258 up mRNA. 3939 LDHA Homo sapiens lactate dehydrogenase A (LDHA), 5.13E-06 4.387206 up mRNA. 3945 LDHB Homo sapiens lactate dehydrogenase B (LDHB), 6.74E-06 2.9264965 down mRNA. 3949 LDLR Homo sapiens low density lipoprotein receptor 2.02E-05 2.6210842 up (familial hypercholesterolemia) (LDLR), mRNA. 26119 LDLRAP1 Homo sapiens low density lipoprotein receptor 3.82E-04 2.3112795 down adaptor protein 1 (LDLRAP1), mRNA. 51176 LEF1 Homo sapiens lymphoid enhancer-binding factor 1 1.17E-05 4.554513 down (LEF1), mRNA. 79143 LENG4 Homo sapiens leukocyte receptor cluster (LRC) 3.87E-05 2.7409623 up member 4 (LENG4), mRNA. 54741 LEPROT Homo sapiens leptin receptor overlapping 4.26E-05 2.0608938 up transcript (LEPROT), mRNA. 3956 LGALS1 Homo sapiens lectin, galactoside-binding, soluble, 5.13E-06 4.3865237 up 1 (galectin 1) (LGALS1), mRNA. 3964 LGALS8 Homo sapiens lectin, galactoside-binding, soluble, 6.74E-06 2.463588 up 8 (galectin 8) (LGALS8), transcript variant 1, mRNA.

3964 LGALS8 Homo sapiens lectin, galactoside-binding, soluble, 5.35E-06 2.5453434 up 8 (galectin 8) (LGALS8), transcript variant 4, mRNA.

64077 LHPP Homo sapiens phospholysine phosphohistidine 7.57E-05 2.0409107 down inorganic pyrophosphate phosphatase (LHPP), mRNA. 11026 LILRA3 Homo sapiens leukocyte immunoglobulin-like 1.17E-05 8.7037945 up receptor, subfamily A (without TM domain), member 3 (LILRA3), mRNA. 11026 LILRA3 Homo sapiens leukocyte immunoglobulin-like 9.37E-05 4.8643785 up receptor, subfamily A (without TM domain), member 3 (LILRA3), mRNA. 353514 LILRA5 Homo sapiens leukocyte immunoglobulin-like 6.17E-06 11.198167 up receptor, subfamily A (with TM domain), member 5 (LILRA5), transcript variant 4, mRNA. 353514 LILRA5 Homo sapiens leukocyte immunoglobulin-like 5.13E-06 11.616155 up receptor, subfamily A (with TM domain), member 5 (LILRA5), transcript variant 1, mRNA.

234

353514 LILRA5 Homo sapiens leukocyte immunoglobulin-like 5.13E-06 5.443482 up receptor, subfamily A (with TM domain), member 5 (LILRA5), transcript variant 1, mRNA. 10859 LILRB1 Homo sapiens leukocyte immunoglobulin-like 3.27E-03 2.1504884 up receptor, subfamily B (with TM and ITIM domains), member 1 (LILRB1), mRNA. 10288 LILRB2 Homo sapiens leukocyte immunoglobulin-like 1.08E-05 2.9256861 up receptor, subfamily B (with TM and ITIM domains), member 2 (LILRB2), transcript variant 2, mRNA.

11025 LILRB3 Homo sapiens leukocyte immunoglobulin-like 7.25E-06 3.2037485 up receptor, subfamily B (with TM and ITIM domains), member 3 (LILRB3), mRNA. 11006 LILRB4 Homo sapiens leukocyte immunoglobulin-like 3.62E-05 2.7389982 up receptor, subfamily B (with TM and ITIM domains), member 4 (LILRB4), transcript variant 2, mRNA.

54923 LIME1 Homo sapiens Lck interacting transmembrane 5.13E-06 4.7727413 down adaptor 1 (LIME1), mRNA. 3985 LIMK2 Homo sapiens LIM domain kinase 2 (LIMK2), 9.35E-05 3.3913217 up transcript variant 1, mRNA. 8825 LIN7A Homo sapiens lin-7 homolog A (C. elegans) (LIN7A), 1.08E-02 5.788392 up mRNA. 4001 LMNB1 Homo sapiens lamin B1 (LMNB1), mRNA. 5.69E-05 2.468467 up 116236 LOC116236 Homo sapiens hypothetical protein LOC116236 1.64E-04 2.068167 down (LOC116236), mRNA. 130355 LOC130355 Homo sapiens hypothetical protein LOC130355 2.39E-04 2.1371233 up (LOC130355), mRNA. 152195 LOC152195 Homo sapiens hypothetical protein LOC152195 7.4E-03 2.180511 up (LOC152195), mRNA. 152485 LOC152485 Homo sapiens hypothetical protein LOC152485 2.18E-05 2.6333103 down (LOC152485), mRNA. 283345 LOC283345 Homo sapiens RPL13-2 pseudogene (LOC283345) 1.52E-05 2.3650246 down on chromosome 12. 349114 LOC349114 Homo sapiens hypothetical protein LOC349114 2.37E-04 2.696288 up (LOC349114), mRNA. 400924 LOC400924 Homo sapiens hypothetical gene supported by 7.82E-03 2.0152562 up AK056895 (LOC400924), mRNA. 400986 LOC400986 Homo sapiens protein immuno-reactive with anti- 5.73E-04 2.4265492 down PTH polyclonal antibodies (LOC400986), mRNA. 401152 LOC401152 Homo sapiens HCV F-transactivated protein 1 5.91E-06 2.2763355 up (LOC401152), mRNA. 401233 LOC401233 Homo sapiens similar to HIV TAT specific factor 1; 1.81E-03 2.783016 up cofactor required for Tat activation of HIV-1 transcription (LOC401233), mRNA. 401357 LOC401357 Homo sapiens hypothetical LOC401357 2.86E-04 2.3805585 up (LOC401357), mRNA. 440348 LOC440348 Homo sapiens similar to nuclear pore complex 1.28E-05 2.0378263 down interacting protein (LOC440348), mRNA. 440686 LOC440686 Homo sapiens similar to histone H2B histone family 4.69E-05 2.5389736 up (LOC440686), mRNA. 440926 LOC440926 Homo sapiens H3 histone, family 3A pseudogene 1.62E-04 2.1827402 up (LOC440926) on chromosome 2. 441743 LOC441743 Homo sapiens similar to C367G8.3 (novel protein 3.14E-04 2.4742877 down similar to RPL23A (60S ribosomal protein L23A)) (LOC441743), mRNA.

235

497661 LOC497661 Homo sapiens chromosome 18 open reading frame 5.27E-06 3.9173088 up 32 (C18orf32), mRNA. 57149 LOC57149 Homo sapiens hypothetical protein A-211C6.1 5.13E-06 3.2778876 up (LOC57149), mRNA. 643940 LOC643940 PREDICTED: Homo sapiens similar to HIG1 domain 5.13E-06 2.5235357 up family member 1A (Hypoxia-inducible gene 1 protein) (LOC643940), mRNA. 648293 LOC648293 PREDICTED: Homo sapiens similar to RNA-binding 5.13E-06 2.9746206 up motif, single-stranded interacting protein 1 (Single- stranded DNA-binding protein MSSP-1) (Suppressor of CDC2 with RNA binding motif 2), transcript variant 2 (LOC648293), mRNA. 649946 LOC649946 Homo sapiens ribosomal protein L23a pseudogene 4.69E-05 2.1075044 down (LOC649946) on chromosome 11. 653745 LOC653745 PREDICTED: Homo sapiens similar to 5.27E-06 2.370234 up phosphoprotein associated with glycosphingolipid microdomains 1 (LOC653745), mRNA.

92017 LOC92017 Homo sapiens similar to RIKEN cDNA 4933437K13 4.47E-05 2.4074538 down (LOC92017), mRNA. 93622 LOC93622 Homo sapiens hypothetical protein BC006130 1.12E-05 2.2445498 down (LOC93622), mRNA. 23175 LPIN1 Homo sapiens lipin 1 (LPIN1), mRNA. 8.44E-06 2.6209936 down 64748 LPPR2 Homo sapiens lipid phosphate phosphatase-related 9.15E-05 2.3464527 up protein type 2 (LPPR2), mRNA. 987 LRBA Homo sapiens LPS-responsive vesicle trafficking, 9.15E-05 2.2243772 down beach and anchor containing (LRBA), mRNA. 79414 LRFN3 Homo sapiens leucine rich repeat and fibronectin 1.54E-02 2.4170325 down type III domain containing 3 (LRFN3), mRNA. 116844 LRG1 Homo sapiens leucine-rich alpha-2-glycoprotein 1 5.13E-06 7.3497386 up (LRG1), mRNA. 26018 LRIG1 Homo sapiens leucine-rich repeats and 1.08E-05 2.5177758 down immunoglobulin-like domains 1 (LRIG1), mRNA. 4043 LRPAP1 Homo sapiens low density lipoprotein receptor- 1.64E-05 3.3948061 up related protein associated protein 1 (LRPAP1), mRNA. 126364 LRRC25 Homo sapiens leucine rich repeat containing 25 8.44E-06 3.1618698 up (LRRC25), mRNA. 9209 LRRFIP2 Homo sapiens leucine rich repeat (in FLII) 5.13E-06 3.2521398 up interacting protein 2 (LRRFIP2), transcript variant 2, mRNA. 9209 LRRFIP2 Homo sapiens leucine rich repeat (in FLII) 5.27E-06 2.8964818 up interacting protein 2 (LRRFIP2), transcript variant 1, mRNA. 54674 LRRN3 Homo sapiens leucine rich repeat neuronal 3 4.60E-03 4.3025613 down (LRRN3), mRNA. 7940 LST1 Homo sapiens leukocyte specific transcript 1 7.68E-06 2.461821 up (LST1), transcript variant 5, mRNA. 7940 LST1 Homo sapiens leukocyte specific transcript 1 2.86E-04 2.084269 up (LST1), transcript variant 1, mRNA. 7940 LST1 Homo sapiens leukocyte specific transcript 1 6.74E-06 2.489791 up (LST1), transcript variant 2, mRNA. 4049 LTA Homo sapiens lymphotoxin alpha (TNF superfamily, 7.25E-06 3.170064 down member 1) (LTA), mRNA. 4048 LTA4H Homo sapiens leukotriene A4 hydrolase (LTA4H), 5.98E-04 2.0738096 up mRNA.

236

4050 LTB Homo sapiens lymphotoxin beta (TNF superfamily, 6.31E-06 2.1910212 down member 3) (LTB), transcript variant 1, mRNA.

1241 LTB4R Homo sapiens leukotriene B4 receptor (LTB4R), 1.46E-05 7.688201 up mRNA. 4054 LTBP3 Homo sapiens latent transforming growth factor 1.68E-04 2.2814333 down beta binding protein 3 (LTBP3), mRNA. 4055 LTBR Homo sapiens lymphotoxin beta receptor (TNFR 1.52E-05 2.398385 up superfamily, member 3) (LTBR), mRNA. 4061 LY6E Homo sapiens lymphocyte antigen 6 complex, 1.28E-05 4.5243306 down locus E (LY6E), mRNA. 4062 LY6H Homo sapiens lymphocyte antigen 6 complex, 6.88E-05 2.0198467 up locus H (LY6H), mRNA. 4063 LY9 Homo sapiens lymphocyte antigen 9 (LY9), 3.38E-04 2.243713 down transcript variant 1, mRNA. 4063 LY9 Homo sapiens lymphocyte antigen 9 (LY9), 4.04E-05 5.2320733 down transcript variant 2, mRNA. 23643 LY96 Homo sapiens lymphocyte antigen 96 (LY96), 1.66E-05 4.751109 up mRNA. 55646 LYAR Homo sapiens Ly1 antibody reactive homolog 5.47E-06 2.1611404 down (mouse) (LYAR), mRNA. 145748 LYSMD4 Homo sapiens LysM, putative peptidoglycan- 1.06E-05 2.0743074 down binding, domain containing 4 (LYSMD4), mRNA. 10226 M6PRBP1 Homo sapiens mannose-6-phosphate receptor 5.13E-06 3.590438 up binding protein 1 (M6PRBP1), mRNA. 10459 MAD2L2 Homo sapiens MAD2 mitotic arrest deficient-like 2 5.47E-06 2.1591587 up (yeast) (MAD2L2), mRNA. 9935 MAFB Homo sapiens v-maf musculoaponeurotic 2.08E-04 2.363313 up fibrosarcoma oncogene homolog B (avian) (MAFB), mRNA. 4097 MAFG Homo sapiens v-maf musculoaponeurotic 3.71E-03 3.8623326 up fibrosarcoma oncogene homolog G (avian) (MAFG), transcript variant 1, mRNA. 9500 MAGED1 Homo sapiens melanoma antigen family D, 1 5.13E-06 3.0279837 down (MAGED1), transcript variant 1, mRNA. 4117 MAK Homo sapiens male germ cell-associated kinase 9.1E-03 2.1963909 up (MAK), mRNA. 4118 MAL Homo sapiens mal, T-cell differentiation protein 1.12E-05 3.9078624 down (MAL), transcript variant a, mRNA. 4118 MAL Homo sapiens mal, T-cell differentiation protein 5.13E-06 4.6572356 down (MAL), transcript variant d, mRNA. 4121 MAN1A1 Homo sapiens mannosidase, alpha, class 1A, 5.13E-06 2.6231341 up member 1 (MAN1A1), mRNA. 4122 MAN2A2 Homo sapiens mannosidase, alpha, class 2A, 7.57E-05 2.2634175 up member 2 (MAN2A2), mRNA. 54682 MANSC1 Homo sapiens MANSC domain containing 1 1.19E-05 4.713355 up (MANSC1), mRNA. 84557 MAP1LC3A Homo sapiens microtubule-associated protein 1 2.63E-05 2.5346806 up light chain 3 alpha (MAP1LC3A), transcript variant 2, mRNA. 81631 MAP1LC3B Homo sapiens microtubule-associated protein 1 5.47E-06 2.3305001 up light chain 3 beta (MAP1LC3B), mRNA. 5604 MAP2K1 Homo sapiens mitogen-activated protein kinase 7.68E-06 2.0153897 up kinase 1 (MAP2K1), mRNA. 8649 MAP2K1IP1 Homo sapiens mitogen-activated protein kinase 5.13E-06 3.926849 up kinase 1 interacting protein 1 (MAP2K1IP1), mRNA.

237

1326 MAP3K8 Homo sapiens mitogen-activated protein kinase 5.27E-06 2.972384 up kinase kinase 8 (MAP3K8), mRNA. 11184 MAP4K1 Homo sapiens mitogen-activated protein kinase 5.13E-06 4.119623 down kinase kinase kinase 1 (MAP4K1), mRNA. 5594 MAPK1 Homo sapiens mitogen-activated protein kinase 1 1.08E-05 2.282172 up (MAPK1), transcript variant 2, mRNA. 5594 MAPK1 Homo sapiens mitogen-activated protein kinase 1 2.20E-05 2.4498398 up (MAPK1), transcript variant 2, mRNA. 1432 MAPK14 Homo sapiens mitogen-activated protein kinase 14 3.55E-04 3.5409217 up (MAPK14), transcript variant 3, mRNA. 1432 MAPK14 Homo sapiens mitogen-activated protein kinase 14 1.18E-03 3.5875697 up (MAPK14), transcript variant 1, mRNA. 5595 MAPK3 Homo sapiens mitogen-activated protein kinase 3 2.37E-04 2.1232176 up (MAPK3), transcript variant 2, mRNA. 9261 MAPKAPK2 Homo sapiens mitogen-activated protein kinase- 7.25E-06 2.4520648 up activated protein kinase 2 (MAPKAPK2), transcript variant 2, mRNA. 7867 MAPKAPK3 Homo sapiens mitogen-activated protein kinase- 5.13E-06 2.0537193 up activated protein kinase 3 (MAPKAPK3), mRNA. 22919 MAPRE1 Homo sapiens microtubule-associated protein, 5.13E-06 2.0557313 up RP/EB family, member 1 (MAPRE1), mRNA. 22924 MAPRE3 Homo sapiens microtubule-associated protein, 6.27E-04 2.0587041 up RP/EB family, member 3 (MAPRE3), mRNA. 4082 MARCKS Homo sapiens myristoylated alanine-rich protein 4.26E-05 2.4990919 up kinase C substrate (MARCKS), mRNA. 65108 MARCKSL1 Homo sapiens MARCKS-like 1 (MARCKSL1), mRNA. 2.37E-04 2.2064352 down 8685 MARCO Homo sapiens macrophage receptor with 3.6E-03 7.2993226 up collagenous structure (MARCO), mRNA. 4145 MATK Homo sapiens megakaryocyte-associated tyrosine 5.13E-06 6.6152873 down kinase (MATK), transcript variant 3, mRNA. 199675 MCEMP1 Homo sapiens mast cell-expressed membrane 5.13E-06 34.225372 up protein 1 (MCEMP1), mRNA. 4172 MCM3 Homo sapiens MCM3 minichromosome 6.74E-06 2.4873135 down maintenance deficient 3 (S. cerevisiae) (MCM3), mRNA. 4176 MCM7 Homo sapiens minichromosome maintenance 1.66E-05 2.0628264 down complex component 7 (MCM7), transcript variant 2, mRNA. 57192 MCOLN1 Homo sapiens mucolipin 1 (MCOLN1), mRNA. 8.52E-04 2.3145444 up 79772 MCTP1 Homo sapiens multiple C2 domains, 2.42E-05 3.7156835 up transmembrane 1 (MCTP1), transcript variant S, mRNA. 55784 MCTP2 Homo sapiens multiple C2 domains, 4.49E-02 2.0433655 up transmembrane 2 (MCTP2), mRNA. 55846 MDS028 Homo sapiens integrin alpha FG-GAP repeat 5.13E-06 2.5022576 down containing 2 (ITFG2), mRNA. 4200 ME2 Homo sapiens malic enzyme 2, NAD(+)-dependent, 7.68E-06 2.3333006 up mitochondrial (ME2), nuclear gene encoding mitochondrial protein, mRNA. 4205 MEF2A Homo sapiens myocyte enhancer factor 2A 02.08E-03 2.0866954 up (MEF2A), mRNA. 4208 MEF2C Homo sapiens myocyte enhancer factor 2C 4.26E-05 2.4912832 down (MEF2C), mRNA. 4209 MEF2D Homo sapiens myocyte enhancer factor 2D 3.90E-05 2.2114096 down (MEF2D), mRNA. 1955 MEGF9 Homo sapiens multiple EGF-like-domains 9 1.39E-05 3.4256182 up (MEGF9), mRNA. 238

284207 METRNL PREDICTED: Homo sapiens meteorin, glial cell 1.08E-04 2.3629737 up differentiation regulator-like (METRNL), mRNA. 51108 METTL9 Homo sapiens methyltransferase like 9 (METTL9), 1.82E-05 3.223023 up transcript variant 2, mRNA. 4242 MFNG Homo sapiens MFNG O-fucosylpeptide 3-beta-N- 5.27E-06 2.3311167 down acetylglucosaminyltransferase (MFNG), mRNA. 64747 MFSD1 Homo sapiens major facilitator superfamily domain 5.13E-06 2.8031132 up containing 1 (MFSD1), mRNA. 8972 MGAM Homo sapiens maltase-glucoamylase (alpha- 2.09E-02 3.1408262 up glucosidase) (MGAM), mRNA. 11320 MGAT4A Homo sapiens mannosyl (alpha-1,3-)-glycoprotein 6.88E-05 2.1486535 up beta-1,4-N-acetylglucosaminyltransferase, isoenzyme A (MGAT4A), mRNA. 84798 MGC13170 Homo sapiens chromosome 19 open reading frame 5.31E-05 2.071528 down 48 (C19orf48), mRNA. 84981 MGC14376 Homo sapiens hypothetical protein MGC14376 4.78E-04 2.395438 up (MGC14376), transcript variant 2, mRNA. 124565 MGC15523 Homo sapiens hypothetical protein MGC15523 5.27E-06 2.5845926 up (MGC15523), mRNA. 84329 MGC15619 Homo sapiens hypothetical protein MGC15619 7.25E-06 2.557482 down (MGC15619), mRNA. 113791 MGC17330 Homo sapiens phosphoinositide-3-kinase 1.66E-05 2.6615613 down interacting protein 1 (PIK3IP1), mRNA. 147015 MGC23280 Homo sapiens dehydrogenase/reductase (SDR 2.78E-02 2.5649884 up family) member 13 (DHRS13), mRNA. 153339 MGC23909 Homo sapiens hypothetical protein MGC23909 5.27E-06 4.853771 up (MGC23909), mRNA. 79037 MGC2463 Homo sapiens poliovirus receptor related 5.13E-06 5.0382767 down immunoglobulin domain containing (PVRIG), mRNA. 80776 MGC4093 Homo sapiens hypothetical protein MGC4093 5.17E-05 2.2752244 up (MGC4093), mRNA. 115752 MGC4562 Homo sapiens DIS3 mitotic control homolog (S. 5.27E-06 2.0363624 down cerevisiae)-like (DIS3L), mRNA. 112597 MGC4677 Homo sapiens hypothetical protein MGC4677 7.25E-06 3.2602782 up (MGC4677), mRNA. 196383 MGC7036 Homo sapiens hypothetical protein MGC7036 7.25E-06 2.4407103 up (MGC7036), mRNA. 4255 MGMT Homo sapiens O-6-methylguanine-DNA 2.02E-05 2.0105166 down methyltransferase (MGMT), mRNA. 4257 MGST1 Homo sapiens microsomal glutathione S- 9.10E-06 3.8062382 up transferase 1 (MGST1), transcript variant 1a, mRNA. 64780 MICAL1 Homo sapiens microtubule associated 1.66E-05 2.2026234 up monoxygenase, calponin and LIM domain containing 1 (MICAL1), mRNA. 57708 MIER1 Homo sapiens mesoderm induction early response 1.39E-05 2.1699524 up 1 homolog (Xenopus laevis) (MIER1), mRNA.

8569 MKNK1 Homo sapiens MAP kinase interacting 5.13E-06 6.2824764 up serine/threonine kinase 1 (MKNK1), transcript variant 1, mRNA. 8079 MLF2 Homo sapiens myeloid leukemia factor 2 (MLF2), 5.69E-05 2.0656548 up mRNA. 197259 MLKL Homo sapiens mixed lineage kinase domain-like 1.17E-05 2.4420292 up (MLKL), mRNA.

239

4302 MLLT6 Homo sapiens myeloid/lymphoid or mixed-lineage 1.28E-05 2.1529853 down leukemia (trithorax homolog, Drosophila); translocated to, 6 (MLLT6), mRNA. 55711 MLSTD1 Homo sapiens male sterility domain containing 1 5.91E-06 5.840736 up (MLSTD1), mRNA. 84188 MLSTD2 Homo sapiens male sterility domain containing 2 2.20E-05 2.1893146 up (MLSTD2), mRNA. 6945 MLX Homo sapiens MAX-like protein X (MLX), transcript 6.05E-05 2.215094 up variant 2, mRNA. 6945 MLX Homo sapiens MAX-like protein X (MLX), transcript 2.09E-05 2.09683 up variant 1, mRNA. 4311 MME Homo sapiens membrane metallo-endopeptidase 1.21E-02 2.605895 down (neutral endopeptidase, enkephalinase, CALLA, CD10) (MME), transcript variant 2a, mRNA.

10893 MMP24 Homo sapiens matrix metallopeptidase 24 3.71E-02 2.0768743 up (membrane-inserted) (MMP24), mRNA. 64386 MMP25 Homo sapiens matrix metallopeptidase 25 2.76E-03 2.3393626 up (MMP25), transcript variant 2, mRNA. 4318 MMP9 Homo sapiens matrix metallopeptidase 9 1.22E-04 7.122397 up (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) (MMP9), mRNA. 64757 MOSC1 Homo sapiens MOCO sulphurase C-terminal 1.47E-04 3.456341 up domain containing 1 (MOSC1), mRNA. 158747 MOSPD2 Homo sapiens motile sperm domain containing 2 1.33E-05 3.45536 up (MOSPD2), mRNA. 4343 MOV10 Homo sapiens Mov10, Moloney leukemia virus 10, 7.73E-04 2.2217736 down homolog (mouse) (MOV10), mRNA. 4354 MPP1 Homo sapiens membrane protein, palmitoylated 1, 2.42E-05 2.498742 up 55kDa (MPP1), mRNA. 9019 MPZL1 Homo sapiens myelin protein zero-like 1 (MPZL1), 2.15E-02 2.0017543 up transcript variant 2, mRNA. 3140 MR1 Homo sapiens major histocompatibility complex, 5.91E-06 2.514702 up class I-related (MR1), mRNA. 10627 MRCL3 Homo sapiens myosin regulatory light chain MRCL3 9.10E-06 2.3504286 up (MRCL3), mRNA. 23164 M-RIP Homo sapiens myosin phosphatase-Rho interacting 6.74E-06 2.3044934 down protein (M-RIP), transcript variant 2, mRNA.

103910 MRLC2 Homo sapiens myosin regulatory light chain MRLC2 5.13E-06 2.2829878 up (MRLC2), mRNA. 9553 MRPL33 Homo sapiens mitochondrial ribosomal protein L33 5.13E-06 2.1818852 up (MRPL33), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA. 84311 MRPL45 Homo sapiens mitochondrial ribosomal protein L45 7.25E-06 2.4148643 down (MRPL45), nuclear gene encoding mitochondrial protein, mRNA. 28973 MRPS18B Homo sapiens mitochondrial ribosomal protein 5.13E-06 2.0923276 down S18B (MRPS18B), nuclear gene encoding mitochondrial protein, mRNA. 10335 MRVI1 Homo sapiens murine retrovirus integration site 1 1.2E-03 2.9061346 up homolog (MRVI1), transcript variant 2, mRNA. 51338 MS4A4A Homo sapiens membrane-spanning 4-domains, 1.00E-04 14.504632 up subfamily A, member 4 (MS4A4A), transcript variant 2, mRNA.

240

64231 MS4A6A Homo sapiens membrane-spanning 4-domains, 2.02E-05 2.68408 up subfamily A, member 6A (MS4A6A), transcript variant 3, mRNA. 64231 MS4A6A Homo sapiens membrane-spanning 4-domains, 3.80E-04 2.3065014 up subfamily A, member 6A (MS4A6A), transcript variant 3, mRNA. 64231 MS4A6A Homo sapiens membrane-spanning 4-domains, 8.52E-04 2.201362 up subfamily A, member 6A (MS4A6A), transcript variant 2, mRNA. 10943 MSL3L1 Homo sapiens male-specific lethal 3-like 1 6.40E-05 2.547183 up (Drosophila) (MSL3L1), transcript variant 2, mRNA. 10943 MSL3L1 Homo sapiens male-specific lethal 3-like 1 1.84E-03 2.0834074 up (Drosophila) (MSL3L1), transcript variant 2, mRNA. 10943 MSL3L1 Homo sapiens male-specific lethal 3-like 1 2.37E-04 2.017259 up (Drosophila) (MSL3L1), transcript variant 4, mRNA. 4482 MSRA Homo sapiens methionine sulfoxide reductase A 7.68E-06 2.7363937 up (MSRA), mRNA. 22921 MSRB2 Homo sapiens methionine sulfoxide reductase B2 7.68E-06 3.0750158 up (MSRB2), mRNA. 9112 MTA1 Homo sapiens metastasis associated 1 (MTA1), 5.13E-06 2.1497588 down mRNA. 4520 MTF1 Homo sapiens metal-regulatory transcription factor 5.35E-06 2.7945697 up 1 (MTF1), mRNA. 10797 MTHFD2 Homo sapiens methylenetetrahydrofolate 1.43E-05 2.7215648 up dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2), nuclear gene encoding mitochondrial protein, transcript variant 2, mRNA. 10797 MTHFD2 Homo sapiens methylenetetrahydrofolate 1.08E-05 3.0921478 up dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2), nuclear gene encoding mitochondrial protein, mRNA. 10588 MTHFS Homo sapiens 5,10-methenyltetrahydrofolate 5.13E-06 3.9467278 up synthetase (5-formyltetrahydrofolate cyclo-ligase) (MTHFS), mRNA. 8897 MTMR3 Homo sapiens myotubularin related protein 3 9.10E-06 2.5437033 up (MTMR3), transcript variant 1, mRNA. 9107 MTMR6 Homo sapiens myotubularin related protein 6 7.68E-06 2.627167 up (MTMR6), mRNA. 4580 MTX1 Homo sapiens metaxin 1 (MTX1), transcript variant 5.27E-06 2.3217897 up 1, mRNA. 4580 MTX1 Homo sapiens metaxin 1 (MTX1), transcript variant 8.44E-06 2.179208 up 1, mRNA. 4599 MX1 Homo sapiens myxovirus (influenza virus) 2.02E-05 4.646171 down resistance 1, interferon-inducible protein p78 (mouse) (MX1), mRNA. 4084 MXD1 Homo sapiens MAX dimerization protein 1 (MXD1), 1.66E-05 2.5458531 up mRNA. 83463 MXD3 Homo sapiens MAX dimerization protein 3 (MXD3), 4.54E-04 2.3566873 up mRNA. 10608 MXD4 Homo sapiens MAX dimerization protein 4 (MXD4), 5.13E-06 2.4440863 down mRNA. 4607 MYBPC3 Homo sapiens myosin binding protein C, cardiac 2.71E-04 2.8825052 up (MYBPC3), mRNA. 4609 MYC Homo sapiens v-myc myelocytomatosis viral 2.61E-04 2.1767058 down oncogene homolog (avian) (MYC), mRNA.

241

4615 MYD88 Homo sapiens myeloid differentiation primary 5.27E-06 2.5324733 up response gene (88) (MYD88), mRNA. 4637 MYL6 Homo sapiens myosin, light chain 6, alkali, smooth 5.27E-06 3.1343985 up muscle and non-muscle (MYL6), transcript variant 1, mRNA. 4637 MYL6 Homo sapiens myosin, light polypeptide 6, alkali, 5.27E-06 3.2141292 up smooth muscle and non-muscle (MYL6), transcript variant 1, mRNA. 27099 NAG8 Homo sapiens nasopharyngeal carcinoma 2.18E-05 2.209735 down associated gene protein-8 (NAG8), mRNA. 4671 NAIP Homo sapiens NLR family, apoptosis inhibitory 1.31E-02 3.9546847 up protein (NAIP), transcript variant 1, mRNA. 91662 NALP12 Homo sapiens NLR family, pyrin domain containing 1.11E-04 2.534172 up 12 (NLRP12), transcript variant 2, mRNA. 91662 NALP12 Homo sapiens NLR family, pyrin domain containing 1.01E-04 2.507363 up 12 (NLRP12), transcript variant 1, mRNA. 54187 NANS Homo sapiens N-acetylneuraminic acid synthase 5.13E-06 2.6940114 up (sialic acid synthase) (NANS), mRNA. 93100 NAPRT1 Homo sapiens nicotinate 6.74E-06 2.6154768 up phosphoribosyltransferase domain containing 1 (NAPRT1), mRNA. 55226 NAT10 Homo sapiens N-acetyltransferase 10 (NAT10), 5.35E-06 2.4969296 down mRNA. 4683 NBN Homo sapiens nibrin (NBN), transcript variant 2, 3.18E-05 2.2353945 up mRNA. 83988 NCALD Homo sapiens neurocalcin delta (NCALD), 6.15E-03 3.7176452 down transcript variant 3, mRNA. 83988 NCALD Homo sapiens neurocalcin delta (NCALD), mRNA. 1.31E-03 3.8601446 down 4688 NCF2 Homo sapiens neutrophil cytosolic factor 2 (65kDa, 9.15E-05 2.9413908 up chronic granulomatous disease, autosomal 2) (NCF2), mRNA. 4689 NCF4 Homo sapiens neutrophil cytosolic factor 4, 40kDa 5.17E-05 2.687491 up (NCF4), transcript variant 2, mRNA. 4689 NCF4 Homo sapiens neutrophil cytosolic factor 4, 40kDa 7.68E-06 3.285344 up (NCF4), transcript variant 1, mRNA. 259197 NCR3 Homo sapiens natural cytotoxicity triggering 2.16E-03 2.9139197 down receptor 3 (NCR3), mRNA. 10397 NDRG1 Homo sapiens N-myc downstream regulated gene 5.35E-06 2.5748284 up 1 (NDRG1), mRNA. 4694 NDUFA1 Homo sapiens NADH dehydrogenase (ubiquinone) 5.47E-06 2.285143 up 1 alpha subcomplex, 1, 7.5kDa (NDUFA1), nuclear gene encoding mitochondrial protein, mRNA.

4697 NDUFA4 Homo sapiens NADH dehydrogenase (ubiquinone) 1.62E-04 2.5491014 up 1 alpha subcomplex, 4, 9kDa (NDUFA4), nuclear gene encoding mitochondrial protein, mRNA.

4709 NDUFB3 Homo sapiens NADH dehydrogenase (ubiquinone) 5.13E-06 6.483008 up 1 beta subcomplex, 3, 12kDa (NDUFB3), mRNA.

4734 NEDD4 Homo sapiens neural precursor cell expressed, 1.09E-02 3.3570197 up developmentally downregulated 4 (NEDD4), transcript variant 2, mRNA. 26012 NELF Homo sapiens nasal embryonic LHRH factor (NELF), 5.43E-05 2.201417 down mRNA. 4753 NELL2 Homo sapiens NEL-like 2 (chicken) (NELL2), mRNA. 1.29E-04 4.6984506 down

242

4758 NEU1 Homo sapiens sialidase 1 (lysosomal sialidase) 5.27E-06 2.4314654 up (NEU1), mRNA. 4778 NFE2 Homo sapiens nuclear factor (erythroid-derived 2), 5.35E-06 3.1501188 up 45kDa (NFE2), mRNA. 4783 NFIL3 Homo sapiens nuclear factor, interleukin 3 1.52E-05 3.5821507 up regulated (NFIL3), mRNA. 64332 NFKBIZ Homo sapiens nuclear factor of kappa light 2.63E-05 2.0495923 up polypeptide gene enhancer in B-cells inhibitor, zeta (NFKBIZ), transcript variant 2, mRNA. 9054 NFS1 Homo sapiens NFS1 nitrogen fixation 1 (S. 5.13E-06 5.309156 down cerevisiae) (NFS1), nuclear gene encoding mitochondrial protein, transcript variant 2, mRNA. 4815 NINJ2 Homo sapiens ninjurin 2 (NINJ2), mRNA. 2.63E-05 3.1039634 up 4824 NKX3-1 Homo sapiens NK3 homeobox 1 (NKX3-1), mRNA. 1.66E-02 2.712998 down 64802 NMNAT1 Homo sapiens nicotinamide nucleotide 4.0E-02 2.0726485 up adenylyltransferase 1 (NMNAT1), mRNA. 10528 NOL5A Homo sapiens nucleolar protein 5A (56kDa with 1.52E-05 2.0307097 down KKE/D repeat) (NOL5A), mRNA. 54433 NOLA1 Homo sapiens nucleolar protein family A, member 2.98E-05 2.2350466 down 1 (H/ACA small nucleolar RNPs) (NOLA1), transcript variant 2, mRNA. 55505 NOLA3 Homo sapiens nucleolar protein family A, member 5.13E-06 3.0532568 up 3 (H/ACA small nucleolar RNPs) (NOLA3), mRNA.

57185 NPAL3 Homo sapiens NIPA-like domain containing 3 5.13E-06 3.2218988 down (NPAL3), mRNA. 4863 NPAT Homo sapiens nuclear protein, ataxia- 1.51E-05 2.1471682 down telangiectasia locus (NPAT), mRNA. 9284 NPIP Homo sapiens nuclear pore complex interacting 7.25E-06 2.1705399 down protein (NPIP), mRNA. 80896 NPL Homo sapiens N-acetylneuraminate pyruvate lyase 7.68E-06 2.8866994 up (dihydrodipicolinate synthase) (NPL), mRNA. 27020 NPTN Homo sapiens neuroplastin (NPTN), transcript 6.31E-06 2.8675702 up variant alpha, mRNA. 4835 NQO2 Homo sapiens NAD(P)H dehydrogenase, quinone 2 3.27E-05 5.2974734 up (NQO2), mRNA. 4898 NRD1 Homo sapiens nardilysin (N-arginine dibasic 5.13E-06 2.2504551 up convertase) (NRD1), mRNA. 79685 NS4ATP2 Homo sapiens SAP30-like (SAP30L), mRNA. 5.35E-06 2.243592 up 79730 NSUN7 Homo sapiens NOL1/NOP2/Sun domain family, 2.20E-04 8.276363 up member 7 (NSUN7), mRNA. 10908 NTE Homo sapiens neuropathy target esterase (NTE), 5.13E-06 3.7707965 up mRNA. 131870 NUDT16 Homo sapiens nudix (nucleoside diphosphate 7.45E-03 2.312262 up linked moiety X)-type motif 16 (NUDT16), mRNA. 11164 NUDT5 Homo sapiens nudix (nucleoside diphosphate 9.97E-06 2.22799 up linked moiety X)-type motif 5 (NUDT5), mRNA. 8650 NUMB Homo sapiens numb homolog (Drosophila) 1.28E-05 2.5121162 up (NUMB), transcript variant 3, mRNA. 8021 NUP214 Homo sapiens nucleoporin 214kDa (NUP214), 1.82E-05 2.5839055 up mRNA. 9688 NUP93 Homo sapiens nucleoporin 93kDa (NUP93), mRNA. 7.68E-06 2.0916955 down 55916 NXT2 Homo sapiens nuclear transport factor 2-like 1.52E-05 2.4291992 up export factor 2 (NXT2), mRNA.

243

10162 OACT5 Homo sapiens membrane bound O-acyltransferase 2.61E-04 2.0393162 up domain containing 5 (MBOAT5), mRNA.

4939 OAS2 Homo sapiens 2'-5'-oligoadenylate synthetase 2, 7.25E-06 4.486012 down 69/71kDa (OAS2), transcript variant 1, mRNA. 4939 OAS2 Homo sapiens 2'-5'-oligoadenylate synthetase 2, 2.04E-03 2.6828973 down 69/71kDa (OAS2), transcript variant 3, mRNA. 4942 OAT Homo sapiens ornithine aminotransferase (gyrate 1.00E-05 3.0850742 up atrophy) (OAT), nuclear gene encoding mitochondrial protein, mRNA. 64859 OBFC2A Homo sapiens oligonucleotide/oligosaccharide- 6.74E-06 3.4310498 up binding fold containing 2A (OBFC2A), transcript variant 2, mRNA. 132299 OCIAD2 Homo sapiens OCIA domain containing 2 (OCIAD2), 1.52E-05 2.9381084 down transcript variant 1, mRNA. 132299 OCIAD2 Homo sapiens OCIA domain containing 2 (OCIAD2), 2.32E-05 2.5999365 down transcript variant 2, mRNA. 10562 OLFM4 Homo sapiens olfactomedin 4 (OLFM4), mRNA. 8.93E-05 46.051617 up 26873 OPLAH Homo sapiens 5-oxoprolinase (ATP-hydrolysing) 1.84E-03 8.579143 up (OPLAH), mRNA. 4987 OPRL1 Homo sapiens opiate receptor-like 1 (OPRL1), 4.26E-05 2.3806522 up transcript variant 2, mRNA. 343169 OR6F1 Homo sapiens olfactory receptor, family 6, 1.43E-02 2.2932305 up subfamily F, member 1 (OR6F1), mRNA. 84418 ORF1-FL49 Homo sapiens putative nuclear protein ORF1-FL49 5.13E-06 22.005098 up (ORF1-FL49), mRNA. 5004 ORM1 Homo sapiens orosomucoid 1 (ORM1), mRNA. 1.62E-04 6.0118136 up 114884 OSBPL10 Homo sapiens oxysterol binding protein-like 10 3.73E-04 2.2006912 down (OSBPL10), mRNA. 114885 OSBPL11 Homo sapiens oxysterol binding protein-like 11 7.68E-06 2.261094 up (OSBPL11), mRNA. 114879 OSBPL5 Homo sapiens oxysterol binding protein-like 5 5.15E-05 2.0881977 down (OSBPL5), transcript variant 1, mRNA. 114881 OSBPL7 Homo sapiens oxysterol binding protein-like 7 8.66E-06 2.5939014 down (OSBPL7), transcript variant 1, mRNA. 114882 OSBPL8 Homo sapiens oxysterol binding protein-like 8 2.17E-04 2.068802 up (OSBPL8), transcript variant 1, mRNA. 126014 OSCAR Homo sapiens osteoclast-associated receptor 5.13E-06 5.1018496 up (OSCAR), transcript variant 3, mRNA. 126014 OSCAR Homo sapiens osteoclast-associated receptor 5.13E-06 4.6895657 up (OSCAR), transcript variant 4, mRNA. 5008 OSM Homo sapiens oncostatin M (OSM), mRNA. 2.99E-04 4.1150975 up 26578 OSTF1 Homo sapiens osteoclast stimulating factor 1 5.47E-06 2.2837226 up (OSTF1), mRNA. 5023 P2RX1 Homo sapiens purinergic receptor P2X, ligand- 1.82E-05 3.342325 up gated ion channel, 1 (P2RX1), mRNA. 27334 P2RY10 Homo sapiens purinergic receptor P2Y, G-protein 3.90E-05 2.5403025 down coupled, 10 (P2RY10), transcript variant 2, mRNA. 53829 P2RY13 Homo sapiens purinergic receptor P2Y, G-protein 2.20E-05 3.1893687 up coupled, 13 (P2RY13), transcript variant 2, mRNA. 53829 P2RY13 Homo sapiens purinergic receptor P2Y, G-protein 6.31E-06 2.8593702 up coupled, 13 (P2RY13), transcript variant 2, mRNA. 286530 P2RY8 Homo sapiens purinergic receptor P2Y, G-protein 5.27E-06 2.86355 down coupled, 8 (P2RY8), mRNA.

244

5033 P4HA1 Homo sapiens procollagen-proline, 2-oxoglutarate 5.47E-06 2.4326131 up 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide I (P4HA1), transcript variant 1, mRNA.

23569 PADI4 Homo sapiens peptidyl arginine deiminase, type IV 5.13E-06 5.0578814 up (PADI4), mRNA. 55824 PAG1 Homo sapiens phosphoprotein associated with 6.74E-06 3.1861682 up glycosphingolipid microdomains 1 (PAG1), mRNA. 23022 PALLD Homo sapiens palladin, cytoskeletal associated 5.77E-04 2.9062388 up protein (PALLD), mRNA. 5066 PAM Homo sapiens peptidylglycine alpha-amidating 1.47E-04 2.491062 up monooxygenase (PAM), transcript variant 3, mRNA. 196743 PAOX Homo sapiens polyamine oxidase (exo-N4-amino) 1.00E-05 2.515569 down (PAOX), transcript variant 5, mRNA. 167153 PAPD4 Homo sapiens PAP associated domain containing 4 8.33E-05 2.0042925 up (PAPD4), mRNA. 9061 PAPSS1 Homo sapiens 3'-phosphoadenosine 5'- 6.74E-06 3.177851 up phosphosulfate synthase 1 (PAPSS1), mRNA. 85315 PAQR8 Homo sapiens progestin and adipoQ receptor 5.13E-06 3.165916 down family member VIII (PAQR8), mRNA. 142 PARP1 Homo sapiens poly (ADP-ribose) polymerase 7.25E-06 2.222497 down family, member 1 (PARP1), mRNA. 64761 PARP12 Homo sapiens poly (ADP-ribose) polymerase 2.20E-05 2.5176356 down family, member 12 (PARP12), mRNA. 54625 PARP14 Homo sapiens poly (ADP-ribose) polymerase 1.03E-04 2.6842163 down family, member 14 (PARP14), mRNA. 23178 PASK Homo sapiens PAS domain containing 2.22E-02 2.0209374 down serine/threonine kinase (PASK), mRNA. 10135 PBEF1 Homo sapiens pre-B-cell colony enhancing factor 1 3.09E-04 3.2584221 up (PBEF1), transcript variant 2, mRNA. 10135 PBEF1 Homo sapiens pre-B-cell colony enhancing factor 1 1.82E-05 3.9533381 up (PBEF1), transcript variant 1, mRNA. 5110 PCMT1 Homo sapiens protein-L-isoaspartate (D-aspartate) 5.13E-06 3.1363792 up O-methyltransferase (PCMT1), mRNA. 5116 PCNT2 Homo sapiens pericentrin 2 (kendrin) (PCNT2), 3.06E-05 2.0909164 down mRNA. 22990 PCNX Homo sapiens pecanex homolog (Drosophila) 8.44E-06 2.7226713 up (PCNX), mRNA. 58488 PCTP Homo sapiens phosphatidylcholine transfer protein 5.35E-06 3.1773274 up (PCTP), mRNA. 11235 PDCD10 Homo sapiens programmed cell death 10 1.52E-05 3.3001914 up (PDCD10), transcript variant 3, mRNA. 11235 PDCD10 Homo sapiens programmed cell death 10 3.15E-04 2.277614 up (PDCD10), transcript variant 3, mRNA. 5144 PDE4D Homo sapiens phosphodiesterase 4D, cAMP- 1.21E-04 3.0916307 up specific (phosphodiesterase E3 dunce homolog, Drosophila) (PDE4D), mRNA. 5150 PDE7A Homo sapiens phosphodiesterase 7A (PDE7A), 7.25E-06 2.3359962 down transcript variant 2, mRNA. 56034 PDGFC Homo sapiens platelet derived growth factor C 3.59E-02 4.8675795 up (PDGFC), mRNA. 5166 PDK4 Homo sapiens pyruvate dehydrogenase kinase, 3.48E-03 2.0584395 up isozyme 4 (PDK4), mRNA. 9260 PDLIM7 Homo sapiens PDZ and LIM domain 7 (enigma) 3.93E-02 2.5412924 up (PDLIM7), transcript variant 4, mRNA.

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9260 PDLIM7 Homo sapiens PDZ and LIM domain 7 (enigma) 8.44E-06 4.006498 up (PDLIM7), transcript variant 4, mRNA. 23590 PDSS1 Homo sapiens prenyl (decaprenyl) diphosphate 4.60E-05 7.2587 up synthase, subunit 1 (PDSS1), mRNA. 57026 PDXP Homo sapiens pyridoxal (pyridoxine, vitamin B6) 1.17E-05 2.230238 down phosphatase (PDXP), mRNA. 118987 PDZD8 Homo sapiens PDZ domain containing 8 (PDZD8), 5.42E-05 2.796286 up mRNA. 5037 PEBP1 Homo sapiens phosphatidylethanolamine binding 5.47E-06 2.9926214 down protein 1 (PEBP1), mRNA. 10455 PECI Homo sapiens peroxisomal D3,D2-enoyl-CoA 5.47E-06 2.3400588 down isomerase (PECI), transcript variant 1, mRNA. 55825 PECR Homo sapiens peroxisomal trans-2-enoyl-CoA 5.13E-06 3.5108263 up reductase (PECR), mRNA. 57162 PELI1 Homo sapiens pellino homolog 1 (Drosophila) 3.80E-04 2.1798205 up (PELI1), mRNA. 53918 PELO Homo sapiens pelota homolog (Drosophila) (PELO), 1.17E-05 2.2285235 up mRNA. 27043 PELP1 Homo sapiens proline, glutamic acid and leucine 7.25E-06 2.1486166 down rich protein 1 (PELP1), mRNA. 93210 PERLD1 Homo sapiens per1-like domain containing 1 1.34E-04 2.728937 down (PERLD1), mRNA. 5209 PFKFB3 Homo sapiens 6-phosphofructo-2-kinase/fructose- 5.13E-06 8.613797 up 2,6-biphosphatase 3 (PFKFB3), mRNA. 5210 PFKFB4 Homo sapiens 6-phosphofructo-2-kinase/fructose- 6.31E-06 2.951753 up 2,6-biphosphatase 4 (PFKFB4), mRNA. 5218 PFTK1 Homo sapiens PFTAIRE protein kinase 1 (PFTK1), 1.35E-02 2.0808308 up mRNA. 5223 PGAM1 Homo sapiens phosphoglycerate mutase 1 (brain) 2.20E-05 2.3780184 up (PGAM1), mRNA. 441531 PGAM4 Homo sapiens phosphoglycerate mutase family 7.32E-05 2.4739196 up member 4 (PGAM4), mRNA. 5226 PGD Homo sapiens phosphogluconate dehydrogenase 5.13E-06 7.489585 up (PGD), mRNA. 5230 PGK1 Homo sapiens phosphoglycerate kinase 1 (PGK1), 5.13E-06 3.5695384 up mRNA. 8993 PGLYRP1 Homo sapiens peptidoglycan recognition protein 1 5.13E-06 7.912276 up (PGLYRP1), mRNA. 5236 PGM1 Homo sapiens phosphoglucomutase 1 (PGM1), 2.63E-05 2.081716 up mRNA. 55276 PGM2 Homo sapiens phosphoglucomutase 2 (PGM2), 5.27E-06 3.8444664 up mRNA. 9489 PGS1 Homo sapiens phosphatidylglycerophosphate 5.13E-06 5.2845798 up synthase 1 (PGS1), mRNA. 9749 PHACTR2 Homo sapiens phosphatase and actin regulator 2 1.67E-05 2.3536258 up (PHACTR2), mRNA. 1912 PHC2 Homo sapiens polyhomeotic-like 2 (Drosophila) 5.35E-06 3.3056862 up (PHC2), transcript variant 1, mRNA. 55331 PHCA Homo sapiens phytoceramidase, alkaline (PHCA), 5.13E-06 7.555555 up mRNA. 23338 PHF15 Homo sapiens PHD finger protein 15 (PHF15), 7.25E-06 2.9041357 down mRNA. 26147 PHF19 Homo sapiens PHD finger protein 19 (PHF19), 8.33E-05 2.2902544 up transcript variant 2, mRNA. 51105 PHF20L1 Homo sapiens PHD finger protein 20-like 1 2.20E-05 2.5001652 up (PHF20L1), transcript variant 1, mRNA.

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10745 PHTF1 Homo sapiens putative homeodomain 7.25E-04 3.4732292 up transcription factor 1 (PHTF1), mRNA. 5266 PI3 Homo sapiens peptidase inhibitor 3, skin-derived 5.17E-05 9.3671 up (SKALP) (PI3), mRNA. 8301 PICALM Homo sapiens phosphatidylinositol binding clathrin 4.44E-03 2.0091095 up assembly protein (PICALM), transcript variant 1, mRNA. 5281 PIGF Homo sapiens phosphatidylinositol glycan, class F 5.13E-06 2.3407953 up (PIGF), transcript variant 2, mRNA. 118788 PIK3AP1 Homo sapiens phosphoinositide-3-kinase adaptor 5.13E-06 3.4233067 up protein 1 (PIK3AP1), mRNA. 5292 PIM1 Homo sapiens pim-1 oncogene (PIM1), mRNA. 7.57E-05 2.1284213 up 415116 PIM3 Homo sapiens pim-3 oncogene (PIM3), mRNA. 5.13E-06 3.4440582 up 57605 PITPNM2 Homo sapiens phosphatidylinositol transfer 2.42E-05 2.345636 up protein, membrane-associated 2 (PITPNM2), mRNA. 9867 PJA2 Homo sapiens praja 2, RING-H2 motif containing 1.82E-05 2.2998512 up (PJA2), mRNA. 5569 PKIA Homo sapiens protein kinase (cAMP-dependent, 2.42E-05 3.220068 down catalytic) inhibitor alpha (PKIA), transcript variant 7, mRNA. 5569 PKIA Homo sapiens protein kinase (cAMP-dependent, 1.71E-03 2.0854526 down catalytic) inhibitor alpha (PKIA), transcript variant 6, mRNA. 5315 PKM2 Homo sapiens pyruvate kinase, muscle (PKM2), 7.25E-06 2.279943 up transcript variant 3, mRNA. 5315 PKM2 Homo sapiens pyruvate kinase, muscle (PKM2), 1.38E-04 2.0353124 up transcript variant 1, mRNA. 8681 PLA2G4B Homo sapiens phospholipase A2, group IVB 4.47E-05 2.3315077 down (cytosolic) (PLA2G4B), mRNA. 51316 PLAC8 Homo sapiens placenta-specific 8 (PLAC8), mRNA. 5.13E-06 3.692069 up 5329 PLAUR Homo sapiens plasminogen activator, urokinase 8.33E-05 2.0735471 up receptor (PLAUR), transcript variant 2, mRNA. 5329 PLAUR Homo sapiens plasminogen activator, urokinase 1.82E-05 2.2523324 up receptor (PLAUR), transcript variant 2, mRNA. 5329 PLAUR Homo sapiens plasminogen activator, urokinase 2.91e-03 2.1392767 up receptor (PLAUR), transcript variant 1, mRNA. 5335 PLCG1 Homo sapiens phospholipase C, gamma 1 (PLCG1), 1.12E-03 2.7355645 down transcript variant 1, mRNA. 59338 PLEKHA1 Homo sapiens pleckstrin homology domain 5.13E-06 3.9215834 down containing, family A (phosphoinositide binding specific) member 1 (PLEKHA1), transcript variant 1, mRNA. 59338 PLEKHA1 Homo sapiens pleckstrin homology domain 1.67E-05 3.0099254 down containing, family A (phosphoinositide binding specific) member 1 (PLEKHA1), transcript variant 2, mRNA. 55041 PLEKHB2 Homo sapiens pleckstrin homology domain 3.42E-05 2.2464712 up containing, family B (evectins) member 2 (PLEKHB2), transcript variant 1, mRNA. 79156 PLEKHF1 Homo sapiens pleckstrin homology domain 1.34E-04 3.2834024 down containing, family F (with FYVE domain) member 1 (PLEKHF1), mRNA. 51177 PLEKHO1 Homo sapiens pleckstrin homology domain 5.47E-06 2.2154317 down containing, family O member 1 (PLEKHO1), mRNA.

247

80301 PLEKHQ1 Homo sapiens pleckstrin homology domain 6.74E-06 2.5815039 up containing, family Q member 1 (PLEKHQ1), mRNA. 5351 PLOD1 Homo sapiens procollagen-lysine 1, 2-oxoglutarate 2.63E-05 2.609169 up 5-dioxygenase 1 (PLOD1), mRNA. 5355 PLP2 Homo sapiens proteolipid protein 2 (colonic 5.13E-06 4.525847 up epithelium-enriched) (PLP2), mRNA. 5359 PLSCR1 Homo sapiens phospholipid scramblase 1 (PLSCR1), 2.25e-02 2.221066 up mRNA. 57048 PLSCR3 Homo sapiens phospholipid scramblase 3 (PLSCR3), 5.13E-06 2.0319176 down mRNA. 10154 PLXNC1 Homo sapiens plexin C1 (PLXNC1), mRNA. 1.39E-03 2.3656461 up 25953 PNKD Homo sapiens paroxysmal nonkinesiogenic 7.25E-06 2.7308388 up dyskinesia (PNKD), transcript variant 2, mRNA. 50640 PNPLA8 Homo sapiens patatin-like phospholipase domain 4.53E-05 2.0974667 up containing 8 (PNPLA8), mRNA. 9533 POLR1C Homo sapiens polymerase (RNA) I polypeptide C, 1.52E-05 2.1059377 down 30kDa (POLR1C), transcript variant 1, mRNA. 64425 POLR1E Homo sapiens polymerase (RNA) I polypeptide E, 5.47E-06 2.5342016 down 53kDa (POLR1E), mRNA. 171568 POLR3H Homo sapiens polymerase (RNA) III (DNA directed) 2.90E-05 2.0133684 down polypeptide H (22.9kD) (POLR3H), transcript variant 1, mRNA. 5442 POLRMT Homo sapiens polymerase (RNA) mitochondrial 1.09E-05 2.1016157 down (DNA directed) (POLRMT), nuclear gene encoding mitochondrial protein, mRNA. 55624 POMGNT1 Homo sapiens protein O-linked mannose beta1,2- 7.25E-06 2.089137 down N-acetylglucosaminyltransferase (POMGNT1), mRNA. 5447 POR Homo sapiens P450 (cytochrome) oxidoreductase 5.13E-06 4.7600846 up (POR), mRNA. 27068 PPA2 Homo sapiens inorganic pyrophosphatase 2 8.44E-06 2.362274 up (PPA2), transcript variant 2, mRNA. 8495 PPFIBP2 Homo sapiens PTPRF interacting protein, binding 5.88E-06 2.0940454 down protein 2 (liprin beta 2) (PPFIBP2), mRNA. 5476 PPGB Homo sapiens protective protein for beta- 3.46E-04 2.035785 up galactosidase (galactosialidosis) (PPGB), mRNA. 5495 PPM1B Homo sapiens protein phosphatase 1B (formerly 2.63E-05 2.0785377 up 2C), magnesium-dependent, beta isoform (PPM1B), transcript variant 2, mRNA. 132160 PPM1M Homo sapiens protein phosphatase 1M (PP2C 5.13E-06 3.082389 up domain containing) (PPM1M), mRNA. 4659 PPP1R12A Homo sapiens protein phosphatase 1, regulatory 1.09E-05 2.7240088 up (inhibitor) subunit 12A (PPP1R12A), mRNA. 26051 PPP1R16B Homo sapiens protein phosphatase 1, regulatory 5.47E-06 2.5043619 down (inhibitor) subunit 16B (PPP1R16B), mRNA. 5509 PPP1R3D Homo sapiens protein phosphatase 1, regulatory 1.39E-05 2.3330824 up (inhibitor) subunit 3D (PPP1R3D), mRNA. 5515 PPP2CA Homo sapiens protein phosphatase 2 (formerly 7.68E-06 2.0384812 up 2A), catalytic subunit, alpha isoform (PPP2CA), mRNA. 55012 PPP2R3C Homo sapiens protein phosphatase 2 (formerly 5.13E-06 2.3775802 up 2A), regulatory subunit B'', gamma (PPP2R3C), mRNA. 5525 PPP2R5A Homo sapiens protein phosphatase 2, regulatory 5.27E-06 2.0492773 up subunit B', alpha isoform (PPP2R5A), mRNA.

248

5533 PPP3CC Homo sapiens protein phosphatase 3 (formerly 2.51E-05 2.3324587 down 2B), catalytic subunit, gamma isoform (PPP3CC), mRNA. 9989 PPP4R1 Homo sapiens protein phosphatase 4, regulatory 6.74E-06 2.503474 up subunit 1 (PPP4R1), mRNA. 9989 PPP4R1 Homo sapiens protein phosphatase 4, regulatory 1.17E-05 2.4836266 up subunit 1 (PPP4R1), mRNA. 23082 PPRC1 Homo sapiens peroxisome proliferator-activated 7.75E-06 2.2690568 down receptor gamma, coactivator-related 1 (PPRC1), mRNA. 84106 PRAM1 Homo sapiens PML-RARA regulated adaptor 1.52E-05 2.7370472 up molecule 1 (PRAM1), mRNA. 9055 PRC1 Homo sapiens protein regulator of cytokinesis 1 1.53E-04 2.332725 up (PRC1), transcript variant 1, mRNA. 5547 PRCP Homo sapiens prolylcarboxypeptidase 5.35E-06 2.48072 up (angiotensinase C) (PRCP), transcript variant 1, mRNA. 5547 PRCP Homo sapiens prolylcarboxypeptidase 5.35E-06 2.284883 up (angiotensinase C) (PRCP), transcript variant 2, mRNA. 10935 PRDX3 Homo sapiens peroxiredoxin 3 (PRDX3), nuclear 3.50E-05 2.2023947 up gene encoding mitochondrial protein, transcript variant 1, mRNA. 25824 PRDX5 Homo sapiens peroxiredoxin 5 (PRDX5), nuclear 5.35E-06 2.0069947 up gene encoding mitochondrial protein, transcript variant 3, mRNA. 5551 PRF1 Homo sapiens perforin 1 (pore forming protein) 7.25E-06 4.4908586 down (PRF1), mRNA. 5562 PRKAA1 Homo sapiens protein kinase, AMP-activated, 9.97E-06 2.0555694 up alpha 1 catalytic subunit (PRKAA1), transcript variant 2, mRNA. 5576 PRKAR2A Homo sapiens protein kinase, cAMP-dependent, 1.68E-04 2.2692022 up regulatory, type II, alpha (PRKAR2A), mRNA. 5578 PRKCA Homo sapiens protein kinase C, alpha (PRKCA), 8.33E-05 2.1191745 down mRNA. 5580 PRKCD Homo sapiens protein kinase C, delta (PRKCD), 6.74E-06 2.4898765 up transcript variant 2, mRNA. 5583 PRKCH Homo sapiens protein kinase C, eta (PRKCH), 6.74E-06 2.521592 down mRNA. 55170 PRMT6 Homo sapiens protein arginine methyltransferase 6 1.06E-04 2.032957 down (PRMT6), mRNA. 29035 PRO0149 Homo sapiens chromosome 16 open reading frame 2.63E-05 2.2502391 up 72 (C16orf72), mRNA. 60675 PROK2 Homo sapiens prokineticin 2 (PROK2), mRNA. 1.66E-05 3.53497 up 27339 PRPF19 Homo sapiens PRP19/PSO4 pre-mRNA processing 5.13E-06 2.4614246 down factor 19 homolog (S. cerevisiae) (PRPF19), mRNA. 5678 PSG9 Homo sapiens pregnancy specific beta-1- 3.81E-05 2.53786 up glycoprotein 9 (PSG9), mRNA. 5682 PSMA1 Homo sapiens proteasome (prosome, macropain) 6.13E-05 2.3995247 up subunit, alpha type, 1 (PSMA1), transcript variant 2, mRNA. 5687 PSMA6 Homo sapiens proteasome (prosome, macropain) 9.97E-06 3.1489394 up subunit, alpha type, 6 (PSMA6), mRNA. 5698 PSMB9 Homo sapiens proteasome (prosome, macropain) 2.86E-04 2.0245085 down subunit, beta type, 9 (large multifunctional peptidase 2) (PSMB9), transcript variant 1, mRNA.

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5718 PSMD12 Homo sapiens proteasome (prosome, macropain) 5.13E-06 2.4008436 up 26S subunit, non-ATPase, 12 (PSMD12), mRNA. XM_946055 XM_946058 5710 PSMD4 Homo sapiens proteasome (prosome, macropain) 5.13E-06 2.008342 up 26S subunit, non-ATPase, 4 (PSMD4), transcript variant 1, mRNA. 5710 PSMD4 Homo sapiens proteasome (prosome, macropain) 5.13E-06 2.055561 up 26S subunit, non-ATPase, 4 (PSMD4), mRNA. 9861 PSMD6 Homo sapiens proteasome (prosome, macropain) 5.13E-06 2.1160266 up 26S subunit, non-ATPase, 6 (PSMD6), mRNA. 9050 PSTPIP2 Homo sapiens proline-serine-threonine 5.47E-06 4.849586 up phosphatase interacting protein 2 (PSTPIP2), mRNA. 5724 PTAFR Homo sapiens platelet-activating factor receptor 5.69E-05 2.4204898 up (PTAFR), mRNA. 23210 PTDSR Homo sapiens phosphatidylserine receptor 1.08E-02 2.7324207 up (PTDSR), mRNA. 5728 PTEN Homo sapiens phosphatase and tensin homolog 5.47E-06 2.9733253 up (mutated in multiple advanced cancers 1) (PTEN), mRNA. 5732 PTGER2 Homo sapiens prostaglandin E receptor 2 (subtype 1.82E-05 2.6291232 up EP2), 53kDa (PTGER2), mRNA. 5747 PTK2 Homo sapiens PTK2 protein tyrosine kinase 2 7.68E-06 2.2727175 down (PTK2), transcript variant 1, mRNA. 5782 PTPN12 Homo sapiens protein tyrosine phosphatase, non- 5.13E-06 3.5356245 up receptor type 12 (PTPN12), mRNA. 26191 PTPN22 Homo sapiens protein tyrosine phosphatase, non- 2.20E-05 2.430728 up receptor type 22 (lymphoid) (PTPN22), transcript variant 1, mRNA. 26191 PTPN22 Homo sapiens protein tyrosine phosphatase, non- 5.81E-05 2.1528652 up receptor type 22 (lymphoid) (PTPN22), transcript variant 2, mRNA. 5775 PTPN4 Homo sapiens protein tyrosine phosphatase, non- 5.35E-06 3.3845642 down receptor type 4 (megakaryocyte) (PTPN4), mRNA. 140885 PTPNS1 Homo sapiens protein tyrosine phosphatase, non- 6.74E-06 3.1488357 up receptor type substrate 1 (PTPNS1), mRNA. 140885 PTPNS1 Homo sapiens protein tyrosine phosphatase, non- 5.27E-06 3.9575012 up receptor type substrate 1 (PTPNS1), mRNA. 5790 PTPRCAP Homo sapiens protein tyrosine phosphatase, 2.02E-05 2.6824536 down receptor type, C-associated protein (PTPRCAP), mRNA. 754 PTTG1IP Homo sapiens pituitary tumor-transforming 1 5.27E-06 2.1541073 up interacting protein (PTTG1IP), mRNA. 80324 PUS1 Homo sapiens pseudouridylate synthase 1 (PUS1), 9.10E-06 2.3368845 down transcript variant 1, mRNA. 54899 PXK Homo sapiens PX domain containing 9.03E-06 2.2016213 up serine/threonine kinase (PXK), mRNA. 5828 PXMP3 Homo sapiens peroxisomal membrane protein 3, 3.10E-05 2.2489338 up 35kDa (Zellweger syndrome) (PXMP3), mRNA. 29108 PYCARD Homo sapiens PYD and CARD domain containing 5.46E-04 2.1096709 up (PYCARD), transcript variant 2, mRNA. 5836 PYGL Homo sapiens phosphorylase, glycogen; liver (Hers 5.27E-06 4.1182103 up disease, glycogen storage disease type VI) (PYGL), mRNA. 149628 PYHIN1 Homo sapiens pyrin and HIN domain family, 2.20E-04 3.2838683 down member 1 (PYHIN1), transcript variant a1, mRNA.

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149628 PYHIN1 Homo sapiens pyrin and HIN domain family, 1.06E-05 5.2302647 down member 1 (PYHIN1), transcript variant b1, mRNA. 27089 QP-C Homo sapiens low molecular mass ubiquinone- 9.29E-04 2.023069 up binding protein (9.5kD) (QP-C), nuclear gene encoding mitochondrial protein, mRNA. 5768 QSCN6 Homo sapiens quiescin Q6 sulfhydryl oxidase 1 5.13E-06 6.234041 up (QSOX1), transcript variant 2, mRNA. 10890 RAB10 Homo sapiens RAB10, member RAS oncogene 6.74E-06 2.2074714 up family (RAB10), mRNA. 22841 RAB11FIP2 Homo sapiens RAB11 family interacting protein 2 7.63E-05 2.080287 up (class I) (RAB11FIP2), mRNA. 9727 RAB11FIP3 Homo sapiens RAB11 family interacting protein 3 5.35E-06 2.5425746 down (class II) (RAB11FIP3), mRNA. 5861 RAB1A Homo sapiens RAB1A, member RAS oncogene 2.95E-05 2.1740088 up family (RAB1A), mRNA. 5862 RAB2 Homo sapiens RAB2A, member RAS oncogene 3.47E-05 2.6897438 up family (RAB2A), mRNA. 55647 RAB20 Homo sapiens RAB20, member RAS oncogene 4.44E-02 3.799992 up family (RAB20), mRNA. 23011 RAB21 Homo sapiens RAB21, member RAS oncogene 1.31E-05 2.2059155 up family (RAB21), mRNA. 53917 RAB24 Homo sapiens RAB24, member RAS oncogene 1.34E-04 2.096354 up family (RAB24), transcript variant 2, mRNA. 53917 RAB24 Homo sapiens RAB24, member RAS oncogene 1.66E-05 2.0745072 up family (RAB24), transcript variant 1, mRNA. 5873 RAB27A Homo sapiens RAB27A, member RAS oncogene 9.10E-06 3.3393779 up family (RAB27A), transcript variant 1, mRNA. 11031 RAB31 Homo sapiens RAB31, member RAS oncogene 5.13E-06 4.293371 up family (RAB31), mRNA. 10981 RAB32 Homo sapiens RAB32, member RAS oncogene 5.13E-06 4.4661 up family (RAB32), mRNA. 83452 RAB33B Homo sapiens RAB33B, member RAS oncogene 3.87E-05 2.075901 up family (RAB33B), mRNA. 5866 RAB3IL1 Homo sapiens RAB3A interacting protein (rabin3)- 3.90E-03 2.310113 up like 1 (RAB3IL1), mRNA. 117177 RAB3IP Homo sapiens RAB3A interacting protein (rabin3) 3.06E-05 2.2733204 down (RAB3IP), transcript variant A, mRNA. 5868 RAB5A Homo sapiens RAB5A, member RAS oncogene 4.22E-05 2.0943434 up family (RAB5A), mRNA. 5878 RAB5C Homo sapiens RAB5C, member RAS oncogene 5.27E-06 2.0098603 up family (RAB5C), transcript variant 2, mRNA. 5870 RAB6A Homo sapiens RAB6A, member RAS oncogene 7.68E-06 2.3078442 up family (RAB6A), transcript variant 2, mRNA. 51762 RAB8B Homo sapiens RAB8B, member RAS oncogene 7.68E-06 2.1526594 up family (RAB8B), mRNA. 10567 RABAC1 Homo sapiens Rab acceptor 1 (prenylated) 5.17E-05 2.112801 up (RABAC1), mRNA. 27342 RABGEF1 Homo sapiens RAB guanine nucleotide exchange 5.13E-06 3.7175806 up factor (GEF) 1 (RABGEF1), mRNA. 11159 RABL2A Homo sapiens RAB, member of RAS oncogene 5.47E-06 2.2879565 down family-like 2A (RABL2A), transcript variant 1, mRNA. 11158 RABL2B Homo sapiens RAB, member of RAS oncogene 5.48E-06 2.1370454 down family-like 2B (RABL2B), transcript variant 2, mRNA. 8480 RAE1 Homo sapiens RAE1 RNA export 1 homolog (S. 5.13E-06 2.2723718 up pombe) (RAE1), transcript variant 1, mRNA.

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23180 RAFTLIN Homo sapiens raftlin, lipid raft linker 1 (RFTN1), 5.13E-06 2.5688217 down mRNA. 5899 RALB Homo sapiens v-ral simian leukemia viral oncogene 2.20E-05 3.0170004 up homolog B (ras related; GTP binding protein) (RALB), mRNA. 5900 RALGDS Homo sapiens ral guanine nucleotide dissociation 3.50E-05 2.470266 down stimulator (RALGDS), transcript variant 1, mRNA.

57826 RAP2C Homo sapiens RAP2C, member of RAS oncogene 5.27E-06 2.45151 up family (RAP2C), mRNA. 5914 RARA Homo sapiens retinoic acid receptor, alpha (RARA), 8.33E-05 2.0344188 up transcript variant 2, mRNA. 5920 RARRES3 Homo sapiens retinoic acid receptor responder 7.68E-06 3.5678754 down (tazarotene induced) 3 (RARRES3), mRNA. 10125 RASGRP1 Homo sapiens RAS guanyl releasing protein 1 5.13E-06 4.654138 down (calcium and DAG-regulated) (RASGRP1), mRNA. 25780 RASGRP3 Homo sapiens RAS guanyl releasing protein 3 2.50E-05 2.5557296 down (calcium and DAG-regulated) (RASGRP3), mRNA. 115727 RASGRP4 Homo sapiens RAS guanyl releasing protein 4 1.53E-02 2.1501565 up (RASGRP4), transcript variant 1, mRNA. 9770 RASSF2 Homo sapiens Ras association (RalGDS/AF-6) 9.97E-06 2.6524665 up domain family 2 (RASSF2), transcript variant 2, mRNA. 9770 RASSF2 Homo sapiens Ras association (RalGDS/AF-6) 9.97E-06 2.30542 up domain family 2 (RASSF2), transcript variant 2, mRNA. 8045 RASSF7 Homo sapiens Ras association (RalGDS/AF-6) 5.47E-06 2.208971 down domain family 7 (RASSF7), mRNA. 23352 RBAF600 Homo sapiens retinoblastoma-associated factor 6.31E-06 2.0767138 up 600 (RBAF600), mRNA. 84549 RBM13 Homo sapiens RNA binding motif protein 13 1.67E-05 2.0225883 down (RBM13), mRNA. 5937 RBMS1 Homo sapiens RNA binding motif, single stranded 5.13E-06 4.021259 up interacting protein 1 (RBMS1), transcript variant 3, mRNA. 3516 RBPSUH Homo sapiens recombining binding protein 4.69E-05 2.2581666 up suppressor of hairless (Drosophila) (RBPSUH), transcript variant 4, mRNA. 9978 RBX1 Homo sapiens ring-box 1 (RBX1), mRNA. 7.57E-05 2.1960974 up 9185 REPS2 Homo sapiens RALBP1 associated Eps domain 1.10E-02 2.1453114 up containing 2 (REPS2), mRNA. 9185 REPS2 PREDICTED: Homo sapiens RALBP1 associated Eps 1.55E-03 2.0997515 up domain containing 2 (REPS2), mRNA. 56729 RETN Homo sapiens resistin (RETN), mRNA. 2.20E-04 112.82527 up 5990 RFX2 Homo sapiens regulatory factor X, 2 (influences 7.70E-03 2.8042586 up HLA class II expression) (RFX2), transcript variant 2, mRNA. 5990 RFX2 Homo sapiens regulatory factor X, 2 (influences 7.57E-03 2.0874906 up HLA class II expression) (RFX2), transcript variant 1, mRNA. 5993 RFX5 Homo sapiens regulatory factor X, 5 (influences 5.13E-06 2.2252 down HLA class II expression) (RFX5), transcript variant 1, mRNA. 23179 RGL1 Homo sapiens ral guanine nucleotide dissociation 2.9E-02 2.059209 up stimulator-like 1 (RGL1), mRNA. 266747 Rgr Homo sapiens Ral-GDS related protein Rgr (Rgr), 5.13E-06 10.970202 up mRNA. 252

64407 RGS18 Homo sapiens regulator of G-protein signalling 18 1.28E-05 3.1929123 up (RGS18), mRNA. 10287 RGS19 Homo sapiens regulator of G-protein signalling 19 5.13E-06 3.0821266 up (RGS19), mRNA. 57414 RHBDD2 Homo sapiens rhomboid domain containing 2 5.47E-06 2.434615 up (RHBDD2), transcript variant 1, mRNA. 57414 RHBDD2 Homo sapiens rhomboid domain containing 2 5.13E-06 2.4721975 up (RHBDD2), mRNA. 391 RHOG Homo sapiens ras homolog gene family, member G 5.91E-06 2.721033 up (rho G) (RHOG), mRNA. 55288 RHOT1 Homo sapiens ras homolog gene family, member 1.39E-05 2.4219975 up T1 (RHOT1), transcript variant 3, mRNA. 55288 RHOT1 Homo sapiens ras homolog gene family, member 9.31E-05 2.163114 up T1 (RHOT1), transcript variant 2, mRNA. 83547 RILP Homo sapiens Rab interacting lysosomal protein 5.13E-06 2.2058845 up (RILP), mRNA. 79890 RIN3 Homo sapiens Ras and Rab interactor 3 (RIN3), 2.86E-04 2.0112429 up mRNA. 8780 RIOK3 Homo sapiens RIO kinase 3 (yeast) (RIOK3), 4.69E-05 2.0572739 up transcript variant 1, mRNA. 6016 RIT1 Homo sapiens Ras-like without CAAX 1 (RIT1), 5.22E-04 3.5757074 up mRNA. 80010 RMI1 Homo sapiens RMI1, RecQ mediated genome 5.47E-06 2.5162008 up instability 1, homolog (S. cerevisiae) (RMI1), mRNA.

6036 RNASE2 Homo sapiens ribonuclease, RNase A family, 2 5.13E-06 5.5336666 up (liver, eosinophil-derived neurotoxin) (RNASE2), mRNA. 6037 RNASE3 Homo sapiens ribonuclease, RNase A family, 3 3.20E-04 3.9439726 up (eosinophil cationic protein) (RNASE3), mRNA. 246243 RNASEH1 Homo sapiens ribonuclease H1 (RNASEH1), mRNA. 5.27E-06 2.2097256 down 6041 RNASEL Homo sapiens ribonuclease L (2',5'- 1.39E-05 2.4552608 up oligoisoadenylate synthetase-dependent) (RNASEL), mRNA. 55658 RNF126 Homo sapiens ring finger protein 126 (RNF126), 5.47E-06 2.113235 down mRNA. 11342 RNF13 Homo sapiens ring finger protein 13 (RNF13), 1.39E-05 2.6303022 up transcript variant 4, mRNA. 84282 RNF135 Homo sapiens ring finger protein 135 (RNF135), 6.65E-03 2.019627 up transcript variant 1, mRNA. 50862 RNF141 Homo sapiens ring finger protein 141 (RNF141), 6.17E-04 3.1523561 up mRNA. 9781 RNF144 Homo sapiens ring finger protein 144 (RNF144), 3.53E-04 2.483058 down mRNA. 81847 RNF146 Homo sapiens ring finger protein 146 (RNF146), 2.88E-05 3.2138133 up mRNA. 284996 RNF149 Homo sapiens ring finger protein 149 (RNF149), 6.88E-05 2.4710698 up mRNA. 494470 RNF165 Homo sapiens ring finger protein 165 (RNF165), 4.38E-02 2.776474 down mRNA. 6093 ROCK1 Homo sapiens Rho-associated, coiled-coil 9.48E-04 2.0793455 up containing protein kinase 1 (ROCK1), mRNA. 83853 ROPN1L Homo sapiens ropporin 1-like (ROPN1L), mRNA. 5.13E-06 6.6726146 up 6095 RORA Homo sapiens RAR-related orphan receptor A 3.01E-03 2.7249856 down (RORA), transcript variant 3, mRNA. 6102 RP2 Homo sapiens retinitis pigmentosa 2 (X-linked 1.27E-04 2.4424632 up recessive) (RP2), mRNA. 253

6137 RPL13 Homo sapiens ribosomal protein L13 (RPL13), 2.42E-05 2.0406044 down transcript variant 2, mRNA. 23521 RPL13A Homo sapiens ribosomal protein L13a (RPL13A), 1.47E-04 2.0675883 down mRNA. 9045 RPL14 Homo sapiens ribosomal protein L14 (RPL14), 1.47E-04 2.2580352 down mRNA. 6146 RPL22 Homo sapiens ribosomal protein L22 (RPL22), 5.91E-06 2.7956874 down mRNA. 56969 RPL23AP13 Homo sapiens ribosomal protein L23a pseudogene 8.06E-05 2.4020362 down 13 (RPL23AP13) on chromosome 2. 6159 RPL29 Homo sapiens ribosomal protein L29 (RPL29), 8.68E-04 2.031099 up mRNA. 6228 RPS23 Homo sapiens ribosomal protein S23 (RPS23), 1.01E-04 2.7326667 down mRNA. 6191 RPS4X Homo sapiens ribosomal protein S4, X-linked 2.90E-05 2.3750894 down (RPS4X), mRNA. 6195 RPS6KA1 Homo sapiens ribosomal protein S6 kinase, 90kDa, 2.63E-05 2.0691285 up polypeptide 1 (RPS6KA1), transcript variant 2, mRNA. 9252 RPS6KA5 Homo sapiens ribosomal protein S6 kinase, 90kDa, 4.18E-05 3.2871196 down polypeptide 5 (RPS6KA5), transcript variant 1, mRNA. 27079 RPUSD2 Homo sapiens RNA pseudouridylate synthase 5.13E-06 2.3987536 down domain containing 2 (RPUSD2), mRNA. 58528 RRAGD Homo sapiens Ras-related GTP binding D (RRAGD), 5.13E-06 3.5632248 up mRNA. 91543 RSAD2 Homo sapiens radical S-adenosyl methionine 1.37E-02 3.4684372 down domain containing 2 (RSAD2), mRNA. 8634 RTCD1 Homo sapiens RNA terminal phosphate cyclase 6.74E-06 2.1010933 up domain 1 (RTCD1), mRNA. 10313 RTN3 Homo sapiens reticulon 3 (RTN3), transcript variant 7.68E-06 2.698501 up 1, mRNA. 10313 RTN3 Homo sapiens reticulon 3 (RTN3), transcript variant 1.82E-05 2.6037283 up 4, mRNA. 57142 RTN4 Homo sapiens reticulon 4 (RTN4), transcript variant 5.13E-06 3.10555 up 1, mRNA. 57142 RTN4 Homo sapiens reticulon 4 (RTN4), transcript variant 5.13E-06 2.8561363 up 3, mRNA. 864 RUNX3 Homo sapiens runt-related transcription factor 3 5.13E-06 2.8488808 down (RUNX3), transcript variant 2, mRNA. 6256 RXRA Homo sapiens retinoid X receptor, alpha (RXRA), 1.52E-05 2.1180124 up mRNA. 6282 S100A11 Homo sapiens S100 calcium binding protein A11 5.27E-06 3.2982779 up (S100A11), mRNA. 6283 S100A12 Homo sapiens S100 calcium binding protein A12 5.13E-06 12.397408 up (S100A12), mRNA. 6277 S100A6 Homo sapiens S100 calcium binding protein A6 5.13E-06 2.9247186 up (S100A6), mRNA. 6279 S100A8 Homo sapiens S100 calcium binding protein A8 5.13E-06 3.185351 up (S100A8), mRNA. 6286 S100P Homo sapiens S100 calcium binding protein P 5.91E-06 12.538133 up (S100P), mRNA. 154075 SAMD3 Homo sapiens sterile alpha motif domain 1.66E-05 5.683149 down containing 3 (SAMD3), transcript variant 1, mRNA. 64092 SAMSN1 Homo sapiens SAM domain, SH3 domain and 9.10E-06 13.222396 up nuclear localization signals 1 (SAMSN1), mRNA.

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8819 SAP30 Homo sapiens sin3-associated polypeptide, 30kDa 2.81E-05 3.1501167 up (SAP30), mRNA. 51128 SAR1B Homo sapiens SAR1 gene homolog B (S. cerevisiae) 5.13E-06 3.7790694 up (SAR1B), transcript variant 2, mRNA. 51128 SAR1B Homo sapiens SAR1 gene homolog B (S. cerevisiae) 2.88E-05 2.3820426 up (SAR1B), transcript variant 1, mRNA. 29940 SART2 Homo sapiens squamous cell carcinoma antigen 5.47E-06 3.3033214 up recognized by T cells 2 (SART2), mRNA. 6303 SAT Homo sapiens spermidine/spermine N1- 7.25E-06 2.8385026 up acetyltransferase 1 (SAT1), mRNA. 81846 SBF2 Homo sapiens SET binding factor 2 (SBF2), mRNA. 1.82E-04 2.2637892 up 388228 SBK1 Homo sapiens SH3-binding domain kinase 1 (SBK1), 5.13E-06 5.0247912 down mRNA. 8631 SCAP1 Homo sapiens src family associated 5.13E-06 5.5414057 down phosphoprotein 1 (SCAP1), mRNA. 59342 SCPEP1 Homo sapiens serine carboxypeptidase 1 (SCPEP1), 5.13E-06 4.6054783 up mRNA. 55681 SCYL2 Homo sapiens SCY1-like 2 (S. cerevisiae) (SCYL2), 7.25E-06 2.2322147 up mRNA. 6386 SDCBP Homo sapiens syndecan binding protein (syntenin) 1.52E-05 3.1811771 up (SDCBP), transcript variant 2, mRNA. 10807 SDCCAG3 Homo sapiens serologically defined colon cancer 3.14E-05 2.0585663 down antigen 3 (SDCCAG3), mRNA. 10194 SDCCAG33 Homo sapiens serologically defined colon cancer 7.66E-05 2.2308984 down antigen 33 (SDCCAG33), mRNA. 6388 SDF2 Homo sapiens stromal cell-derived factor 2 (SDF2), 5.13E-06 2.6602075 up mRNA. 27020 SDFR1 Homo sapiens stromal cell derived factor receptor 6.74E-06 2.806331 up 1 (SDFR1), transcript variant beta, mRNA. 9117 SEC22C Homo sapiens SEC22 vesicle trafficking protein 2.25E-05 2.3320997 up homolog C (S. cerevisiae) (SEC22C), transcript variant 2, mRNA. 10802 SEC24A Homo sapiens SEC24 related gene family, member 1.09E-05 2.5405312 up A (S. cerevisiae) (SEC24A), mRNA. 6402 SELL Homo sapiens selectin L (lymphocyte adhesion 1.78E-04 2.008816 up molecule 1) (SELL), mRNA. 51714 SELT Homo sapiens selenoprotein T (SELT), mRNA. 6.15E-05 2.498092 up 64218 SEMA4A Homo sapiens sema domain, immunoglobulin 1.52E-05 3.6353498 up domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4A (SEMA4A), mRNA. 51734 SEPX1 Homo sapiens selenoprotein X, 1 (SEPX1), mRNA. 6.74E-06 2.9104857 up 57515 SERINC1 Homo sapiens serine incorporator 1 (SERINC1), 1.28E-05 2.5843377 up mRNA. 5265 SERPINA1 Homo sapiens serpin peptidase inhibitor, clade A 1.26E-02 2.581325 up (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), transcript variant 1, mRNA. 5265 SERPINA1 Homo sapiens serpin peptidase inhibitor, clade A 5.27E-06 3.6783197 up (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), transcript variant 3, mRNA. 5265 SERPINA1 Homo sapiens serpin peptidase inhibitor, clade A 5.27E-06 3.6435215 up (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), transcript variant 2, mRNA. 5265 SERPINA1 Homo sapiens serpin peptidase inhibitor, clade A 1.0E-02 2.7376792 up (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), transcript variant 2, mRNA.

255

1992 SERPINB1 Homo sapiens serpin peptidase inhibitor, clade B 5.13E-06 7.584304 up (ovalbumin), member 1 (SERPINB1), mRNA. 5271 SERPINB8 Homo sapiens serpin peptidase inhibitor, clade B 4.90E-05 3.092629 up (ovalbumin), member 8 (SERPINB8), transcript variant 1, mRNA. 5271 SERPINB8 Homo sapiens serpin peptidase inhibitor, clade B 9.10E-06 3.0228734 up (ovalbumin), member 8 (SERPINB8), transcript variant 3, mRNA. 5270 SERPINE2 Homo sapiens serpin peptidase inhibitor, clade E 6.66E-04 2.0740278 down (nexin, plasminogen activator inhibitor type 1), member 2 (SERPINE2), mRNA. 29946 SERTAD3 Homo sapiens SERTA domain containing 3 4.69E-05 2.0235457 up (SERTAD3), transcript variant 2, mRNA. 387893 SETD8 Homo sapiens SET domain containing (lysine 5.35E-06 2.3038507 up methyltransferase) 8 (SETD8), mRNA. 10946 SF3A3 Homo sapiens splicing factor 3a, subunit 3, 60kDa 6.74E-06 2.0442965 down (SF3A3), mRNA. 51639 SF3B14 Homo sapiens splicing factor 3B, 14 kDa subunit 7.25E-06 2.85155 up (SF3B14), mRNA. 23450 SF3B3 Homo sapiens splicing factor 3b, subunit 3, 130kDa 9.21E-06 2.2218034 down (SF3B3), mRNA. 113402 SFT2D1 Homo sapiens SFT2 domain containing 1 (SFT2D1), 5.13E-06 2.701378 up mRNA. 94081 SFXN1 Homo sapiens sideroflexin 1 (SFXN1), mRNA. 1.08E-05 3.7567773 up 119559 SFXN4 Homo sapiens sideroflexin 4 (SFXN4), transcript 1.39E-05 2.0434752 up variant 2, mRNA. 94097 SFXN5 Homo sapiens sideroflexin 5 (SFXN5), mRNA. 8.95E-05 2.9185047 up 4068 SH2D1A Homo sapiens SH2 domain protein 1A, Duncan's 1.28E-05 3.318976 down disease (lymphoproliferative syndrome) (SH2D1A), mRNA. 51100 SH3GLB1 Homo sapiens SH3-domain GRB2-like endophilin 5.13E-06 4.4182706 up B1 (SH3GLB1), mRNA. 30011 SH3KBP1 Homo sapiens SH3-domain kinase binding protein 2.70E-04 3.7459664 down 1 (SH3KBP1), transcript variant 2, mRNA. 26751 SH3YL1 Homo sapiens SH3 domain containing, Ysc84-like 1 6.31E-06 2.408518 down (S. cerevisiae) (SH3YL1), mRNA. 92799 SHKBP1 Homo sapiens SH3KBP1 binding protein 1 3.18E-05 2.62271 up (SHKBP1), mRNA. 8036 SHOC2 Homo sapiens soc-2 suppressor of clear homolog 1.39E-05 2.5338082 up (C. elegans) (SHOC2), mRNA. 54847 SIDT1 Homo sapiens SID1 transmembrane family, 3.55E-02 2.239598 down member 1 (SIDT1), mRNA. 89790 SIGLEC10 Homo sapiens sialic acid binding Ig-like lectin 10 5.13E-06 5.184889 up (SIGLEC10), mRNA. 8778 SIGLEC5 Homo sapiens sialic acid binding Ig-like lectin 5 5.13E-06 5.2311654 up (SIGLEC5), mRNA. 27036 SIGLEC7 Homo sapiens sialic acid binding Ig-like lectin 7 3.76E-02 3.021048 up (SIGLEC7), transcript variant 1, mRNA. 64374 SIL1 Homo sapiens SIL1 homolog, endoplasmic 1.26E-05 2.7031844 up reticulum chaperone (S. cerevisiae) (SIL1), transcript variant 1, mRNA. 64374 SIL1 Homo sapiens SIL1 homolog, endoplasmic 1.28E-05 2.7401364 up reticulum chaperone (S. cerevisiae) (SIL1), mRNA. 57568 SIPA1L2 Homo sapiens signal-induced proliferation- 5.91E-06 5.884139 up associated 1 like 2 (SIPA1L2), mRNA. 10326 SIRPB1 Homo sapiens signal-regulatory protein beta 1 1.54E-04 2.47641 up (SIRPB1), mRNA. 256

128646 SIRPD Homo sapiens signal-regulatory protein delta 5.12E-03 2.7034366 up (SIRPD), mRNA. 55423 SIRPG Homo sapiens signal-regulatory protein gamma 6.15E-03 3.2933543 down (SIRPG), transcript variant 2, mRNA. 23408 SIRT5 Homo sapiens sirtuin (silent mating type 1.47E-04 2.4177494 up information regulation 2 homolog) 5 (S. cerevisiae) (SIRT5), transcript variant 1, mRNA. 8631 SKAP1 Homo sapiens src kinase associated 1.34E-04 3.3015227 down phosphoprotein 1 (SKAP1), transcript variant 2, mRNA. 8935 SKAP2 Homo sapiens src kinase associated 5.13E-06 3.7175558 up phosphoprotein 2 (SKAP2), mRNA. 6503 SLA Homo sapiens Src-like-adaptor (SLA), transcript 1.79E-05 3.3963377 up variant 1, mRNA. 6503 SLA Homo sapiens Src-like-adaptor (SLA), transcript 5.13E-06 4.604537 up variant 2, mRNA. 114836 SLAMF6 Homo sapiens SLAM family member 6 (SLAMF6), 5.13E-06 2.5163615 down mRNA. 6556 SLC11A1 Homo sapiens solute carrier family 11 (proton- 3.87E-05 2.799787 up coupled divalent metal ion transporters), member 1 (SLC11A1), transcript variant 2, mRNA. 9990 SLC12A6 Homo sapiens solute carrier family 12 1.28E-05 2.38768 up (potassium/chloride transporters), member 6 (SLC12A6), mRNA. 9123 SLC16A3 Homo sapiens solute carrier family 16, member 3 5.91E-06 3.2691417 up (monocarboxylic acid transporter 4) (SLC16A3), transcript variant 2, mRNA. 9120 SLC16A6 Homo sapiens solute carrier family 16 6.38E-05 3.6259203 up (monocarboxylic acid transporters), member 6 (SLC16A6), mRNA. 6573 SLC19A1 Homo sapiens solute carrier family 19 (folate 8.78E-04 2.0835345 up transporter), member 1 (SLC19A1), transcript variant 1, mRNA. 55356 SLC22A15 Homo sapiens solute carrier family 22 (organic 4.19E-03 2.4700074 up cation transporter), member 15 (SLC22A15), mRNA. 6583 SLC22A4 Homo sapiens solute carrier family 22 (organic 1.28E-05 3.9389577 up cation transporter), member 4 (SLC22A4), mRNA. 55972 SLC25A40 Homo sapiens solute carrier family 25, member 40 1.36E-03 5.8174376 up (SLC25A40), mRNA. 65010 SLC26A6 Homo sapiens solute carrier family 26, member 6 8.98E-03 2.2475884 up (SLC26A6), transcript variant 3, mRNA. 66035 SLC2A11 Homo sapiens solute carrier family 2 (facilitated 1.09E-03 12.1210575 up glucose transporter), member 11 (SLC2A11), transcript variant 3, mRNA. 144195 SLC2A14 Homo sapiens solute carrier family 2 (facilitated 1.33E-05 5.573804 up glucose transporter), member 14 (SLC2A14), mRNA. 6515 SLC2A3 Homo sapiens solute carrier family 2 (facilitated 5.13E-06 3.8878593 up glucose transporter), member 3 (SLC2A3), mRNA. 1318 SLC31A2 Homo sapiens solute carrier family 31 (copper 1.39E-05 2.8930454 up transporters), member 2 (SLC31A2), mRNA. 10559 SLC35A1 Homo sapiens solute carrier family 35 (CMP-sialic 1.52E-05 2.2966766 up acid transporter), member A1 (SLC35A1), mRNA. 80255 SLC35F5 Homo sapiens solute carrier family 35, member F5 1.71E-03 2.2013497 up (SLC35F5), mRNA.

257

206358 SLC36A1 Homo sapiens solute carrier family 36 5.27E-06 4.276486 up (proton/amino acid symporter), member 1 (SLC36A1), mRNA. 120103 SLC36A4 Homo sapiens solute carrier family 36 7.25E-06 2.5944667 up (proton/amino acid symporter), member 4 (SLC36A4), mRNA. 81539 SLC38A1 Homo sapiens solute carrier family 38, member 1 5.47E-06 3.1893635 down (SLC38A1), mRNA. 54407 SLC38A2 Homo sapiens solute carrier family 38, member 2 5.91E-06 2.5765672 up (SLC38A2), mRNA. 27173 SLC39A1 Homo sapiens solute carrier family 39 (zinc 5.13E-06 2.3091557 up transporter), member 1 (SLC39A1), mRNA. 64116 SLC39A8 Homo sapiens solute carrier family 39 (zinc 1.81E-04 4.246782 up transporter), member 8 (SLC39A8), mRNA. 30061 SLC40A1 Homo sapiens solute carrier family 40 (iron- 4.15E-04 2.5611005 up regulated transporter), member 1 (SLC40A1), mRNA. 54946 SLC41A3 Homo sapiens solute carrier family 41, member 3 5.62E-06 2.17463 down (SLC41A3), transcript variant 2, mRNA. 54946 SLC41A3 Homo sapiens solute carrier family 41, member 3 9.10E-06 2.2424684 down (SLC41A3), transcript variant 1, mRNA. 23446 SLC44A1 Homo sapiens solute carrier family 44, member 1 1.11E-04 2.156885 up (SLC44A1), transcript variant 2, mRNA. 23446 SLC44A1 Homo sapiens solute carrier family 44, member 1 5.13E-06 2.4726274 up (SLC44A1), transcript variant 1, mRNA. 9057 SLC7A6 Homo sapiens solute carrier family 7 (cationic 6.09E-06 2.7540555 down amino acid transporter, y+ system), member 6 (SLC7A6), mRNA. 10479 SLC9A6 Homo sapiens solute carrier family 9 5.35E-06 2.1164196 up (sodium/hydrogen exchanger), member 6 (SLC9A6), mRNA. 353189 SLCO4C1 Homo sapiens solute carrier organic anion 3.81E-02 2.4337308 up transporter family, member 4C1 (SLCO4C1), mRNA. 9748 SLK Homo sapiens STE20-like kinase (yeast) (SLK), 8.44E-06 3.3453143 up mRNA. 6590 SLPI Homo sapiens secretory leukocyte peptidase 2.20E-04 8.641318 up inhibitor (SLPI), mRNA. 11039 SMA4 Homo sapiens SMA4 (SMA4), mRNA. 1.03E-05 2.3718147 down 4088 SMAD3 Homo sapiens SMAD family member 3 (SMAD3), 5.13E-06 2.3363004 down mRNA. 10924 SMPDL3A Homo sapiens sphingomyelin phosphodiesterase, 7.17E-04 11.779018 up acid-like 3A (SMPDL3A), mRNA.

6525 SMTN Homo sapiens smoothelin (SMTN), transcript 2.84E-04 2.0053816 up variant 3, mRNA. 8773 SNAP23 Homo sapiens synaptosomal-associated protein, 6.88E-05 2.0804262 up 23kDa (SNAP23), transcript variant 2, mRNA. 55509 SNFT Homo sapiens Jun dimerization protein p21SNFT 1.86E-02 2.6653688 up (SNFT), mRNA. 6637 SNRPG Homo sapiens small nuclear ribonucleoprotein 6.55E-03 2.0086162 up polypeptide G (SNRPG), mRNA. 6638 SNRPN Homo sapiens small nuclear ribonucleoprotein 5.27E-06 2.903949 down polypeptide N (SNRPN), transcript variant 2, mRNA. 8926 SNURF Homo sapiens SNRPN upstream reading frame 8.93E-06 2.9593873 down (SNURF), transcript variant 1, mRNA. 29887 SNX10 Homo sapiens sorting nexin 10 (SNX10), mRNA. 1.82E-05 2.7822063 up

258

29916 SNX11 Homo sapiens sorting nexin 11 (SNX11), transcript 6.31E-06 2.0708773 up variant 1, mRNA. 23161 SNX13 Homo sapiens sorting nexin 13 (SNX13), mRNA. 3.87E-05 2.4602334 up 57231 SNX14 Homo sapiens sorting nexin 14 (SNX14), transcript 5.47E-06 2.4378521 up variant 2, mRNA. 81609 SNX27 Homo sapiens sorting nexin family member 27 2.02E-05 2.0202353 up (SNX27), mRNA. 8724 SNX3 Homo sapiens sorting nexin 3 (SNX3), transcript 5.13E-06 3.4656262 up variant 3, mRNA. 8835 SOCS2 Homo sapiens suppressor of cytokine signaling 2 5.70E-04 2.4129548 down (SOCS2), mRNA. 6648 SOD2 Homo sapiens superoxide dismutase 2, 6.88E-05 2.7007437 up mitochondrial (SOD2), nuclear gene encoding mitochondrial protein, transcript variant 3, mRNA. 6648 SOD2 Homo sapiens superoxide dismutase 2, 2.63E-05 2.4178562 up mitochondrial (SOD2), nuclear gene encoding mitochondrial protein, transcript variant 2, mRNA. 6272 SORT1 Homo sapiens sortilin 1 (SORT1), mRNA. 5.13E-06 7.3709874 up 6672 SP100 Homo sapiens SP100 nuclear antigen (SP100), 1.34E-04 2.216641 up transcript variant 1, mRNA. 11262 SP140 Homo sapiens SP140 nuclear body protein (SP140), 7.68E-06 2.5960333 down transcript variant 1, mRNA. 60559 SPCS3 Homo sapiens signal peptidase complex subunit 3 4.60E-05 2.8208823 up homolog (S. cerevisiae) (SPCS3), mRNA. 11160 SPFH2 Homo sapiens SPFH domain family, member 2 2.29E-05 2.4365819 up (SPFH2), transcript variant 1, mRNA. 6688 SPI1 Homo sapiens spleen focus forming virus (SFFV) 1.66E-05 2.9030843 up proviral integration oncogene spi1 (SPI1), mRNA. 6688 SPI1 Homo sapiens spleen focus forming virus (SFFV) 5.35E-06 3.2189314 up proviral integration oncogene spi1 (SPI1), transcript variant 2, mRNA. 6689 SPIB Homo sapiens Spi-B transcription factor (Spi- 5.78E-05 2.4692757 down 1/PU.1 related) (SPIB), mRNA. 9806 SPOCK2 Homo sapiens sparc/osteonectin, cwcv and kazal- 5.13E-06 4.714466 down like domains proteoglycan (testican) 2 (SPOCK2), mRNA. 84888 SPPL2A Homo sapiens signal peptide peptidase-like 2A 5.13E-06 4.0812507 up (SPPL2A), mRNA. 9517 SPTLC2 Homo sapiens serine palmitoyltransferase, long 1.43E-03 2.195914 up chain base subunit 2 (SPTLC2), mRNA. 58472 SQRDL Homo sapiens sulfide quinone reductase-like 7.68E-06 3.2714407 up (yeast) (SQRDL), mRNA. 5552 SRGN Homo sapiens serglycin (SRGN), mRNA. 6.74E-06 2.9109418 up 390284 SRP14P1 Homo sapiens signal recognition particle 14kDa 2.02E-05 2.058656 up (homologous Alu RNA binding protein) pseudogene 1 (SRP14P1) on chromosome 12. 6732 SRPK1 Homo sapiens SFRS protein kinase 1 (SRPK1), 1.96E-04 2.5812087 up mRNA. 140809 SRXN1 Homo sapiens sulfiredoxin 1 homolog (S. 4.68E-05 2.3871 up cerevisiae) (SRXN1), mRNA. 23648 SSBP3 Homo sapiens single stranded DNA binding protein 1.83E-04 2.1193433 down 3 (SSBP3), transcript variant 1, mRNA. 170463 SSBP4 Homo sapiens single stranded DNA binding protein 5.27E-06 2.2562535 down 4 (SSBP4), transcript variant 1, mRNA. 54434 SSH1 Homo sapiens slingshot homolog 1 (Drosophila) 7.19E-04 2.279053 up (SSH1), mRNA.

259

85464 SSH2 Homo sapiens slingshot homolog 2 (Drosophila) 6.74E-06 2.4406815 up (SSH2), mRNA. 6484 ST3GAL4 Homo sapiens ST3 beta-galactoside alpha-2,3- 2.86E-03 2.4027174 up sialyltransferase 4 (ST3GAL4), mRNA. 8869 ST3GAL5 Homo sapiens ST3 beta-galactoside alpha-2,3- 5.35E-06 2.7190828 down sialyltransferase 5 (ST3GAL5), mRNA. 6480 ST6GAL1 Homo sapiens ST6 beta-galactosamide alpha-2,6- 6.31E-06 2.2134047 down sialyltranferase 1 (ST6GAL1), transcript variant 3, mRNA. 256435 ST6GALNAC3 Homo sapiens ST6 (alpha-N-acetyl-neuraminyl-2,3- 3.60E-03 8.124919 up beta-galactosyl-1, 3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 3 (ST6GALNAC3), mRNA. 30815 ST6GALNAC6 Homo sapiens ST6 (alpha-N-acetyl-neuraminyl-2,3- 9.95E-05 2.0022986 down beta-galactosyl-1, 3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 6 (ST6GALNAC6), mRNA. 23166 STAB1 Homo sapiens stabilin 1 (STAB1), mRNA. 6.88E-05 2.6656153 up 83930 STARD3NL Homo sapiens STARD3 N-terminal like (STARD3NL), 2.51E-05 2.5439224 up mRNA. 6772 STAT1 Homo sapiens signal transducer and activator of 1.47E-04 2.320497 down transcription 1, 91kDa (STAT1), transcript variant alpha, mRNA. 6772 STAT1 Homo sapiens signal transducer and activator of 1.34E-04 2.5204864 down transcription 1, 91kDa (STAT1), transcript variant beta, mRNA. 6772 STAT1 Homo sapiens signal transducer and activator of 9.15E-05 3.1338727 down transcription 1, 91kDa (STAT1), transcript variant beta, mRNA. 6775 STAT4 Homo sapiens signal transducer and activator of 1.17E-05 3.587767 down transcription 4 (STAT4), mRNA. 79689 STEAP4 Homo sapiens STEAP family member 4 (STEAP4), 1.89E-03 2.6824038 up mRNA. 8576 STK16 Homo sapiens serine/threonine kinase 16 (STK16), 3.41E-03 2.0608761 up transcript variant 1, mRNA. 9262 STK17B Homo sapiens serine/threonine kinase 17b 2.3E-03 2.107266 up (STK17B), mRNA. 6788 STK3 Homo sapiens serine/threonine kinase 3 (STE20 1.09E-05 4.4723954 up homolog, yeast) (STK3), mRNA. 23012 STK38L Homo sapiens serine/threonine kinase 38 like 1.17E-03 2.07433 up (STK38L), mRNA. 27347 STK39 Homo sapiens serine threonine kinase 39 1.67E-05 2.5048876 down (STE20/SPS1 homolog, yeast) (STK39), mRNA. 2040 STOM Homo sapiens stomatin (STOM), transcript variant 4.90E-05 6.5691223 up 1, mRNA. 2040 STOM Homo sapiens stomatin (STOM), transcript variant 5.13E-06 7.167354 up 1, mRNA. 6809 STX3A Homo sapiens syntaxin 3 (STX3), mRNA. 1.39E-05 3.2399926 up 8417 STX7 Homo sapiens syntaxin 7 (STX7), mRNA. 1.32E-02 2.061515 up 6813 STXBP2 Homo sapiens syntaxin binding protein 2 (STXBP2), 5.13E-06 6.9053655 up mRNA. 6814 STXBP3 Homo sapiens syntaxin binding protein 3 (STXBP3), 5.17E-05 2.184866 up mRNA. 134957 STXBP5 Homo sapiens syntaxin binding protein 5 8.44E-06 2.893183 up (tomosyn) (STXBP5), mRNA.

260

10923 SUB1 Homo sapiens SUB1 homolog (S. cerevisiae) 1.1E-03 2.1046803 up (SUB1), mRNA. 55959 SULF2 Homo sapiens sulfatase 2 (SULF2), transcript 1.12E-02 2.018624 down variant 1, mRNA. 55959 SULF2 Homo sapiens sulfatase 2 (SULF2), transcript 1.66E-04 2.5312667 down variant 1, mRNA. 285362 SUMF1 Homo sapiens sulfatase modifying factor 1 3.87E-05 2.0271306 up (SUMF1), mRNA. 6821 SUOX Homo sapiens sulfite oxidase (SUOX), nuclear gene 2.58E-03 2.2054517 up encoding mitochondrial protein, transcript variant 2, mRNA. 6821 SUOX Homo sapiens sulfite oxidase (SUOX), nuclear gene 5.47E-06 2.2596295 up encoding mitochondrial protein, transcript variant 2, mRNA. 64420 SUSD1 Homo sapiens sushi domain containing 1 (SUSD1), 5.13E-06 2.4038653 up mRNA. 6840 SVIL Homo sapiens supervillin (SVIL), transcript variant 5.91E-06 2.8247247 up 1, mRNA. 6845 SYBL1 Homo sapiens synaptobrevin-like 1 (SYBL1), mRNA. 5.35E-06 2.481822 up

6850 SYK Homo sapiens spleen tyrosine kinase (SYK), mRNA. 7.68E-06 2.0453272 up

23224 SYNE2 Homo sapiens spectrin repeat containing, nuclear 2.29E-04 2.319495 down envelope 2 (SYNE2), transcript variant 1, mRNA. 8867 SYNJ1 Homo sapiens synaptojanin 1 (SYNJ1), transcript 2.90E-05 2.0787117 up variant 2, mRNA. 54843 SYTL2 Homo sapiens synaptotagmin-like 2 (SYTL2), 1.69E-05 3.6670816 down transcript variant e, mRNA. 6879 TAF7 Homo sapiens TAF7 RNA polymerase II, TATA box 2.20E-05 2.3324485 up binding protein (TBP)-associated factor, 55kDa (TAF7), mRNA. 117289 TAGAP Homo sapiens T-cell activation GTPase activating 9.10E-06 2.5182037 down protein (TAGAP), transcript variant 3, mRNA. 117289 TAGAP Homo sapiens T-cell activation GTPase activating 7.68E-06 2.3894684 down protein (TAGAP), transcript variant 2, mRNA. 10010 TANK Homo sapiens TRAF family member-associated 5.69E-05 2.4050903 up NFKB activator (TANK), transcript variant 1, mRNA. 6891 TAP2 Homo sapiens transporter 2, ATP-binding cassette, 1.11E-04 2.161089 down sub-family B (MDR/TAP) (TAP2), transcript variant 1, mRNA. 8887 TAX1BP1 Homo sapiens Tax1 (human T-cell leukemia virus 9.10E-06 2.3963335 up type I) binding protein 1 (TAX1BP1), mRNA. 8887 TAX1BP1 Homo sapiens Tax1 (human T-cell leukemia virus 5.27E-06 2.4154434 up type I) binding protein 1 (TAX1BP1), transcript variant 2, mRNA. 57533 TBC1D14 Homo sapiens TBC1 domain family, member 14 5.13E-06 2.5938985 up (TBC1D14), mRNA. 55357 TBC1D2 Homo sapiens TBC1 domain family, member 2 5.13E-06 3.0845976 up (TBC1D2), mRNA. 9882 TBC1D4 Homo sapiens TBC1 domain family, member 4 3.81E-03 2.0122747 down (TBC1D4), mRNA. 51256 TBC1D7 Homo sapiens TBC1 domain family, member 7 7.25E-06 2.1340473 up (TBC1D7), mRNA. 6902 TBCA Homo sapiens tubulin-specific chaperone a (TBCA), 5.35E-06 2.2097712 up mRNA. 29110 TBK1 Homo sapiens TANK-binding kinase 1 (TBK1), 4.26E-05 2.145113 up mRNA.

261

6916 TBXAS1 Homo sapiens thromboxane A synthase 1 (platelet, 5.13E-06 4.053732 up cytochrome P450, family 5, subfamily A) (TBXAS1), transcript variant TXS-I, mRNA. 6919 TCEA2 Homo sapiens transcription elongation factor A 5.13E-06 2.2153099 down (SII), 2 (TCEA2), transcript variant 1, mRNA. 6920 TCEA3 Homo sapiens transcription elongation factor A 1.31E-03 2.8047628 down (SII), 3 (TCEA3), mRNA. 6921 TCEB1 Homo sapiens transcription elongation factor B 5.35E-06 2.0938437 up (SIII), polypeptide 1 (15kDa, elongin C) (TCEB1), mRNA. 6932 TCF7 Homo sapiens transcription factor 7 (T-cell specific, 1.34E-02 2.2970107 down HMG-box) (TCF7), transcript variant 2, mRNA.

6932 TCF7 Homo sapiens transcription factor 7 (T-cell specific, 1.65E-02 2.2085178 down HMG-box) (TCF7), transcript variant 3, mRNA.

10312 TCIRG1 Homo sapiens T-cell, immune regulator 1, ATPase, 2.42E-05 2.1058588 up H+ transporting, lysosomal V0 subunit A3 (TCIRG1), transcript variant 2, mRNA. 6947 TCN1 Homo sapiens transcobalamin I (vitamin B12 8.44E-06 13.522709 up binding protein, R binder family) (TCN1), mRNA. 6948 TCN2 Homo sapiens transcobalamin II; macrocytic 9.97E-03 2.5073173 up anemia (TCN2), mRNA. 122402 TDRD9 Homo sapiens tudor domain containing 9 (TDRD9), 1.45E-04 9.490689 up mRNA. 7008 TEF Homo sapiens thyrotrophic embryonic factor (TEF), 9.10E-06 2.4008708 up mRNA. 54997 TESC Homo sapiens tescalcin (TESC), mRNA. 5.61E-03 2.0003784 up 7037 TFRC Homo sapiens transferrin receptor (p90, CD71) 7.15E-04 2.4574177 up (TFRC), mRNA. 7039 TGFA Homo sapiens transforming growth factor, alpha 1.00E-05 4.844284 up (TGFA), mRNA. 7045 TGFBI Homo sapiens transforming growth factor, beta- 3.72E-03 2.1941187 down induced, 68kDa (TGFBI), mRNA. 7049 TGFBR3 Homo sapiens transforming growth factor, beta 1.34E-04 4.7717967 down receptor III (TGFBR3), mRNA. 60436 TGIF2 Homo sapiens TGFB-induced factor 2 (TALE family 6.85E-04 2.0344095 down homeobox) (TGIF2), mRNA. 7057 THBS1 Homo sapiens thrombospondin 1 (THBS1), mRNA. 5.48E-04 14.143862 up 90459 THEX1 Homo sapiens three prime histone mRNA 4.1E-02 2.9041352 up exonuclease 1 (THEX1), mRNA. 7076 TIMP1 Homo sapiens TIMP metallopeptidase inhibitor 1 5.27E-06 3.5237439 up (TIMP1), mRNA. 7077 TIMP2 Homo sapiens TIMP metallopeptidase inhibitor 2 3.60E-05 3.552546 up (TIMP2), mRNA. 7077 TIMP2 Homo sapiens TIMP metallopeptidase inhibitor 2 5.47E-06 3.2073245 up (TIMP2), mRNA. 25976 TIPARP Homo sapiens TCDD-inducible poly(ADP-ribose) 1.39E-05 2.9189072 up polymerase (TIPARP), mRNA. 7084 TK2 Homo sapiens thymidine kinase 2, mitochondrial 6.88E-04 2.0480692 up (TK2), mRNA. 7086 TKT Homo sapiens transketolase (Wernicke-Korsakoff 1.22E-04 2.030949 up syndrome) (TKT), mRNA. 7096 TLR1 Homo sapiens toll-like receptor 1 (TLR1), mRNA. 1.11E-04 2.3303494 up 7097 TLR2 Homo sapiens toll-like receptor 2 (TLR2), mRNA. 2.20E-04 5.9200134 up

262

7099 TLR4 Homo sapiens toll-like receptor 4 (TLR4), transcript 6.74E-06 3.5023856 up variant 4, mRNA. 7100 TLR5 Homo sapiens toll-like receptor 5 (TLR5), mRNA. 5.27E-06 5.570661 up 51284 TLR7 Homo sapiens toll-like receptor 7 (TLR7), mRNA. 3.18E-05 2.8307898 down 51311 TLR8 Homo sapiens toll-like receptor 8 (TLR8), transcript 9.10E-06 4.0001364 up variant 1, mRNA. 51311 TLR8 Homo sapiens toll-like receptor 8 (TLR8), transcript 8.44E-06 3.8374279 up variant 2, mRNA. 53346 TM6SF1 Homo sapiens transmembrane 6 superfamily 7.68E-06 3.1814644 up member 1 (TM6SF1), mRNA. 9375 TM9SF2 Homo sapiens transmembrane 9 superfamily 6.74E-06 2.3152728 up member 2 (TM9SF2), mRNA. 64114 TMBIM1 Homo sapiens transmembrane BAX inhibitor motif 1.39E-05 2.0240154 up containing 1 (TMBIM1), mRNA. 51643 TMBIM4 Homo sapiens transmembrane BAX inhibitor motif 5.13E-06 2.1981583 up containing 4 (TMBIM4), mRNA. 54499 TMCO1 Homo sapiens transmembrane and coiled-coil 6.88E-05 2.277675 up domains 1 (TMCO1), mRNA. 55002 TMCO3 Homo sapiens transmembrane and coiled-coil 1.09E-05 4.236887 up domains 3 (TMCO3), mRNA. 50999 TMED5 Homo sapiens transmembrane emp24 protein 5.47E-06 3.1973817 up transport domain containing 5 (TMED5), mRNA. 79073 TMEM109 Homo sapiens transmembrane protein 109 5.27E-06 2.2828548 down (TMEM109), mRNA. 79134 TMEM185B Homo sapiens transmembrane protein 185B 1.08E-05 2.634556 up (TMEM185B) on chromosome 2. 23670 TMEM2 Homo sapiens transmembrane protein 2 (TMEM2), 9.10E-06 2.0314133 up mRNA. 120224 TMEM45B Homo sapiens transmembrane protein 45B 4.51E-02 2.1744459 up (TMEM45B), mRNA. 55529 TMEM55A Homo sapiens transmembrane protein 55A 3.09E-04 2.226089 up (TMEM55A), mRNA. 54968 TMEM70 Homo sapiens transmembrane protein 70 2.62E-03 2.0122697 up (TMEM70), mRNA. 58986 TMEM8 Homo sapiens transmembrane protein 8 (five 5.46E-04 2.111879 up membrane-spanning domains) (TMEM8), mRNA. 641649 TMEM91 Homo sapiens transmembrane protein 91 1.16E-02 2.068093 up (TMEM91), mRNA. 147184 TMEM99 Homo sapiens transmembrane protein 99 8.18E-05 2.0539787 down (TMEM99), mRNA. 83862 TMPIT Homo sapiens transmembrane protein 120A 5.13E-06 5.330161 up (TMEM120A), mRNA. 79089 TMUB2 Homo sapiens transmembrane and ubiquitin-like 5.27E-06 2.0301595 up domain containing 2 (TMUB2), transcript variant 1, mRNA. 7128 TNFAIP3 Homo sapiens tumor necrosis factor, alpha- 5.13E-06 2.535046 up induced protein 3 (TNFAIP3), mRNA. 126282 TNFAIP8L1 Homo sapiens tumor necrosis factor, alpha- 4.02E-05 2.3987746 down induced protein 8-like 1 (TNFAIP8L1), mRNA. 7132 TNFRSF1A Homo sapiens tumor necrosis factor receptor 6.88E-05 2.2245054 up superfamily, member 1A (TNFRSF1A), mRNA. 8718 TNFRSF25 Homo sapiens tumor necrosis factor receptor 7.25E-06 5.231462 down superfamily, member 25 (TNFRSF25), transcript variant 10, mRNA. 939 TNFRSF7 Homo sapiens tumor necrosis factor receptor 5.27E-06 6.002582 down superfamily, member 7 (TNFRSF7), mRNA.

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54472 TOLLIP Homo sapiens toll interacting protein (TOLLIP), 5.13E-06 2.223728 up mRNA. 10043 TOM1 Homo sapiens target of myb1 (chicken) (TOM1), 1.39E-05 2.5397213 up mRNA. 1861 TOR1A Homo sapiens torsin family 1, member A (torsin A) 9.97E-06 2.355419 up (TOR1A), mRNA. 26092 TOR1AIP1 Homo sapiens torsin A interacting protein 1 2.42E-05 2.074896 up (TOR1AIP1), mRNA. 9540 TP53I3 Homo sapiens tumor protein p53 inducible protein 1.08E-02 5.992417 up 3 (TP53I3), transcript variant 2, mRNA. 55858 TPARL Homo sapiens transmembrane protein 165 3.13E-04 2.313302 up (TMEM165), mRNA. 7167 TPI1 Homo sapiens triosephosphate isomerase 1 (TPI1), 6.31E-06 2.151335 up mRNA. 27010 TPK1 Homo sapiens thiamin pyrophosphokinase 1 3.08E-03 3.0417762 up (TPK1), transcript variant 2, mRNA. 27010 TPK1 Homo sapiens thiamin pyrophosphokinase 1 5.13E-06 3.9716504 up (TPK1), mRNA. 8460 TPST1 Homo sapiens tyrosylprotein sulfotransferase 1 8.12E-04 5.207051 up (TPST1), mRNA. 8459 TPST2 Homo sapiens tyrosylprotein sulfotransferase 2 5.13E-06 2.8909817 up (TPST2), transcript variant 1, mRNA. 7188 TRAF5 Homo sapiens TNF receptor-associated factor 5 5.62E-06 3.0638025 down (TRAF5), transcript variant 1, mRNA. 79865 TREML2 Homo sapiens triggering receptor expressed on 1.82E-05 2.6735244 up myeloid cells-like 2 (TREML2), mRNA. 10221 TRIB1 Homo sapiens tribbles homolog 1 (Drosophila) 5.27E-06 4.1030045 up (TRIB1), mRNA. 28951 TRIB2 Homo sapiens tribbles homolog 2 (Drosophila) 8.62E-05 2.7836354 down (TRIB2), mRNA. 7706 TRIM25 Homo sapiens tripartite motif-containing 25 2.90E-05 2.73022 up (TRIM25), mRNA. 23650 TRIM29 Homo sapiens tripartite motif-containing 29 3.64E-05 2.2921526 up (TRIM29), transcript variant 1, mRNA. 54765 TRIM44 Homo sapiens tripartite motif-containing 44 5.13E-06 2.1404412 down (TRIM44), mRNA. 6738 TROVE2 Homo sapiens TROVE domain family, member 2 7.50E-04 2.1647313 up (TROVE2), mRNA. 140803 TRPM6 Homo sapiens transient receptor potential cation 1.60E-04 2.230239 up channel, subfamily M, member 6 (TRPM6), mRNA. 79042 TSEN34 Homo sapiens tRNA splicing endonuclease 34 1.17E-05 2.6112914 up homolog (S. cerevisiae) (TSEN34), mRNA. 79042 TSEN34 Homo sapiens tRNA splicing endonuclease 34 2.90E-05 2.4600291 up homolog (S. cerevisiae) (TSEN34), transcript variant 2, mRNA. 283989 TSEN54 Homo sapiens tRNA splicing endonuclease 54 4.33E-02 2.2225332 down homolog (S. cerevisiae) (TSEN54), mRNA. 81619 TSPAN14 Homo sapiens tetraspanin 14 (TSPAN14), mRNA. 1.52E-05 2.302972 up 10100 TSPAN2 Homo sapiens tetraspanin 2 (TSPAN2), mRNA. 9.65E-04 2.7537334 up 10077 TSPAN32 Homo sapiens tetraspanin 32 (TSPAN32), transcript 5.35E-06 2.1602862 down variant 1, mRNA. 706 TSPO Homo sapiens translocator protein (18kDa) (TSPO), 5.13E-06 6.5941825 up transcript variant PBR-S, mRNA. 706 TSPO Homo sapiens translocator protein (18kDa) (TSPO), 5.13E-06 3.5931153 up transcript variant PBR, mRNA.

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706 TSPO Homo sapiens translocator protein (18kDa) (TSPO), 5.13E-06 6.5472555 up transcript variant PBR, mRNA. 7267 TTC3 Homo sapiens tetratricopeptide repeat domain 3 9.10E-06 2.5195348 down (TTC3), transcript variant 1, mRNA. 7294 TXK Homo sapiens TXK tyrosine kinase (TXK), mRNA. 1.31E-04 2.4760602 down 7295 TXN Homo sapiens thioredoxin (TXN), mRNA. 5.13E-06 5.347241 up 81542 TXNDC Homo sapiens thioredoxin domain containing 2.90E-05 2.2423642 up (TXNDC), mRNA. 51314 TXNDC3 Homo sapiens thioredoxin domain containing 3 5.65E-03 2.644698 up (spermatozoa) (TXNDC3), mRNA. 84817 TXNL5 Homo sapiens thioredoxin-like 5 (TXNL5), mRNA. 5.13E-06 3.4908164 up 7296 TXNRD1 Homo sapiens thioredoxin reductase 1 (TXNRD1), 5.13E-06 2.4054728 up transcript variant 1, mRNA. 7305 TYROBP Homo sapiens TYRO protein tyrosine kinase 9.97E-06 2.0423567 up binding protein (TYROBP), transcript variant 1, mRNA. 219743 TYSND1 Homo sapiens trypsin domain containing 1 5.13E-06 2.4522114 down (TYSND1), mRNA. 51271 UBAP1 Homo sapiens ubiquitin associated protein 1 5.35E-06 2.5244715 up (UBAP1), mRNA. 53347 UBASH3A Homo sapiens ubiquitin associated and SH3 2.34E-02 2.0363843 down domain containing, A (UBASH3A), transcript variant 2, mRNA. 55236 UBE1L2 Homo sapiens ubiquitin-activating enzyme E1-like 1.39E-05 2.293835 up 2 (UBE1L2), mRNA. 7319 UBE2A Homo sapiens ubiquitin-conjugating enzyme E2A 7.68E-06 2.0290892 up (RAD6 homolog) (UBE2A), transcript variant 2, mRNA. 11065 UBE2C Homo sapiens ubiquitin-conjugating enzyme E2C 1.99E-03 2.1636586 up (UBE2C), transcript variant 6, mRNA. 140739 UBE2F Homo sapiens ubiquitin-conjugating enzyme E2F 5.27E-06 2.6726508 up (putative) (UBE2F), mRNA. 7328 UBE2H Homo sapiens ubiquitin-conjugating enzyme E2H 1.00E-05 4.5497627 up (UBC8 homolog, yeast) (UBE2H), transcript variant 1, mRNA. 7328 UBE2H Homo sapiens ubiquitin-conjugating enzyme E2H 1.08E-05 2.8271759 up (UBC8 homolog, yeast) (UBE2H), transcript variant 2, mRNA. 51465 UBE2J1 Homo sapiens ubiquitin-conjugating enzyme E2, J1 6.74E-06 2.4162953 up (UBC6 homolog, yeast) (UBE2J1), mRNA. 9246 UBE2L6 Homo sapiens ubiquitin-conjugating enzyme E2L 6 8.33E-05 2.174446 down (UBE2L6), transcript variant 1, mRNA. 59286 UBL5 Homo sapiens ubiquitin-like 5 (UBL5), transcript 5.47E-06 2.7284927 up variant 2, mRNA. 29979 UBQLN1 Homo sapiens ubiquilin 1 (UBQLN1), transcript 2.20E-05 2.063639 up variant 2, mRNA. 80019 UBTD1 Homo sapiens ubiquitin domain containing 1 9.10E-06 4.6596165 up (UBTD1), mRNA. 7357 UGCG Homo sapiens UDP-glucose ceramide 5.13E-06 5.3274803 up glucosyltransferase (UGCG), mRNA. 7360 UGP2 Homo sapiens UDP-glucose pyrophosphorylase 2 1.08E-05 2.282324 up (UGP2), transcript variant 1, mRNA. 7360 UGP2 Homo sapiens UDP-glucose pyrophosphorylase 2 8.44E-06 2.6670218 up (UGP2), transcript variant 1, mRNA. 7360 UGP2 Homo sapiens UDP-glucose pyrophosphorylase 2 5.91E-06 2.4629483 up (UGP2), transcript variant 1, mRNA.

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23353 UNC84A Homo sapiens unc-84 homolog A (C. elegans) 3.90E-05 2.0030198 down (UNC84A), mRNA. 7374 UNG Homo sapiens uracil-DNA glycosylase (UNG), 2.20E-05 2.3123088 down transcript variant 2, mRNA. 284415 UNQ3033 Homo sapiens LAIR hlog (UNQ3033), mRNA. 1.62E-05 5.5203085 up 65110 UPF3A Homo sapiens UPF3 regulator of nonsense 4.99E-05 2.1988344 down transcripts homolog A (yeast) (UPF3A), transcript variant 1, mRNA. 7378 UPP1 Homo sapiens uridine phosphorylase 1 (UPP1), 5.13E-06 7.378657 up transcript variant 2, mRNA. 7390 UROS Homo sapiens uroporphyrinogen III synthase 7.25E-06 2.0056314 down (congenital erythropoietic porphyria) (UROS), mRNA. 83706 URP2 Homo sapiens UNC-112 related protein 2 (URP2), 1.66E-05 2.1923494 up transcript variant URP2SF, mRNA. 83706 URP2 Homo sapiens UNC-112 related protein 2 (URP2), 1.39E-05 2.1220028 up transcript variant URP2SF, mRNA. 9958 USP15 Homo sapiens ubiquitin specific peptidase 15 3.40E-04 2.256709 up (USP15), mRNA. 9098 USP6 Homo sapiens ubiquitin specific peptidase 6 (Tre-2 2.51E-04 2.4982307 up oncogene) (USP6), mRNA. 9341 VAMP3 Homo sapiens vesicle-associated membrane 6.31E-06 2.7583616 up protein 3 (cellubrevin) (VAMP3), mRNA. 8674 VAMP4 Homo sapiens vesicle-associated membrane 8.93E-05 2.3848705 up protein 4 (VAMP4), transcript variant 2, mRNA. 7407 VARS Homo sapiens valyl-tRNA synthetase (VARS), 1.92E-05 2.0157428 down mRNA. 7408 VASP Homo sapiens vasodilator-stimulated 8.44E-06 3.081069 up phosphoprotein (VASP), transcript variant 2, mRNA. 10493 VAT1 Homo sapiens vesicle amine transport protein 1 5.28E-05 2.1259196 up homolog (T californica) (VAT1), mRNA. 7423 VEGFB PREDICTED: Homo sapiens vascular endothelial 8.44E-06 2.5253143 down growth factor B (VEGFB), mRNA. 7431 VIM Homo sapiens vimentin (VIM), mRNA. 1.52E-05 2.125174 up 8876 VNN1 Homo sapiens vanin 1 (VNN1), mRNA. 5.13E-06 6.509467 up 8875 VNN2 Homo sapiens vanin 2 (VNN2), transcript variant 1, 1.30E-03 3.028327 up mRNA. 8875 VNN2 Homo sapiens vanin 2 (VNN2), transcript variant 1, 2.42E-05 2.9667041 up mRNA. 29802 VPREB3 Homo sapiens pre-B lymphocyte gene 3 (VPREB3), 7.27E-05 2.9298446 down mRNA. 54832 VPS13C Homo sapiens vacuolar protein sorting 13 homolog 9.55E-04 2.0049055 down C (S. cerevisiae) (VPS13C), transcript variant 1B, mRNA. 84313 VPS25 Homo sapiens vacuolar protein sorting 25 homolog 7.25E-06 2.0142643 up (S. cerevisiae) (VPS25), mRNA. 51699 VPS29 Homo sapiens vacuolar protein sorting 29 (yeast) 6.74E-06 2.2137716 up (VPS29), transcript variant 1, mRNA. 55737 VPS35 Homo sapiens vacuolar protein sorting 35 homolog 6.31E-06 2.0989456 up (S. cerevisiae) (VPS35), mRNA. 84277 WBSCR18 Homo sapiens Williams Beuren syndrome 5.13E-06 2.7013147 down chromosome region 18 (WBSCR18), mRNA. 114049 WBSCR22 Homo sapiens Williams Beuren syndrome 5.13E-06 2.18183 down chromosome region 22 (WBSCR22), mRNA.

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23001 WDFY3 Homo sapiens WD repeat and FYVE domain 8.29E-03 2.4394045 up containing 3 (WDFY3), transcript variant 2, mRNA. 55339 WDR33 Homo sapiens WD repeat domain 33 (WDR33), 3.00E-04 2.155574 down transcript variant 3, mRNA. 282809 WDR51B Homo sapiens WD repeat domain 51B (WDR51B), 2.81E-05 2.2347162 up mRNA. 84058 WDR54 Homo sapiens WD repeat domain 54 (WDR54), 5.13E-06 2.8456492 down mRNA. 54663 WDR74 PREDICTED: Homo sapiens WD repeat domain 74 4.58E-05 2.2829664 down (WDR74), mRNA. 55062 WIPI1 Homo sapiens WD repeat domain, 5.27E-06 4.774687 up phosphoinositide interacting 1 (WIPI1), mRNA. 7485 WRB Homo sapiens tryptophan rich basic protein (WRB), 9.10E-06 2.1208797 up mRNA. 26118 WSB1 Homo sapiens WD repeat and SOCS box-containing 5.35E-06 3.253631 up 1 (WSB1), transcript variant 3, mRNA. 26118 WSB1 Homo sapiens WD repeat and SOCS box-containing 5.27E-06 3.7353961 up 1 (WSB1), transcript variant 2, mRNA. 55884 WSB2 Homo sapiens WD repeat and SOCS box-containing 5.35E-06 2.6757147 up 2 (WSB2), mRNA. 11060 WWP2 Homo sapiens WW domain containing E3 ubiquitin 1.55E-3 2.622582 up protein ligase 2 (WWP2), transcript variant 2, mRNA. 54739 XAF1 Homo sapiens XIAP associated factor-1 (XAF1), 6.37E-05 4.115252 down transcript variant 2, mRNA. 54739 XAF1 Homo sapiens XIAP associated factor-1 (XAF1), 8.17E-03 2.0057201 down transcript variant 2, mRNA. 7504 XK Homo sapiens X-linked Kx blood group (McLeod 4.04E-04 2.7637672 up syndrome) (XK), mRNA. 64328 XPO4 Homo sapiens exportin 4 (XPO4), mRNA. 1.85E-05 2.0293672 down 54432 YIPF1 Homo sapiens Yip1 domain family, member 1 6.74E-06 2.4743662 up (YIPF1), mRNA. 7532 YWHAG Homo sapiens tyrosine 3- 5.13E-06 2.2790608 up monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide (YWHAG), mRNA. 7533 YWHAH Homo sapiens tyrosine 3- 7.68E-06 2.1762502 up monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide (YWHAH), mRNA. 51776 ZAK Homo sapiens sterile alpha motif and leucine 1.67E-04 2.360896 up zipper containing kinase AZK (ZAK), transcript variant 2, mRNA. 7535 ZAP70 Homo sapiens zeta-chain (TCR) associated protein 8.66E-06 4.574251 down kinase 70kDa (ZAP70), transcript variant 2, mRNA.

81030 ZBP1 Homo sapiens Z-DNA binding protein 1 (ZBP1), 2.86E-04 2.3077092 down mRNA. 7704 ZBTB16 Homo sapiens zinc finger and BTB domain 5.78E-05 4.2436647 up containing 16 (ZBTB16), transcript variant 1, mRNA. 9841 ZBTB24 Homo sapiens zinc finger and BTB domain 8.44E-06 2.0458806 down containing 24 (ZBTB24), mRNA. 57659 ZBTB4 Homo sapiens zinc finger and BTB domain 8.44E-06 2.0443172 down containing 4 (ZBTB4), mRNA. 285381 ZCSL2 Homo sapiens DPH3, KTI11 homolog (S. cerevisiae) 9.10E-06 3.1996632 up (DPH3), transcript variant 1, mRNA.

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84885 ZDHHC12 Homo sapiens zinc finger, DHHC-type containing 12 7.57E-05 2.0542245 up (ZDHHC12), mRNA. 131540 ZDHHC19 Homo sapiens zinc finger, DHHC-type containing 19 5.22E-04 33.226856 up (ZDHHC19), mRNA. 51304 ZDHHC3 Homo sapiens zinc finger, DHHC-type containing 3 6.17E-06 3.297276 up (ZDHHC3), mRNA. 29801 ZDHHC8 Homo sapiens zinc finger, DHHC-type containing 8 5.47E-06 2.30029 down (ZDHHC8), mRNA. 9839 ZFHX1B Homo sapiens zinc finger homeobox 1b (ZFHX1B), 1.17E-05 2.8815064 up mRNA. 64397 ZFP106 Homo sapiens zinc finger protein 106 homolog 5.47E-06 2.404434 up (mouse) (ZFP106), mRNA. 7538 ZFP36 Homo sapiens zinc finger protein 36, C3H type, 1.28E-05 2.074414 up homolog (mouse) (ZFP36), mRNA. 678 ZFP36L2 Homo sapiens zinc finger protein 36, C3H type-like 5.42E-05 2.1886992 down 2 (ZFP36L2), mRNA. 9765 ZFYVE16 Homo sapiens zinc finger, FYVE domain containing 1.08E-05 2.2273862 up 16 (ZFYVE16), mRNA. 10269 ZMPSTE24 Homo sapiens zinc metallopeptidase (STE24 5.13E-06 2.5700939 up homolog, yeast) (ZMPSTE24), mRNA. 10838 ZNF275 Homo sapiens zinc finger protein 275 (ZNF275), 5.13E-06 3.1497006 down mRNA. 7580 ZNF32 Homo sapiens zinc finger protein 32 (ZNF32), 5.50E-03 2.4229715 up transcript variant 1, mRNA. 26152 ZNF337 Homo sapiens zinc finger protein 337 (ZNF337), 9.21E-06 2.4192097 down mRNA. 162979 ZNF342 Homo sapiens zinc finger protein 342 (ZNF342), 7.25E-06 2.2081544 down mRNA. 55893 ZNF395 Homo sapiens zinc finger protein 395 (ZNF395), 5.35E-06 2.7907915 down mRNA. 220929 ZNF438 Homo sapiens zinc finger protein 438 (ZNF438), 9.97E-06 5.368193 up mRNA. 168544 ZNF467 Homo sapiens zinc finger protein 467 (ZNF467), 5.98E-04 2.5720587 up mRNA. 57711 ZNF529 Homo sapiens zinc finger protein 529 (ZNF529), 1.17E-05 2.1962404 down mRNA. 57616 ZNF537 Homo sapiens zinc finger protein 537 (ZNF537), 2.12E-03 3.029457 up mRNA. 91120 ZNF682 Homo sapiens zinc finger protein 682 (ZNF682), 9.69E-03 2.1394207 up mRNA. 257101 ZNF683 Homo sapiens zinc finger protein 683 (ZNF683), 2.09E-02 3.0123246 down mRNA. 7644 ZNF91 Homo sapiens zinc finger protein 91 (HPF7, HTF10) 3.87E-05 2.5146646 down (ZNF91), mRNA. 7791 ZYX Homo sapiens zyxin (ZYX), transcript variant 1, 3.14E-04 2.1189222 up mRNA. 7791 ZYX Homo sapiens zyxin (ZYX), transcript variant 1, 8.33E-05 2.4799135 up mRNA.

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Appendix 8.2: Signalling pathways from 1780 SDE gene transcripts

Ingenuity Canonical B H Ratio % Molecules No. of Pathways p value molecule CD28 Signaling in T 2.63E+09 2.40E-01 24 CD247,FYN,HLA- 31 Helper Cells DOA,CD3E,CDC42,CD4,ARPC5,HLA- DQA1,HLA-DMB,LCK,ACTR3,HLA- DRA,ARPC3,ARPC1A,CHUK,MAP2K1,ITK,AT M,HLA- DMA,ACTR2,CHP,PPP3CC,CD3D,CD3G,SYK,Z AP70,LAT,ITPR3,FCER1G,CD86,CARD11 Role of NFAT in 8.71E+06 1.74E-01 17.4 CD247,FYN,HLA- 34 Regulation of the DOA,CD3E,MAPK1,CD4,HLA-DQA1,HLA- Immune Response DMB,KRAS,CD79A,LCK,GNA15,HLA- DRA,CHUK,MAP2K1,ITK,ATM,HLA- DMA,CD79B,CHP,PPP3CC,CD3D,GNG5,GNAI 3,CD3G,SYK,MEF2D,ZAP70,LAT,ITPR3,GNB2, FCER1G,CD86,MEF2C Calcium-induced T 8.71E+06 2.88E-01 28.8 CD247,HLA-DMA,HLA- 19 Lymphocyte Apoptosis DOA,CD3E,CD4,CHP,HLA-DQA1,HLA- DMB,PPP3CC,CD3D,CD3G,LCK,PRKCD,MEF2 D,ZAP70,HLA-DRA,ITPR3,FCER1G,PRKCH Phospholipase C 5.89E+06 1.56E-01 15.6 PEBP1,CD247,FYN,MPRIP,CD3E,MAPK1,MYL 40 Signaling 6,KRAS,CD79A,HMOX1,LCK,RHOG,AHNAK,R HOT1,MARCKS,MAP2K1,RALGDS,ITK,HDAC4, CD79B,CHP,RALB,PPP3CC,CD3D,GNG5,CD3G ,MYL12A,MYL12B,SYK,PRKCD,MEF2D,LAT,J MJD7- PLA2G4B,ZAP70,ITPR3,GNB2,FCER1G,PPP1R 12A,MEF2C,PRKCH CTLA4 Signaling in 3.63E+06 2.40E-01 24 CD247,HLA-DMA,FYN,HLA- 23 Cytotoxic T Lymphocytes DOA,CD3E,PPP2CA,CD4,CLTC,HLA- DQA1,HLA- DMB,JAK2,CD3D,PPP2R5A,CD3G,LCK,SYK,LA T,HLA- DRA,ZAP70,FCER1G,CD86,PTPN22,ATM Cdc42 Signaling 3.63E+06 1.92E-01 19.2 CD247,HLA- 28 DOA,MPRIP,MYL6,MAPK1,CD3E,CDC42,ARP C5,HLA-DQA1,HLA- DMB,EXOC6,IQGAP1,HLA- DPA1,CLIP1,ACTR3,HLA- DRA,ARPC3,ARPC1A,ITK,HLA- DMA,ACTR2,HLA-DRB3 (includes others),CD3D,CD3G,MYL12A,MYL12B,FCER1 G,PPP1R12A Nur77 Signaling in T 3.63E+06 2.88E-01 28.8 CD247,HLA-DMA,HLA-DOA,CD3E,CHP,HLA- 17 Lymphocytes DQA1,APAF1,HLA- DMB,PPP3CC,CD3D,BCL2,CD3G,MEF2D,HLA- DRA,FCER1G,CD86,RXRA Glycolysis/Gluconeogene 3.98E+05 1.61E-01 16.1 PGK1,PKM2,ACSL3,PGM2,ACSS1,GAPDH,PG 20 sis M1,TPI1,LDHB,HK2,DHRS9,PGAM1,ACSS2,DL D,ALDOA,ALDH3B1,PECR,HK3,LDHA,ACSL1 iCOS-iCOSL Signaling in T 3.98E+05 1.93E-01 19.3 CD247,HLA-DMA,HLA- 23 Helper Cells DOA,CD3E,CD4,CHP,HLA-DQA1,HLA- DMB,PPP3CC,CD3D,IL2RB,PTEN,CD3G,LCK,L AT,HLA- DRA,ZAP70,ITPR3,FCER1G,CHUK,PLEKHA1,A TM,ITK PKCθ Signaling in T 3.09E+05 1.76E-01 17.6 CD247,HLA-DMA,FYN,HLA- 24 Lymphocytes DOA,MAPK1,CD3E,CD4,CHP,HLA-DQA1,HLA- DMB,KRAS,PPP3CC,CD3D,CD3G,LCK,LAT,HLA - DRA,ZAP70,FCER1G,CD86,MAP3K8,CHUK,CA RD11,ATM Cytotoxic T Lymphocyte- 1.95E+05 2.83E-01 28.3 CD247,HLA-DMA,HLA-DOA,CD3E,HLA- 15 mediated Apoptosis of DQA1,APAF1,HLA-DRB3 (includes Target Cells others),HLA-DMB,CD3D,HLA- DPA1,BCL2,CD3G,PRF1,HLA-DRA,FCER1G

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Type I Diabetes Mellitus 5.89E+04 1.86E-01 18.6 CD247,HLA-DMA,HLA- 22 Signaling DOA,MAPK1,CD3E,MYD88,IFNGR2,HLA- DQA1,APAF1,HLA- DMB,IFNGR1,JAK2,CD3D,BCL2,CD3G,PRF1,L TA,HLA-DRA,FCER1G,CD86,CHUK,IL1RAP OX40 Signaling Pathway 5.62E+04 2.38E-01 23.8 CD247,HLA-DMA,HLA-DOA,CD3E,CD4,HLA- 15 DQA1,HLA-DRB3 (includes others),HLA- DMB,CD3D,HLA-DPA1,BCL2,CD3G,HLA- DRA,FCER1G,TRAF5 T Cell Receptor Signaling 2.63E+04 1.85E-01 18.5 CD247,FYN,MAPK1,CD3E,CD4,KRAS,PPP3CC, 20 CD8A,CD3D,CD3G,LCK,RASGRP1,PAG1,LAT,Z AP70,CHUK,MAP2K1,CARD11,ATM,ITK fMLP Signaling in 1.86E+04 1.65E-01 16.5 ACTR2,MAPK1,CDC42,CHP,ARPC5,FPR2,KRA 21 Neutrophils S,PPP3CC,GNG5,FPR1,GNAI3,ACTR3,PRKCD,I TPR3,CYBB,GNB2,ARPC3,ARPC1A,PRKCH,MA P2K1,ATM T Helper Cell 1.78E+04 2.29E-01 22.9 HLA-DMA,HLA-DOA,IL12RB1,IL21R,HLA- 16 Differentiation DQA1,IFNGR2,IFNGR1,HLA- DMB,BCL6,IL18R1,STAT4,HLA- DRA,IL10RB,FCER1G,CD86,CXCR5 Natural Killer Cell 2.75E+03 1.73E-01 17.3 CD247,FYN,MAPK1,LAIR1,TYROBP,KLRD1,KR 19 Signaling AS,LCK,SH2D1A,KLRB1,PRKCD,SYK,ZAP70,LA T,FCER1G,PRKCH,HCST,MAP2K1,ATM Altered T Cell and B Cell 1.55E+03 1.84E-01 18.4 HLA-DMA,HLA-DOA,CD79B,TLR8,HLA- 16 Signaling in Rheumatoid DQA1,LTB,HLA- Arthritis DMB,CD79A,TLR4,TLR5,LTA,HLA- DRA,TLR7,FCER1G,CD86,CHUK B Cell Development 1.20E+03 3.00E-01 30 IL7R,HLA-DMA,HLA-DOA,CD79B,HLA- 9 DRA,HLA-DQA1,CD86,HLA-DMB,CD79A Fcγ Receptor-mediated 1.20E+03 1.68E-01 16.8 ACTR2,FYN,MAPK1,CDC42,ARPC5,PTEN,HM 17 Phagocytosis in OX1,ACTR3,PRKCD,SYK,HCK,VAMP3,ARPC3, Macrophages and ARPC1A,PRKCH,VASP,FGR Monocytes Regulation of IL-2 7.41E+02 1.70E-01 17 CD247,FYN,CD3E,MAPK1,SMAD3,CHP,KRAS, 15 Expression in Activated PPP3CC,CD3D,CD3G,LAT,ZAP70,CHUK,CARD and Anergic T 11,MAP2K1 Lymphocytes Primary 6.76E+02 2.00E-01 20 IL7R,LCK,CD3E,CD4,ZAP70,ADA,RFX5,CD8A,C 11 Immunodeficiency D3D,UNG,CD79A Signaling Toll-like Receptor 3.80E+02 2.00E-01 20 TLR4,LY96 (includes 11 Signaling EG:17087),TOLLIP,TLR5,MAPK1,MYD88,TLR8 ,TLR7,CD14,CHUK,IRAK3 Integrin Signaling 3.80E+02 1.27E-01 12.7 FYN,MPRIP,MAPK1,CDC42,ARPC5,KRAS,ITGB 26 7,PTEN,PTK2 (includes EG:14083),RHOG,ACTR3,RHOT1,ARF4,ARPC 3,ARPC1A,MAP2K1,VASP,ATM,ACTR2,ASAP1 ,RALB,MYL12A,ITGAM (includes EG:16409),MYL12B,PPP1R12A,CAPN3 Dendritic Cell Maturation 3.80E+02 1.23E-01 12.3 HLA-DMA,HLA- 22 DOA,MAPK1,TYROBP,MYD88,HLA- DQA1,CD58,LTB,HLA-DMB,HLA-DRB3 (includes others),JAK2,STAT4,TLR4,LTA,HLA- DRA,FCER1G,CD86,LTBR,CHUK,CCR7,IFNAR1 ,ATM CCR5 Signaling in 3.39E+02 1.48E-01 14.8 CD247,CD3E,MAPK1,CD4,CCL5,CD3D,GNG5, 13 Macrophages GNAI3,CD3G,PRKCD,GNB2,FCER1G,PRKCH Role of Macrophages, 2.75E+02 1.06E-01 10.6 IL18RAP,MAPK1,IL32,TLR8,LTB,KRAS,JAK2,C 34 Fibroblasts and CL5,IL18R1,IL1R2,C5AR1,GNA15,CEBPA,TLR7 Endothelial Cells in ,LTBR,CHUK,TRAF5,MAPKAPK2,MAP2K1,IL1 Rheumatoid Arthritis RAP,ATM,VEGFB,MYD88,CHP,CEBPB (includes EG:1051),PPP3CC,IRAK3,TLR4,TLR5,CEBPD,P RKCD,LTA,LEF1,PRKCH

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Production of Nitric 2.75E+02 1.14E-01 11.4 MAPK1,PPP2CA,IFNGR2,IFNGR1,JAK2,NCF4, 24 Oxide and Reactive SPI1 (includes Oxygen Species in EG:20375),PPP2R5A,TLR4,PPP1R3D,RHOG,R Macrophages HOT1,PRKCD,CYBA,CYBB,PPP1R12A,SERPIN A1,S100A8,MAP3K8,PRKCH,CHUK,MAP2K1,S IRPA,ATM IL-8 Signaling 2.09E+02 1.20E-01 12 VEGFB,MAPK1,KRAS,IRAK3,CSTB,GNG5,BCL2 23 ,PTK2 (includes EG:14083),HMOX1,GNAI3,ITGAM (includes EG:16409),CCND2,RHOG,CCND3,RHOT1,MY L12B,PRKCD,GNB2,CYBB,PRKCH,CHUK,MAP 2K1,ATM NF-κB Signaling 1.74E+02 1.29E-01 12.9 AZI2,MYD88,TLR8,TNFAIP3,TBK1,KRAS,IRAK 22 3,IL1R2,TLR4,LCK,TLR5,LTA,ZAP70,TGFA,TLR 7,FCER1G,MAP3K8,LTBR,CHUK,TRAF5,CARD 11,ATM Molecular Mechanisms 1.74E+02 9.95E-02 9.95 FYN,APH1B,CDC42,MAPK1,SMAD3,CTNNA1, 37 of Cancer KRAS,HIF1A,JAK2,E2F3,BCL2,CDC25B,PTK2 (includes EG:14083),LAMTOR3,RHOG,GNA15,RHOT1, MAP2K1,RALGDS,ATM,HAT1,CDKN2D,RALB, CDK6,APAF1,NBN,GNAI3,CCND2,CCND3,FOX O1,PRKCD,CDK4,RASGRP1,RBPJ,LEF1,PRKCH, BIRC2 Role of Pattern 1.74E+02 1.59E-01 15.9 OAS2,MAPK1,MYD88,TLR8,CCL5,RNASEL,TL 13 Recognition Receptors in R4,C5AR1,TLR5,SYK,TLR7,NLRC4,ATM Recognition of Bacteria and Viruses Actin Nucleation by ARP- 1.58E+02 1.67E-01 16.7 ACTR2,ACTR3,RHOG,CDC42,RHOT1,ARPC5,P 11 WASP Complex PP1R12A,ARPC3,ARPC1A,KRAS,VASP Allograft Rejection 1.51E+02 1.67E-01 16.7 HLA-DMA,PRF1,HLA-DOA,HLA-DRA,HLA- 10 Signaling DQA1,FCER1G,CD86,HLA-DRB3 (includes others),HLA-DMB,HLA-DPA1 Antigen Presentation 1.51E+02 2.09E-01 20.9 HLA-DMA,HLA-DOA,HLA-DRA,HLA- 9 Pathway DQA1,HLA-DRB3 (includes others),HLA- DMB,CD74,HLA-DPA1,MR1 Pyruvate Metabolism 9.55E+01 8.53E-02 8.53 PKM2,ACSL3,ACSS2,DLD,ACSS1,ME2,ACOT9, 11 AKR1B1,ACSL1,LDHA,LDHB PI3K Signaling in B 8.91E+01 1.27E-01 12.7 FYN,MAPK1,CD79B,CHP,KRAS,ATF6,PPP3CC, 18 Lymphocytes CD79A,PTEN,TLR4,GNA15,SYK,ITPR3,IRS2,PI K3AP1,CHUK,PLEKHA1,MAP2K1 Fructose and Mannose 7.94E+01 6.98E-02 6.98 NUDT5,PFKFB3,PGM2,HK2,PFKFB4,ALDOA,H 9 Metabolism K3,TPI1,AKR1B1 Regulation of Actin- 6.92E+01 1.46E-01 14.6 ACTR2,MPRIP,CDC42,MYL6,ARPC5,MYL12A, 13 based Motility by Rho ACTR3,RHOG,RHOT1,MYL12B,PPP1R12A,AR PC3,ARPC1A Hepatic Fibrosis / Hepatic 5.37E+01 1.25E-01 12.5 IL18RAP,VEGFB,MYL6,SMAD3,IFNGR2,IFNGR 18 Stellate Cell Activation 1,CCL5,BCL2,IL1R2,TLR4,LY96 (includes EG:17087),TIMP1,TGFA,CD14,IL1RAP,CCR7,I FNAR1,TIMP2 (includes EG:21858) Role of Tissue Factor in 5.01E+01 1.35E-01 13.5 FYN,CDC42,MAPK1,PLAUR,KRAS,JAK2,PDXP, 15 Cancer PTEN,LCK,ARRB2,HCK,RPS6KA5,RPS6KA1 (includes EG:20111),FGR,ATM Signaling by Rho Family 5.01E+01 1.03E-01 10.3 MYL6,MAPK1,CDC42,ARPC5,IQGAP1,SEPT9, 26 GTPases CLIP1,PTK2 (includes EG:14083),RHOG,ACTR3,GNA15,RHOT1,CYB B,ARPC3,ARPC1A,MAP2K1,ATM,ACTR2,VIM, GNG5,GNAI3,MYL12A,MYL12B,GNB2,PPP1R 12A,SEPT6 Glioma Invasiveness 4.79E+01 1.67E-01 16.7 PTK2 (includes 10 Signaling EG:14083),RHOG,MAPK1,RHOT1,TIMP1,CD4 4 (includes EG:100330801),PLAUR,KRAS,TIMP2 (includes EG:21858),ATM

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IL-12 Signaling and 4.27E+01 1.12E-01 11.2 MAPK1,IL12RB1,MYD88,IFNGR1,CEBPB 17 Production in (includes EG:1051),SPI1 (includes Macrophages EG:20375),STAT4,TLR4,PRKCD,PRKCH,MAP3 K8,SERPINA1,S100A8,CHUK,RXRA,MAP2K1,A TM Nicotinate and 4.27E+01 1.04E-01 10.4 MAPK1,CDK6,NAPRT1,VNN1,VNN2,PRKCD,C 14 Nicotinamide DK4,PRKAA1,GRK6,PRKCH,NAMPT,MAP3K8, Metabolism MAP2K1,BST1 (includes EG:12182) Crosstalk between 3.89E+01 1.37E-01 13.7 KLRD1,TYROBP,LTB,HLA-DRB3 (includes 13 Dendritic Cells and others),IL2RB,TLR4,PRF1,LTA,HLA- Natural Killer Cells DRA,TLR7,CD86,LTBR,CCR7 p70S6K Signaling 3.89E+01 1.22E-01 12.2 YWHAG,CD79B,MAPK1,YWHAH,PPP2CA,KR 16 AS,PPP2R5A,CD79A,GNAI3,GNA15,PRKCD,SY K,PRKCH,BCAP31,MAP2K1,ATM Oxidative 3.63E+01 1.14E-01 11.4 ATP6V0E2,COX17 (includes 17 Phosphorylation EG:10063),NDUFB3,COX7A2,ATP6V1D (includes EG:299159),ATP6V0B,ATP6V1C1,TCIRG1,CO X10 (includes EG:1352),ATP6V1A,ATP6AP1,NDUFA1,ATP6 V1E1,PPA2 (includes EG:27068),ATP6V0D1,ATP6V0E1,ATP6V1B2 TREM1 Signaling 3.55E+01 1.45E-01 14.5 TLR4,TLR5,MAPK1,TYROBP,TLR8,TLR7,LAT2, 9 CD86,JAK2 Autoimmune Thyroid 3.55E+01 1.45E-01 14.5 HLA-DMA,PRF1,HLA-DOA,HLA-DRA,HLA- 8 Disease Signaling DQA1,FCER1G,CD86,HLA-DMB IL-15 Signaling 3.47E+01 1.49E-01 14.9 PTK2 (includes 10 EG:14083),LCK,MAPK1,SYK,KRAS,JAK2,MAP2 K1,IL2RB,ATM,BCL2 Role of JAK1 and JAK3 in 3.47E+01 1.52E-01 15.2 IL7R,FES,MAPK1,SYK,IL21R,IRS2,KRAS,JAK2,I 10 γc Cytokine Signaling L2RB,ATM Small Cell Lung Cancer 3.47E+01 1.25E-01 12.5 PTK2 (includes 11 Signaling EG:14083),CDK4,APAF1,CDK6,TRAF5,CHUK, RXRA,PTEN,ATM,BCL2,BIRC2 Clathrin-mediated 3.47E+01 1.09E-01 10.9 ACTR2,VEGFB,RAB5A,CDC42,CHP,CLTC,ARPC 21 Endocytosis Signaling 5,NUMB,SH3GLB1,PPP3CC,ITGB7,ARRB2,AC TR3,LDLR,SYNJ1,RAB5C,ARPC3,ARPC1A,SERP INA1,S100A8,ATM Rac Signaling 3.39E+01 1.15E-01 11.5 ACTR2,CDC42,MAPK1,ARPC5,KRAS,IQGAP1, 14 PTK2 (includes EG:14083),ACTR3,CD44 (includes EG:100330801),CYBB,ARPC3,ARPC1A,MAP2 K1,ATM Communication between 3.39E+01 1.21E-01 12.1 TLR4,TLR5,CD4,HLA- 12 Innate and Adaptive DRA,TLR8,TLR7,FCER1G,CD86,HLA-DRB3 Immune Cells (includes others),CCL5,CD8A,CCR7 Graft-versus-Host 3.09E+01 1.67E-01 16.7 HLA-DMA,PRF1,HLA-DOA,HLA-DRA,HLA- 8 Disease Signaling DQA1,FCER1G,CD86,HLA-DMB ERK/MAPK Signaling 3.09E+01 1.03E-01 10.3 ETS1,FYN,YWHAG,YWHAH,MAPK1,PPP2CA,E 21 TS2,KRAS,PPP2R5A,PTK2 (includes EG:14083),LAMTOR3,PPP1R3D,DUSP1,PRKC D,MKNK1,JMJD7- PLA2G4B,PPP1R12A,RPS6KA5,RPS6KA1 (includes EG:20111),MAP2K1,ATM Pancreatic 3.02E+01 1.19E-01 11.9 VEGFB,MAPK1,CDC42,SMAD3,KRAS,JAK2,E2 14 Adenocarcinoma F3,BCL2,HMOX1,CDK4,TGFA,MAP2K1,RALG Signaling DS,ATM Lymphotoxin β Receptor 2.95E+01 1.50E-01 15 MAPK1,LTA,APAF1,LTB,TRAF5,CHUK,LTBR,A 9 Signaling TM,BIRC2 CXCR4 Signaling 2.63E+01 1.07E-01 10.7 MYL6,MAPK1,CD4,KRAS,GNG5,PTK2 18 (includes EG:14083),GNAI3,MYL12A,RHOG,GNA15,MY L12B,RHOT1,PRKCD,ITPR3,GNB2,PRKCH,MA P2K1,ATM

272

RhoA Signaling 2.24E+01 1.25E-01 12.5 ACTR2,MPRIP,MYL6,ARPC5,SEPT9,PTK2 14 (includes EG:14083),MYL12A,ACTR3,MYL12B,LPAR5,P PP1R12A,ARPC3,ARPC1A,SEPT6 Galactose Metabolism 2.14E+01 7.14E-02 7.14 PGM2,GLA,HK2,UGP2,PGM1,HK3,AKR1B1 7 VDR/RXR Activation 2.04E+01 1.36E-01 13.6 SERPINB1,FOXO1,GADD45A,PRKCD,MXD1,C 11 EBPA,CD14,PRKCH,CEBPB (includes EG:1051),CCL5,RXRA Ephrin Receptor Signaling 1.78E+01 9.60E-02 9.6 ACTR2,FYN,VEGFB,MAPK1,CDC42,ARPC5,KR 19 AS,JAK2,GNG5,GRINA,PTK2 (includes EG:14083),GNAI3,ACTR3,SDCBP,GNA15,GNB 2,ARPC3,ARPC1A,MAP2K1 Inositol Phosphate 1.74E+01 8.90E-02 8.9 MAPK1,CDK6,PTEN,GNAI3,GNA15,SYNJ1,CD 17 Metabolism K4,PRKCD,PRKAA1,GRK6,IMPA2,MAP3K8,PR KCH,IRS2,MAP2K1,ATM,MTMR3 Glucocorticoid Receptor 1.70E+01 8.90E-02 8.9 CD247,YWHAH,MAPK1,CD3E,HSPA1A/HSPA 26 Signaling 1B,SMAD3,KRAS,CCL5,JAK2,TAF7,CD163,BCL 2,IL1R2,ANXA1,CEBPA,PRKAA1,CHUK,FKBP5, MAP2K1,ATM,CHP,CEBPB (includes EG:1051),PPP3CC,CD3D,CD3G,DUSP1 IL-3 Signaling 1.55E+01 1.35E-01 13.5 FOXO1,MAPK1,PRKCD,CHP,KRAS,PRKCH,PPP 10 3CC,JAK2,MAP2K1,ATM CD40 Signaling 1.38E+01 1.30E-01 13 MAPK1,LTA,TNFAIP3,TRAF5,CHUK,MAPKAPK 9 2,MAP2K1,ATM,FCER2 GM-CSF Signaling 1.38E+01 1.34E-01 13.4 ETS1,MAPK1,CSF2RA,HCK,KRAS,PPP3CC,JAK 9 2,MAP2K1,ATM p38 MAPK Signaling 1.38E+01 1.23E-01 12.3 IL18RAP,MAPKAPK3,IRAK3,IL1R2,DUSP1,ME 13 F2D,MKNK1,JMJD7- PLA2G4B,MAP4K1,MEF2C,RPS6KA5,MAPKA PK2,IL1RAP LPS-stimulated MAPK 1.38E+01 1.22E-01 12.2 TLR4,CDC42,MAPK1,PRKCD,CD14,KRAS,PRK 10 Signaling CH,CHUK,MAP2K1,ATM MIF-mediated 1.35E+01 1.43E-01 14.3 TLR4,LY96 (includes 6 Glucocorticoid EG:17087),MAPK1,JMJD7- Regulation PLA2G4B,CD14,CD74 B Cell Receptor Signaling 1.35E+01 1.04E-01 10.4 ETS1,MAPK1,CD79B,CDC42,KRAS,PPP3CC,BC 16 L6,CD79A,PTEN,PAG1,SYK,MAP3K8,PIK3AP1, CHUK,MAP2K1,ATM Starch and Sucrose 1.23E+01 5.52E-02 5.52 NUDT5,PGM2,HK2,UGP2,GBA,PGM1,GBE1,P 9 Metabolism YGL,HK3 Interferon Signaling 1.23E+01 1.67E-01 16.7 IFNGR2,MX1,IFNGR1,JAK2,IFNAR1,BCL2 6 Cell Cycle: G1/S 1.20E+01 1.36E-01 13.6 CCND3,CCND2,HDAC4,CDK4,SMAD3,CDK6,E 8 Checkpoint Regulation 2F3,ATM Aryl Hydrocarbon 1.17E+01 9.68E-02 9.68 MGST1,MAPK1,NQO2,APAF1,CDK6,CYP1B1, 15 Receptor Signaling GSTO1,CTSD,CCND3,CCND2,CDK4,ALDH3B1, RXRA,MCM7,ATM Non-Small Cell Lung 1.17E+01 1.14E-01 11.4 MAPK1,CDK4,ITPR3,TGFA,CDK6,KRAS,RXRA, 9 Cancer Signaling MAP2K1,ATM Hypoxia Signaling in the 1.17E+01 1.36E-01 13.6 UBE2H,UBE2A,COPS5,HIF1A,UBE2J1,LDHA,U 9 Cardiovascular System BE2F,PTEN,ATM Cardiac Hypertrophy 1.17E+01 9.13E-02 9.13 MYL6,MAPK1,MAPKAPK3,CHP,KRAS,ATF6,P 22 Signaling PP3CC,GNG5,GNAI3,MYL12A,RHOG,GNA15, RHOT1,MYL12B,MEF2D,GNB2,MAP3K8,MEF 2C,RPS6KA1 (includes EG:20111),MAPKAPK2,MAP2K1,ATM IL-17A Signaling in 1.17E+01 1.50E-01 15 MAPK1,CEBPD,LCN2,CHUK,CEBPB (includes 6 Fibroblasts EG:1051),NFKBIZ Thrombopoietin 1.17E+01 1.27E-01 12.7 MAPK1,PRKCD,IRS2,KRAS,PRKCH,JAK2,MAP 8 Signaling 2K1,ATM Acute Myeloid Leukemia 1.15E+01 1.22E-01 12.2 CSF3R,MAPK1,CSF2RA,CEBPA,LEF1,KRAS,MA 10 Signaling P2K1,SPI1 (includes EG:20375),FLT3LG,ATM Cyclins and Cell Cycle 1.15E+01 1.15E-01 11.5 CCND3,CCND2,HDAC4,PPP2CA,CDK4,CDKN2 10 Regulation D,CDK6,E2F3,PPP2R5A,ATM 273

PPARα/RXRα Activation 1.10E+01 9.60E-02 9.6 IL18RAP,MAPK1,ACAA1,SMAD3,ACOX1,KRA 17 S,JAK2,GK,ABCA1,ACVR1B,IL1R2,GNA15,PRK AA1,CHUK,RXRA,IL1RAP,MAP2K1 IL-4 Signaling 1.07E+01 1.25E-01 12.5 HLA-DMA,HLA-DOA,HLA-DRA,HLA- 9 DQA1,KRAS,HLA-DMB,JAK2,ATM,FCER2 Glioblastoma Multiforme 1.07E+01 9.58E-02 9.58 MAPK1,CDC42,CDK6,KRAS,E2F3,PTEN,RHOG 16 Signaling ,GNA15,FOXO1,RHOT1,PRKCD,CDK4,ITPR3,L EF1,MAP2K1,ATM CCR3 Signaling in 1.07E+01 1.04E-01 10.4 MPRIP,MAPK1,KRAS,GNG5,GNAI3,PRKCD,J 13 Eosinophils MJD7- PLA2G4B,ITPR3,GNB2,PPP1R12A,PRKCH,MA P2K1,ATM IL-10 Signaling 1.02E+01 1.15E-01 11.5 IL1R2,CCR1,HMOX1,IL18RAP,MAPK1,IL10RB, 9 CD14,CHUK,IL1RAP Thrombin Signaling 1.00E+01 9.31E-02 9.31 MPRIP,MYL6,MAPK1,KRAS,GNG5,PTK2 19 (includes EG:14083),GNAI3,MYL12A,RHOG,GNA15,MY L12B,RHOT1,PRKCD,ITPR3,GNB2,PPP1R12A, PRKCH,MAP2K1,ATM Synaptic Long Term 9.79E+00 1.06E-01 10.6 PPP1R3D,MAPK1,PRKCD,ITPR3,CHP,PPP1R1 12 Potentiation 2A,KRAS,PRKCH,PPP3CC,RPS6KA1 (includes EG:20111),MAP2K1,GRINA PI3K/AKT Signaling 9.79E+00 9.56E-02 9.56 YWHAG,MAPK1,YWHAH,PPP2CA,KRAS,JAK2, 13 PPP2R5A,PTEN,BCL2,FOXO1,MAP3K8,CHUK, MAP2K1 Renal Cell Carcinoma 9.79E+00 1.23E-01 12.3 ETS1,CDC42,MAPK1,TGFA,KRAS,HIF1A,MAP 9 Signaling 2K1,TCEB1,ATM TNFR2 Signaling 9.59E+00 1.52E-01 15.2 LTA,TNFAIP3,TBK1,CHUK,BIRC2 5 LXR/RXR Activation 9.48E+00 9.92E-02 9.92 IL18RAP,ABCA1,IL1R2,TLR4,LY96 (includes 13 EG:17087),LDLR,CD14,SERPINA1,S100A8,H MGCR,RXRA,IL1RAP,HADH NRF2-mediated Oxidative 9.48E+00 9.47E-02 9.47 MGST1,MAPK1,NQO2,KRAS,JUNB,GSTO1,TX 18 Stress Response NRD1,HMOX1,SOD2,DNAJC5,PRKCD,DNAJA3 ,PRKCH,DNAJB6,TXN (includes EG:116484),FKBP5,MAP2K1,ATM ILK Signaling 9.48E+00 9.68E-02 9.68 VEGFB,MYL6,MAPK1,CDC42,PPP2CA,VIM,HI 18 F1A,PPP2R5A,ITGB7,PTEN,PTK2 (includes EG:14083),RHOG,RHOT1,PPP1R12A,IRS2,LEF 1,RPS6KA5,ATM HMGB1 Signaling 9.35E+00 1.11E-01 11.1 TLR4,RHOG,CDC42,MAPK1,RHOT1,HAT1,IFN 11 GR2,IFNGR1,KRAS,MAP2K1,ATM Actin Cytoskeleton 9.16E+00 8.70E-02 8.7 ACTR2,MPRIP,MYL6,MAPK1,CDC42,ARPC5,K 20 Signaling RAS,IQGAP1,PTK2 (includes EG:14083),MYL12A,ACTR3,MYL12B,CD14,PP P1R12A,ARPC3,ARPC1A,SSH2,MAP2K1,ATM, MATK CNTF Signaling 9.16E+00 1.35E-01 13.5 MAPK1,KRAS,RPS6KA5,JAK2,RPS6KA1 7 (includes EG:20111),MAP2K1,ATM Glioma Signaling 9.16E+00 9.91E-02 9.91 MAPK1,PRKCD,CDK4,CDKN2D,CDK6,KRAS,P 11 RKCH,E2F3,MAP2K1,PTEN,ATM IL-6 Signaling 9.16E+00 1.10E-01 11 IL1R2,IL18RAP,MAPK1,CD14,KRAS,CHUK,CE 11 BPB (includes EG:1051),JAK2,MAPKAPK2,MAP2K1,IL1RAP Pentose Phosphate 9.16E+00 6.33E-02 6.33 PGM2,PGD,G6PD,PGM1,ALDOA 5 Pathway Gα12/13 Signaling 9.16E+00 1.02E-01 10.2 CDC42,MAPK1,MYL6,KRAS,PTK2 (includes 13 EG:14083),MYL12A,MYL12B,MEF2D,LPAR5, MEF2C,CHUK,MAP2K1,ATM Inositol Metabolism 8.61E+00 1.18E-01 11.8 ALDOA,TPI1 2 ERK5 Signaling 8.61E+00 1.27E-01 12.7 YWHAG,YWHAH,MEF2D,KRAS,MAP3K8,MEF 8 2C,RPS6KA5,RPS6KA1 (includes EG:20111) NF-κB Activation by 8.24E+00 1.10E-01 11 LCK,MAPK1,PRKCD,CD4,KRAS,PRKCH,CXCR5, 9 Viruses CHUK,ATM

274

Prolactin Signaling 8.24E+00 1.12E-01 11.2 FYN,MAPK1,PRKCD,KRAS,PRKCH,CEBPB 9 (includes EG:1051),JAK2,MAP2K1,ATM MIF Regulation of Innate 7.66E+00 1.20E-01 12 TLR4,LY96 (includes 6 Immunity EG:17087),MAPK1,JMJD7- PLA2G4B,CD14,CD74 Endometrial Cancer 7.50E+00 1.23E-01 12.3 MAPK1,CTNNA1,LEF1,KRAS,MAP2K1,PTEN,A 7 Signaling TM Leukocyte Extravasation 7.46E+00 9.23E-02 9.23 MYL6,MAPK1,CDC42,CTNNA1,NCF4,PTK2 18 Signaling (includes EG:14083),GNAI3,ITGAM (includes EG:16409),TIMP1,CYBA,PRKCD,CD44 (includes EG:100330801),CYBB,PRKCH,VASP,ATM,TIM P2 (includes EG:21858),ITK RhoGDI Signaling 7.33E+00 8.54E-02 8.54 ACTR2,MYL6,CDC42,ARPC5,GNG5,GNAI3,M 17 YL12A,ACTR3,RHOG,GNA15,MYL12B,RHOT1, GNB2,CD44 (includes EG:100330801),ARPC3,PPP1R12A,ARPC1A α-Adrenergic Signaling 7.33E+00 9.52E-02 9.52 GNAI3,MAPK1,PRKCD,ITPR3,GNB2,PYGL,KR 10 AS,PRKCH,MAP2K1,GNG5 HGF Signaling 7.33E+00 1.05E-01 10.5 PTK2 (includes 11 EG:14083),ETS1,CDC42,MAPK1,PRKCD,ETS2, KRAS,MAP3K8,PRKCH,MAP2K1,ATM Melanoma Signaling 7.29E+00 1.30E-01 13 MAPK1,CDK4,KRAS,MAP2K1,PTEN,ATM 6 Polyamine Regulation in 7.29E+00 1.38E-01 13.8 AZIN1,MXD1,SAT1,KRAS 4 Colon Cancer IL-2 Signaling 7.29E+00 1.21E-01 12.1 LCK,MAPK1,SYK,KRAS,MAP2K1,IL2RB,ATM 7 Apoptosis Signaling 7.13E+00 1.06E-01 10.6 MAPK1,APAF1,KRAS,CHUK,RPS6KA1 10 (includes EG:20111),MAP2K1,PARP1,BCL2,CAPN3,BIR C2 Type II Diabetes Mellitus 6.95E+00 7.79E-02 7.79 PKM2,ACSL3,MAPK1,PRKCD,PRKAA1,ACSL4,I 12 Signaling RS2,PRKCH,CHUK,CEBPB (includes EG:1051),ACSL1,ATM Cell Cycle: G2/M DNA 6.95E+00 1.25E-01 12.5 CDC25B,YWHAG,GADD45A,YWHAH,RPS6KA 6 Damage Checkpoint 1 (includes EG:20111),ATM Regulation Cholecystokinin/Gastrin- 6.95E+00 1.04E-01 10.4 PTK2 (includes 11 mediated Signaling EG:14083),RHOG,MAPK1,RHOT1,PRKCD,ME F2D,ITPR3,KRAS,MEF2C,PRKCH,MAP2K1 Fatty Acid Metabolism 6.95E+00 6.75E-02 6.75 ACAD11,ACSL3,DHRS9,ACAA1,ECI2,ACOX1,A 11 CSL4,PECR,ACSL1,HADH,CYP1B1 mTOR Signaling 6.93E+00 8.72E-02 8.72 VEGFB,MAPK1,PPP2CA,FKBP1A,KRAS,HIF1A, 17 PPP2R5A,HMOX1,RPS4X,RHOG,RHOT1,PRKC D,PRKAA1,RPS6KA5,PRKCH,RPS6KA1 (includes EG:20111),ATM DNA Double-Strand 6.64E+00 1.58E-01 15.8 PARP1,ATM,NBN 3 Break Repair by Non- Homologous End Joining Fc Epsilon RI Signaling 6.34E+00 9.91E-02 9.91 FYN,MAPK1,PRKCD,SYK,LAT,JMJD7- 11 PLA2G4B,FCER1G,KRAS,PRKCH,MAP2K1,AT M Prostate Cancer Signaling 6.34E+00 9.47E-02 9.47 FOXO1,MAPK1,LEF1,KRAS,CHUK,MAP2K1,PT 9 EN,ATM,BCL2 Propanoate Metabolism 6.34E+00 5.79E-02 5.79 ACAD11,ACSL3,ACSS2,ACSS1,ACSL1,LDHA,L 7 DHB Protein Kinase A 6.34E+00 8.07E-02 8.07 YWHAH,MYL6,MAPK1,ANAPC1/LOC1002869 26 Signaling 79,PDE7A,SMAD3,PTK2 (includes EG:14083),GNA15,CHUK,MAP2K1,VASP,YW HAG,CHP,PYGL,PPP3CC,GNG5,GNAI3,MYL12 A,PPP1R3D,MYL12B,PRKCD,ITPR3,GNB2,PPP 1R12A,PRKCH,LEF1 NGF Signaling 6.34E+00 9.48E-02 9.48 RHOG,CDC42,MAPK1,PRKCD,KRAS,MAP3K8, 11 RPS6KA5,CHUK,RPS6KA1 (includes EG:20111),MAP2K1,ATM

275

Role of JAK1, JAK2 and 6.34E+00 1.48E-01 14.8 IFNGR2,IFNGR1,JAK2,IFNAR1 4 TYK2 in Interferon Signaling Growth Hormone 6.34E+00 1.07E-01 10.7 MAPK1,PRKCD,CEBPA,RPS6KA5,PRKCH,JAK2 8 Signaling ,RPS6KA1 (includes EG:20111),ATM Chemokine Signaling 6.34E+00 1.11E-01 11.1 PTK2 (includes 8 EG:14083),GNAI3,MPRIP,MAPK1,PPP1R12A, KRAS,CCL5,MAP2K1 Macropinocytosis 6.34E+00 1.05E-01 10.5 RAB5A,CDC42,PRKCD,CD14,KRAS,PRKCH,ITG 8 Signaling B7,ATM Chronic Myeloid 6.34E+00 9.71E-02 9.71 HDAC4,MAPK1,CDK4,SMAD3,CDK6,KRAS,CH 10 Leukemia Signaling UK,E2F3,MAP2K1,ATM IL-1 Signaling 6.34E+00 9.35E-02 9.35 GNAI3,TOLLIP,MAPK1,GNA15,MYD88,GNB2, 10 CHUK,IRAK3,IL1RAP,GNG5 14-3-3-mediated 6.32E+00 9.92E-02 9.92 YWHAG,FOXO1,MAPK1,YWHAH,GNA15,PRK 12 Signaling CD,VIM,KRAS,PRKCH,RPS6KA1 (includes EG:20111),MAP2K1,ATM Nitrogen Metabolism 6.12E+00 4.17E-02 4.17 VNN1,VNN2,CA4,CA5B,CCDC92 5 FLT3 Signaling in 6.12E+00 1.13E-01 11.3 STAT4,MAPK1,KRAS,RPS6KA5,RPS6KA1 8 Hematopoietic (includes EG:20111),MAP2K1,FLT3LG,ATM Progenitor Cells Aminosugars Metabolism 6.12E+00 6.72E-02 6.72 POR,CYB5R4,HK2,PDE7A,CYB5R1,HK3,NANS, 8 NPL Sphingolipid Metabolism 6.12E+00 8.18E-02 8.18 NEU1,GLA,LPIN1,GALC,VNN1,GBA,PIGF,VNN 9 2,UGCG Axonal Guidance 6.08E+00 7.44E-02 7.44 FYN,CDC42,MAPK1,MYL6,ARPC5,KRAS,ABLI 32 Signaling M1,PTK2 (includes EG:14083),MICAL1,ACTR3,GNA15,MKNK1,A RPC3,ARPC1A,SEMA4A,MAP2K1,VASP,ATM, ACTR2,VEGFB,FES,CHP,PPP3CC,GNG5,GNAI3 ,MYL12A,SDCBP,MYL12B,PRKCD,GNB2,RTN4 (includes EG:57142),PRKCH Eicosanoid Signaling 5.89E+00 9.09E-02 9.09 LTB4R,JMJD7- 7 PLA2G4B,FPR2,ALOX5AP,PTGER2,ALOX5,TB XAS1 Granzyme B Signaling 5.45E+00 1.88E-01 18.8 PRF1,APAF1,PARP1 3 Telomerase Signaling 5.37E+00 1.01E-01 10.1 ETS1,HDAC4,MAPK1,PPP2CA,ETS2,KRAS,MA 10 P2K1,IL2RB,PPP2R5A,ATM Renin-Angiotensin 5.31E+00 8.73E-02 8.73 PTK2 (includes 11 Signaling EG:14083),MAPK1,PRKCD,ITPR3,KRAS,PRKC H,PTGER2,JAK2,CCL5,MAP2K1,ATM Notch Signaling 5.31E+00 1.22E-01 12.2 FURIN,APH1B,MFNG,NUMB,RBPJ 5 Caveolar-mediated 5.31E+00 9.64E-02 9.64 FYN,CD55,FLOT2,ITGAM (includes 8 Endocytosis Signaling EG:16409),RAB5A,RAB5C,FLOT1,ITGB7 IL-17 Signaling 5.31E+00 1.08E-01 10.8 MAPK1,TIMP1,KRAS,CEBPB (includes 8 EG:1051),JAK2,MAPKAPK2,MAP2K1,ATM IGF-1 Signaling 5.26E+00 9.52E-02 9.52 PTK2 (includes 10 EG:14083),YWHAG,FOXO1,MAPK1,YWHAH,I RS2,KRAS,JAK2,MAP2K1,ATM Hepatic Cholestasis 5.18E+00 7.56E-02 7.56 IL18RAP,MYD88,IRAK3,IL1R2,TLR4,LY96 13 (includes EG:17087),PRKCD,PPRC1,CD14,PRKCH,CHUK ,RXRA,IL1RAP VEGF Signaling 5.18E+00 9.28E-02 9.28 PTK2 (includes 9 EG:14083),VEGFB,FOXO1,MAPK1,KRAS,HIF1 A,MAP2K1,ATM,BCL2 Role of PI3K/AKT 5.11E+00 1.00E-01 10 GNAI3,MAPK1,CCL5,MAP2K1,PLAC8,IFNAR1, 7 Signaling in the ATM Pathogenesis of Influenza Cell Cycle Control of 5.11E+00 1.29E-01 12.9 MCM3,CDK4,CDK6,MCM7 4 Chromosomal Replication

276

Granzyme A Signaling 5.06E+00 1.67E-01 16.7 GZMA,PRF1,HMGB2 3 FAK Signaling 5.06E+00 8.91E-02 8.91 PTK2 (includes 9 EG:14083),FYN,MAPK1,ASAP1,KRAS,MAP2K 1,PTEN,ATM,CAPN3 RANK Signaling in 5.06E+00 9.57E-02 9.57 MAPK1,CHP,MAP3K8,PPP3CC,TRAF5,CHUK, 9 Osteoclasts MAP2K1,ATM,BIRC2 LPS/IL-1 Mediated 4.71E+00 7.98E-02 7.98 IL18RAP,MGST1,ACSL3,MYD88,MGMT,ACOX 17 Inhibition of RXR 1,GSTO1,ABCA1,IL1R2,TLR4,LY96 (includes Function EG:17087),ACSL4,CD14,ALDH3B1,RXRA,IL1R AP,ACSL1 HER-2 Signaling in Breast 4.71E+00 1.01E-01 10.1 CDC42,FOXO1,PRKCD,CDK6,KRAS,PRKCH,ITG 8 Cancer B7,ATM Role of MAPK Signaling in 4.71E+00 1.06E-01 10.6 MAPK1,JMJD7- 7 the Pathogenesis of PLA2G4B,KRAS,CCL5,MAP2K1,RABGEF1,BCL Influenza 2 Role of NFAT in Cardiac 4.71E+00 7.88E-02 7.88 HDAC4,MAPK1,CHP,KRAS,PPP3CC,GNG5,GN 16 Hypertrophy AI3,GNA15,MEF2D,PRKCD,ITPR3,GNB2,MEF 2C,PRKCH,MAP2K1,ATM CD27 Signaling in 4.71E+00 1.07E-01 10.7 APAF1,MAP3K8,TRAF5,CHUK,CD27,MAP2K1 6 Lymphocytes PAK Signaling 4.70E+00 8.49E-02 8.49 PTK2 (includes 9 EG:14083),MYL12A,CDC42,MAPK1,MYL6,MY L12B,KRAS,MAP2K1,ATM HIF1α Signaling 4.69E+00 9.43E-02 9.43 VEGFB,MAPK1,COPS5,KRAS,HIF1A,TCEB1,LD 10 HA,SLC2A3,LDHB,ATM Fatty Acid Elongation in 4.69E+00 6.52E-02 6.52 ACAA1,PECR,HADH 3 Mitochondria Mechanisms of Viral Exit 4.59E+00 1.11E-01 11.1 CHMP2A,PRKCD,SH3GLB1,PRKCH,VPS25 5 from Host Cells (includes EG:28084) Virus Entry via Endocytic 4.59E+00 9.09E-02 9.09 FYN,CD55,CDC42,PRKCD,CLTC,KRAS,PRKCH,I 9 Pathways TGB7,ATM G Beta Gamma Signaling 4.59E+00 7.96E-02 7.96 GNAI3,CDC42,MAPK1,GNA15,PRKCD,GNB2, 9 KRAS,PRKCH,GNG5 Semaphorin Signaling in 4.57E+00 1.15E-01 11.5 PTK2 (includes 6 Neurons EG:14083),FYN,RHOG,FES,MAPK1,RHOT1 Pathogenesis of Multiple 4.53E+00 2.22E-01 22.2 CCR1,CCL5 2 Sclerosis FcγRIIB Signaling in B 4.35E+00 9.09E-02 9.09 CD79B,SYK,KRAS,CD79A,ATM 5 Lymphocytes Glutathione Metabolism 4.35E+00 7.14E-02 7.14 MGST1,PGD,G6PD,GLRX,GSTO1,IDH1 6 Role of IL-17A in Arthritis 4.08E+00 9.52E-02 9.52 MAPK1,CCL5,RPS6KA1 (includes 6 EG:20111),MAPKAPK2,MAP2K1,ATM Glycerolipid Metabolism 4.08E+00 6.25E-02 6.25 GLA,ABHD5,LPIN1,DHRS9,AGPAT2,PECR,AG 9 PAT9,GK,AKR1B1 Erythropoietin Signaling 3.98E+00 8.97E-02 8.97 MAPK1,PRKCD,KRAS,PRKCH,JAK2,MAP2K1,A 7 TM G Protein Signaling 3.96E+00 1.03E-01 10.3 LCK,GNB2,JAK2,GNG5 4 Mediated by Tubby DNA Methylation and 3.96E+00 1.30E-01 13 MTA1,SAP30 (includes EG:60406),DNMT1 3 Transcriptional Repression Signaling One Carbon Pool by 3.96E+00 8.33E-02 8.33 MTHFS,MTHFD2,ATIC 3 Folate Systemic Lupus 3.94E+00 7.48E-02 7.48 CD247,PRPF19,MAPK1,CD79B,CD3E,KRAS,C 16 Erythematosus Signaling D3D,CD79A,SNRPN,CD3G,LCK,LAT,TLR7,FCE R1G,CD86,ATM Thyroid Cancer Signaling 3.94E+00 1.11E-01 11.1 MAPK1,LEF1,KRAS,RXRA,MAP2K1 5 PTEN Signaling 3.89E+00 8.13E-02 8.13 PTK2 (includes 10 EG:14083),CDC42,FOXO1,MAPK1,YWHAH,K RAS,CHUK,MAP2K1,PTEN,BCL2 277

Biosynthesis of Steroids 3.63E+00 2.48E-02 2.48 IDI1,COX10 (includes EG:1352),HMGCR 3 Colorectal Cancer 3.57E+00 7.51E-02 7.51 VEGFB,MAPK1,SMAD3,TLR8,KRAS,IFNGR1,J 19 Metastasis Signaling AK2,GNG5,TLR4,RHOG,TLR5,RHOT1,GNB2,T LR7,LEF1,PTGER2,MAP2K1,RALGDS,ATM Insulin Receptor 3.56E+00 8.27E-02 8.27 FYN,PPP1R3D,FOXO1,MAPK1,PPP1R12A,IRS 11 Signaling 2,KRAS,JAK2,MAP2K1,PTEN,ATM TGF-β Signaling 3.56E+00 8.99E-02 8.99 RUNX3,MAPK1,SMAD3,MAP4K1,KRAS,MAP2 8 K1,ACVR1B,BCL2 TWEAK Signaling 3.54E+00 1.05E-01 10.5 TNFRSF25,APAF1,CHUK,BIRC2 4 Lysine Biosynthesis 3.54E+00 3.08E-02 3.08 VNN1,VNN2 2 Breast Cancer Regulation 3.54E+00 7.66E-02 7.66 CDC42,MAPK1,PPP2CA,KRAS,E2F3,PPP2R5A, 16 by Stathmin1 GNG5,GNAI3,PPP1R3D,PRKCD,ITPR3,GNB2,P PP1R12A,PRKCH,MAP2K1,ATM Amyotrophic Lateral 3.54E+00 7.76E-02 7.76 VEGFB,RAB5A,RAB5C,APAF1,GRINA,ATM,BC 9 Sclerosis Signaling L2,CAPN3,BIRC2 Synaptic Long Term 3.54E+00 7.86E-02 7.86 GNAI3,MAPK1,GNA15,PPP2CA,PRKCD,JMJD 11 Depression 7- PLA2G4B,ITPR3,KRAS,PRKCH,MAP2K1,PPP2 R5A Myc Mediated Apoptosis 3.54E+00 1.00E-01 10 YWHAG,YWHAH,APAF1,KRAS,ATM,BCL2 6 Signaling Death Receptor Signaling 3.54E+00 9.52E-02 9.52 TNFRSF25,APAF1,TBK1,CHUK,BCL2,BIRC2 6 Oncostatin M Signaling 3.37E+00 1.14E-01 11.4 MAPK1,KRAS,JAK2,MAP2K1 4 Retinoic acid Mediated 3.37E+00 9.38E-02 9.38 APAF1,PARP12,RXRA,TIPARP,IFNAR1,PARP1 6 Apoptosis Signaling Neuregulin Signaling 3.27E+00 8.00E-02 8 MAPK1,PRKCD,TGFA,KRAS,PRKCH,MAP2K1, 8 PTEN,MATK TNFR1 Signaling 3.27E+00 9.62E-02 9.62 CDC42,APAF1,TNFAIP3,CHUK,BIRC2 5 Cell Cycle Regulation by 3.20E+00 1.11E-01 11.1 PPP2CA,CDK4,E2F3,PPP2R5A 4 BTG Family Proteins Aldosterone Signaling in 3.16E+00 7.51E-02 7.51 MAPK1,HSPA1A/HSPA1B,KRAS,DNAJC5,GNA 13 Epithelial Cells 15,DUSP1,PRKCD,ITPR3,PRKCH,DNAJB6,DNA JC30,MAP2K1,ATM JAK/Stat Signaling 3.10E+00 9.38E-02 9.38 STAT4,MAPK1,KRAS,JAK2,MAP2K1,ATM 6 Aminophosphonate 3.01E+00 5.26E-02 5.26 PIGF,MGMT,CHPT1 3 Metabolism Tumoricidal Function of 3.01E+00 1.25E-01 12.5 PRF1,SRGN,APAF1 3 Hepatic Natural Killer Cells Antiproliferative Role of 2.99E+00 8.57E-02 8.57 MAPK1,GNB2,KRAS,MAP2K1,GNG5,ATM 6 Somatostatin Receptor 2 AMPK Signaling 2.92E+00 6.67E-02 6.67 CAB39,PFKFB3,MAPK1,PFKFB4,PPP2CA,PPM 11 1B,PRKAA1,IRS2,HMGCR,PPP2R5A,ATM IL-17A Signaling in Airway 2.88E+00 8.33E-02 8.33 MAPK1,CHUK,JAK2,MAP2K1,PTEN,ATM 6 Cells Dopamine-DARPP32 2.80E+00 7.10E-02 7.1 KCNJ2,PPP2CA,CHP,PPP3CC,PPP2R5A,GRINA 13 Feedback in cAMP ,GNAI3,PPP1R3D,GNA15,PRKCD,ITPR3,PPP1 Signaling R12A,PRKCH PPAR Signaling 2.79E+00 7.84E-02 7.84 IL1R2,IL18RAP,MAPK1,KRAS,CHUK,RXRA,MA 8 P2K1,IL1RAP Docosahexaenoic Acid 2.74E+00 8.33E-02 8.33 FOXO1,APAF1,ATM,BCL2 4 (DHA) Signaling Ceramide Signaling 2.74E+00 8.05E-02 8.05 CTSD,PPP2CA,KRAS,MAP2K1,PPP2R5A,ATM, 7 BCL2 P2Y Purigenic Receptor 2.72E+00 7.19E-02 7.19 GNAI3,MAPK1,GNA15,PRKCD,GNB2,KRAS,P 10 Signaling Pathway RKCH,MAP2K1,GNG5,ATM DNA Double-Strand 2.72E+00 1.18E-01 11.8 ATM,NBN 2 Break Repair by Homologous

278

Recombination

Folate Biosynthesis 2.72E+00 2.78E-02 2.78 ALPP/ALPPL2,ALPL 2 Antiproliferative Role of 2.70E+00 1.15E-01 11.5 MAPK1,SMAD3,RPS6KA1 (includes 3 TOB in T Cell Signaling EG:20111) p53 Signaling 2.68E+00 8.33E-02 8.33 CCND2,GADD45A,CDK4,APAF1,DRAM1,PTE 8 N,ATM,BCL2 SAPK/JNK Signaling 2.68E+00 7.92E-02 7.92 LCK,CDC42,GADD45A,FCER1G,MAP4K1,KRA 8 S,GNG5,ATM Role of PKR in Interferon 2.54E+00 8.89E-02 8.89 APAF1,TRAF5,CHUK,RNASEL 4 Induction and Antiviral Response Role of IL-17F in Allergic 2.54E+00 9.30E-02 9.3 MAPK1,RPS6KA5,RPS6KA1 (includes 4 Inflammatory Airway EG:20111),MAP2K1 Diseases Corticotropin Releasing 2.54E+00 6.92E-02 6.92 GNAI3,MAPK1,PRKCD,MEF2D,ARPC5,ITPR3, 9 Hormone Signaling MEF2C,PRKCH,MAP2K1 GNRH Signaling 2.48E+00 7.04E-02 7.04 PTK2 (includes 10 EG:14083),GNAI3,CDC42,MAPK1,PRKCD,ITP R3,KRAS,MAP3K8,PRKCH,MAP2K1 Neurotrophin/TRK 2.45E+00 7.89E-02 7.89 CDC42,MAPK1,KRAS,RPS6KA1 (includes 6 Signaling EG:20111),MAP2K1,ATM Sulfur Metabolism 2.32E+00 3.64E-02 3.64 PAPSS1,SUOX 2 Germ Cell-Sertoli Cell 2.31E+00 7.27E-02 7.27 PTK2 (includes 12 Junction Signaling EG:14083),RHOG,CDC42,MAPK1,RHOT1,CTN NA1,KRAS,MAP3K8,IQGAP1,MAP2K1,RAB8B ,ATM Role of BRCA1 in DNA 2.31E+00 8.20E-02 8.2 C17orf70,GADD45A,E2F3,ATM,NBN 5 Damage Response 4-1BB Signaling in T 2.31E+00 8.82E-02 8.82 MAPK1,CHUK,MAP2K1 3 Lymphocytes Melatonin Signaling 2.31E+00 7.69E-02 7.69 GNAI3,MAPK1,GNA15,PRKCD,PRKCH,MAP2 6 K1 Hereditary Breast Cancer 2.31E+00 7.26E-02 7.26 C17orf70,HDAC4,GADD45A,CDK4,CDK6,KRA 9 Signaling S,PTEN,ATM,NBN Paxillin Signaling 2.29E+00 7.27E-02 7.27 PTK2 (includes EG:14083),ITGAM (includes 8 EG:16409),CDC42,MAPK1,KRAS,PTPN12,ITG B7,ATM Melanocyte 2.27E+00 7.61E-02 7.61 MAPK1,KRAS,RPS6KA5,RPS6KA1 (includes 7 Development and EG:20111),MAP2K1,ATM,BCL2 Pigmentation Signaling MSP-RON Signaling 2.17E+00 8.16E-02 8.16 TLR4,ITGAM (includes EG:16409),JAK2,ATM 4 Pathway Mitotic Roles of Polo-Like 2.17E+00 8.33E-02 8.33 CDC25B,SLK,ANAPC1/LOC100286979,PPP2C 5 Kinase A,PPP2R5A CDK5 Signaling 2.15E+00 7.45E-02 7.45 PPP1R3D,MAPK1,PPP2CA,PPP1R12A,KRAS, 7 MAP2K1,PPP2R5A Ovarian Cancer Signaling 2.15E+00 7.09E-02 7.09 VEGFB,MAPK1,CDK4,CD44 (includes 10 EG:100330801),LEF1,KRAS,MAP2K1,PTEN,A TM,BCL2 Mitochondrial 2.10E+00 5.81E-02 5.81 COX7A2,NDUFB3,COX17 (includes 10 Dysfunction EG:10063),PRDX3,FURIN,SOD2,APH1B,PRDX 5,COX10 (includes EG:1352),GLRX2 Stilbene, Coumarine and 2.08E+00 2.74E-02 2.74 GBA,PRDX5 2 Lignin Biosynthesis EGF Signaling 2.02E+00 7.69E-02 7.69 MAPK1,ITPR3,MAP2K1,ATM 4 Xenobiotic Metabolism 1.98E+00 6.69E-02 6.69 MGST1,MAPK1,PPP2CA,NQO2,MGMT,KRAS, 18 Signaling PPP2R5A,CYP1B1,GSTO1,HMOX1,PRKCD,MA P3K8,PRKCH,ALDH3B1,RXRA,PNPLA6,MAP2 K1,ATM 279

Lipid Antigen 1.96E+00 9.09E-02 9.09 CD3E,FCER1G 2 Presentation by CD1 Role of CHK Proteins in 1.95E+00 8.57E-02 8.57 E2F3,ATM,NBN 3 Cell Cycle Checkpoint Control Role of JAK2 in Hormone- 1.95E+00 8.57E-02 8.57 IRS2,JAK2,SIRPA 3 like Cytokine Signaling cAMP-mediated signaling 1.94E+00 6.88E-02 6.88 RGS18,PDE7A,MAPK1,FPR2,PPP3CC,OPRL1,F 15 PR1,P2RY13,LAMTOR3,LTB4R,DUSP1,RPS6K A1 (includes EG:20111),PKIA,PTGER2,MAP2K1 RAR Activation 1.90E+00 6.52E-02 6.52 DHRS9,MAPK1,DUSP1,PRKCD,SMAD3,PRKC 12 H,JAK2,RXRA,MAPKAPK2,MAP2K1,PTEN,PAR P1 Huntington's Disease 1.90E+00 6.41E-02 6.41 HDAC4,MAPK1,HSPA1A/HSPA1B,CLTC,APAF 15 Signaling 1,GNG5,CTSD,DNAJC5,PRKCD,GNB2,VAMP3, PRKCH,GOSR2,CAPN3,ATM IL-9 Signaling 1.90E+00 7.50E-02 7.5 IRS2,BCL3,ATM 3 Pyrimidine Metabolism 1.90E+00 4.67E-02 4.67 NUDT5,POLR1C,POLR3H,POLR1E,UPP1,TXN 10 (includes EG:116484),PUS1,POLRMT,UNG,TXNRD1 Glycerophospholipid 1.90E+00 4.86E-02 4.86 HMOX1,LPIN1,GNA15,PIGF,AGPAT2,JMJD7- 9 Metabolism PLA2G4B,CHKA,CHPT1,AGPAT9 Leptin Signaling in 1.90E+00 6.90E-02 6.9 FOXO1,MAPK1,GNA15,JAK2,MAP2K1,ATM 6 Obesity CREB Signaling in 1.86E+00 6.12E-02 6.12 GNAI3,MAPK1,GNA15,PRKCD,ITPR3,GNB2,K 12 Neurons RAS,PRKCH,RPS6KA1 (includes EG:20111),MAP2K1,GNG5,ATM Reelin Signaling in 1.86E+00 7.32E-02 7.32 FYN,LCK,HCK,MAP4K1,FGR,ATM 6 Neurons Role of Osteoblasts, 1.84E+00 6.33E-02 6.33 IL18RAP,MAPK1,CHP,PPP3CC,BCL2,IL18R1,IL 15 Osteoclasts and 1R2,FOXO1,LEF1,TRAF5,CHUK,IL1RAP,ALPL, Chondrocytes in BIRC2,ATM Rheumatoid Arthritis G-Protein Coupled 1.84E+00 6.42E-02 6.42 FYN,RGS18,PDE7A,MAPK1,KRAS,CX3CR1,OP 34 Receptor Signaling RL1,LAMTOR3,P2RY10,LTB4R,C5AR1,GPR11 4,CHUK,MAP2K1,ATM,CCR1,GPR97,FPR2,PC NX,FPR1,GPR183,GPR56,P2RY13,DUSP1,RAS GRP1,GPR65,LPAR5,MAP3K8,GPR160,PTGER 2,EMR1,RPS6KA1 (includes EG:20111),EMR2,CCR7 Angiopoietin Signaling 1.82E+00 6.76E-02 6.76 PTK2 (includes 5 EG:14083),FOXO1,KRAS,CHUK,ATM Androgen Signaling 1.80E+00 5.76E-02 5.76 GNAI3,MAPK1,GNA15,PRKCD,SMAD3,GNB2, 8 PRKCH,GNG5 Nucleotide Sugars 1.74E+00 1.54E-02 1.54 UGP2 1 Metabolism Differential Regulation of 1.66E+00 8.70E-02 8.7 LCN2,CCL5 2 Cytokine Production in Intestinal Epithelial Cells by IL-17A and IL-17F April Mediated Signaling 1.66E+00 6.98E-02 6.98 MAPK1,TRAF5,CHUK 3 TR/RXR Activation 1.62E+00 6.25E-02 6.25 SLC16A3,LDLR,BCL3,HIF1A,RXRA,ATM 6 Assembly of RNA 1.61E+00 7.69E-02 7.69 POLR1C 1 Polymerase I Complex Arginine and Proline 1.61E+00 2.87E-02 2.87 P4HA1,VNN1,VNN2,OAT,SAT1 5 Metabolism IL-22 Signaling 1.61E+00 8.00E-02 8 MAPK1,IL10RB 2 Endothelin-1 Signaling 1.59E+00 6.01E-02 6.01 HMOX1,GNAI3,MAPK1,GNA15,PRKCD,JMJD 11 7-PLA2G4B,ITPR3,KRAS,PRKCH,PTGER2,ATM PDGF Signaling 1.59E+00 6.33E-02 6.33 MAPK1,KRAS,JAK2,MAP2K1,ATM 5

280

B Cell Activating Factor 1.59E+00 6.67E-02 6.67 MAPK1,TRAF5,CHUK 3 Signaling N-Glycan Biosynthesis 1.59E+00 3.85E-02 3.85 ST6GAL1,MAN1A1,B4GALT5 3 Bladder Cancer Signaling 1.59E+00 6.67E-02 6.67 VEGFB,MAPK1,CDK4,KRAS,RPS6KA5,MAP2K 6 1 Neuropathic Pain 1.59E+00 6.31E-02 6.31 MAPK1,GNA15,PRKCD,ITPR3,PRKCH,GRINA, 7 Signaling In Dorsal Horn ATM Neurons N-Glycan Degradation 1.59E+00 8.00E-02 8 NEU1,MAN1A1 2 IL-17A Signaling in 1.59E+00 8.00E-02 8 MAPK1,CCL5 2 Gastric Cells Role of JAK family kinases 1.59E+00 7.41E-02 7.41 MAPK1,JAK2 2 in IL-6-type Cytokine Signaling Acute Phase Response 1.59E+00 6.18E-02 6.18 HMOX1,SOD2,MAPK1,MYD88,SERPINA1,KR 11 Signaling AS,CHUK,CEBPB (includes EG:1051),JAK2,MAP2K1,IL1RAP Metabolism of 1.59E+00 3.53E-02 3.53 MGST1,DHRS9,PECR,ALDH3B1,GSTO1,CYP1 6 Xenobiotics by B1 Cytochrome P450 Role of RIG1-like 1.59E+00 6.52E-02 6.52 TBK1,CHUK,TRIM25 3 Receptors in Antiviral Innate Immunity Valine, Leucine and 1.59E+00 2.44E-02 2.44 VARS 1 Isoleucine Biosynthesis Fatty Acid Biosynthesis 1.59E+00 2.04E-02 2.04 ACSL3 1 Induction of Apoptosis by 1.50E+00 6.25E-02 6.25 APAF1,CHUK,BCL2,BIRC2 4 HIV1 IL-15 Production 1.50E+00 6.45E-02 6.45 PTK2 (includes EG:14083),JAK2 2 Activation of IRF by 1.47E+00 5.80E-02 5.8 LTA,TBK1,CHUK,IFNAR1 4 Cytosolic Pattern Recognition Receptors Citrate Cycle 1.45E+00 3.57E-02 3.57 DLD,IDH1 2 Gap Junction Signaling 1.44E+00 5.56E-02 5.56 GNAI3,MAPK1,GNA15,PRKCD,ITPR3,KRAS,P 10 RKCH,PPP3CC,MAP2K1,ATM Role of Oct4 in 1.44E+00 6.67E-02 6.67 SH3GLB1,HOXB1,PARP1 3 Mammalian Embryonic Stem Cell Pluripotency Sphingosine-1-phosphate 1.40E+00 5.69E-02 5.69 PTK2 (includes 7 Signaling EG:14083),GNAI3,RHOG,MAPK1,GNA15,RH OT1,ATM Role of IL-17A in Psoriasis 1.37E+00 7.69E-02 7.69 S100A8 1 Complement System 1.30E+00 5.88E-02 5.88 CD55,C5AR1 2 Telomere Extension by 1.27E+00 5.88E-02 5.88 NBN 1 Telomerase Methane Metabolism 1.27E+00 1.54E-02 1.54 PRDX5 1 Phenylalanine, Tyrosine 1.27E+00 1.54E-02 1.54 FARS2 1 and Tryptophan Biosynthesis RAN Signaling 1.23E+00 4.35E-02 4.35 KPNA4 1

281

Appendix 8.3: Signalling pathways from 933 SDE upregulated gene transcripts

Ingenuity Canonical B H Ratio % Molecules No. of Pathways p value molecule Glycolysis/ 2.82E+06 1.69E-01 16.9 PGK1,PKM2,ACSL3,PGM2,GAPDH,PGM1, 21 Gluconeogenesis TPI1,PGAM4,GPI,HK2,DHRS9,PGAM1,ACS S2,DLD,FBP1,ALDOA,ALDH3B1,PECR,HK3, LDHA,ACSL1 Toll-like Receptor Signaling 2.24E+05 2.73E-01 27.3 TLR1,TOLLIP,MAPK1,MYD88,TLR8,IRAK3, 15 TLR2,TLR4,FOS,LY96 (includes EG:17087),MAPK14,TLR5,CD14,CHUK,IRA K2 Production of Nitric Oxide 8.51E+04 1.38E-01 13.8 MAPK1,PPP2CA,MAPK3,JAK2,SPI1 29 and Reactive Oxygen (includes Species in Macrophages EG:20375),RHOG,RHOT1,CYBA,CYBB,S10 0A8,SERPINA1,CHUK,MAP2K1,ORM1/OR M2,TNFRSF1A,IFNGR2,IFNGR1,NCF4,PPP 2R5A,TLR2,TLR4,FOS,PPP1R3D,MAPK14,P RKCD,NCF2,PPP1R12A,MAP3K8,SIRPA fMLP Signaling in 1.07E+04 1.57E-01 15.7 ACTR2,MAPK1,CDC42,MAPK3,CHP,ARPC5 20 Neutrophils ,FPR2,KRAS,GNG5,FPR1,GNG10,GNAI3,A CTR3,PRKCD,NCF2,GNB2,CYBB,ARPC3,AR PC1A,MAP2K1 Role of Macrophages, 1.82E+03 1.09E-01 10.9 IL18RAP,TLR1,MAPK1,MAPK3,TLR8,KRAS, 35 Fibroblasts and Endothelial JAK2,IL17RA,IL18R1,IL1R2,C5AR1,GNA15, Cells in Rheumatoid CEBPA,OSM,LTBR,CHUK,MAPKAPK2,MAP Arthritis 2K1,IL1RAP,TNFRSF1A,MYD88,CHP,GNAQ ,CEBPB (includes EG:1051),IRAK3,TLR2,TLR4,FOS,MAPK14, TLR5,IL1RN,CEBPD,PRKCD,IL1B,IRAK2 IL-6 Signaling 1.78E+03 1.70E-01 17 IL18RAP,MAPK1,TNFRSF1A,MAPK3,KRAS, 17 CEBPB (includes EG:1051),JAK2,IL1R2,FOS,MAPK14,IL1RN, IL1B,CD14,CHUK,MAPKAPK2,IL1RAP,MAP 2K1 IL-17A Signaling in 1.78E+03 2.50E-01 25 FOS,MAPK14,MAPK1,MAPK3,CEBPD,LCN 10 Fibroblasts 2,CHUK,CEBPB (includes EG:1051),NFKBIZ,IL17RA IL-10 Signaling 1.66E+03 1.79E-01 17.9 CCR1,IL18RAP,IL4R,MAPK1,IL1R2,HMOX1 14 ,FOS,MAPK14,IL1RN,IL10RB,IL1B,CD14,C HUK,IL1RAP Signaling by Rho Family 1.26E+03 1.15E-01 11.5 MYL6,MAPK1,CDC42,MAPK3,ARPC5,LIMK 29 GTPases 2,CDC42EP2,IQGAP1,CLIP1,RHOG,ACTR3, GNA15,RHOT1,CYBB,ARPC3,ARPC1A,MA P2K1,ACTR2,GNAQ,VIM,GNG5,GNG10,G NAI3,FOS,MYL12A,MYL12B,NCF2,GNB2,P PP1R12A Fcγ Receptor-mediated 1.07E+03 1.58E-01 15.8 ACTR2,MAPK1,CDC42,MAPK3,ARPC5,PTE 16 Phagocytosis in N,HMOX1,ACTR3,PRKCD,SYK,HCK,VAMP3 Macrophages and ,ARPC3,ARPC1A,VASP,FGR Monocytes IL-1 Signaling 1.07E+03 1.50E-01 15 TOLLIP,MAPK1,MYD88,GNAQ,PRKAR2A,I 16 RAK3,GNG5,GNG10,GNAI3,FOS,MAPK14, GNA15,GNB2,CHUK,IL1RAP,IRAK2 Role of Pattern Recognition 1.07E+03 1.71E-01 17.1 TLR1,MAPK1,MYD88,MAPK3,TLR8,RNASE 14 Receptors in Recognition of L,TLR2,TLR4,C5AR1,TLR5,SYK,IL1B,C3AR1, Bacteria and Viruses NLRC4 LXR/RXR Activation 7.94E+02 1.37E-01 13.7 IL18RAP,ORM1/ORM2,TNFRSF1A,CD36,A 18 BCA1,IL1R2,TLR4,LY96 (includes EG:17087),LDLR,IL1RN,IL1B,CD14,S100A8 ,SERPINA1,RXRA,HMGCR,IL1RAP,MMP9

282

IL-8 Signaling 7.94E+02 1.20E-01 12 MAPK1,MAPK3,LIMK2,KRAS,IRAK3,CSTB, 23 GNG5,GNG10,HMOX1,GNAI3,RHOG,CCN D3,ITGAM (includes EG:16409),RHOT1,MYL12B,PRKCD,NCF2, GNB2,CYBB,CHUK,MAP2K1,MMP9,IRAK2 PPARα/RXRα Activation 7.94E+02 1.24E-01 12.4 IL18RAP,MAPK1,ACAA1,MAPK3,ACOX1,G 22 NAQ,CD36,PRKAR2A,KRAS,JAK2,GK,ABCA 1,ACVR1B,IL1R2,MAPK14,GNA15,PRKAA1 ,IL1B,CHUK,RXRA,IL1RAP,MAP2K1 Cdc42 Signaling 6.76E+02 1.30E-01 13 ACTR2,MYL6,MAPK1,CDC42,ARPC5,LIMK 19 2,EXOC6,CDC42EP2,IQGAP1,CLIP1,FOS,M YL12A,MAPK14,ACTR3,MYL12B,FCER1G, ARPC3,PPP1R12A,ARPC1A TREM1 Signaling 6.61E+02 1.77E-01 17.7 TLR2,TLR4,TLR1,TLR5,MAPK1,TYROBP,M 11 APK3,TLR8,LAT2,IL1B,JAK2 Pentose Phosphate 4.57E+02 1.01E-01 10.1 PGM2,GPI,PGD,TKT,G6PD,PGM1,FBP1,AL 8 Pathway DOA Starch and Sucrose 3.72E+02 7.36E-02 7.36 NUDT5,PGM2,GPI,HK2,UGP2,GBA,PGM1, 12 Metabolism GBE1,HPSE,PYGL,HK3,CSGALNACT1 Actin Nucleation by ARP- 3.24E+02 1.67E-01 16.7 ACTR2,ACTR3,RHOG,CDC42,RHOT1,ARPC 11 WASP Complex 5,PPP1R12A,ARPC3,ARPC1A,KRAS,VASP NF-κB Signaling 2.95E+02 1.23E-01 12.3 TLR1,AZI2,TNFRSF1A,MYD88,TLR8,TNFAI 21 P3,TBK1,KRAS,IRAK3,TANK,TLR2,IL1R2,TL R4,TLR5,IL1RN,TGFA,FCER1G,IL1B,MAP3K 8,LTBR,CHUK IL-12 Signaling and 2.75E+02 1.18E-01 11.8 ORM1/ORM2,MAPK1,MYD88,MAPK3,IFN 18 Production in Macrophages GR1,CEBPB (includes EG:1051),SPI1 (includes EG:20375),TLR2,FOS,TLR4,MAPK14,PRKC D,S100A8,SERPINA1,MAP3K8,CHUK,RXRA ,MAP2K1 Nicotinate and 2.63E+02 1.12E-01 11.2 MAPK1,MAPK3,LIMK2,NAPRT1,VNN1,CD 15 Nicotinamide Metabolism K5,PIM1,VNN2,PRKCD,PRKAA1,GRK6,NA MPT,MAP3K8,MAP2K1,BST1 (includes EG:12182) Oxidative Phosphorylation 2.24E+02 1.21E-01 12.1 NDUFA4,COX17 (includes 18 EG:10063),NDUFB3,COX7B,COX7A2,ATP6 V1D (includesEG:299159),ATP6V0B,ATP6V1C1 ,TCIRG1,ATP6V1A,ATP6AP1,NDUFA1,ATP 6V1E1,ATP5C1,PPA2 (includes EG:27068), ATP6V0D1, ATP6V0E1,ATP6V1B2 Integrin Signaling 2.00E+02 1.12E-01 11.2 ACTR2,MAPK1,CDC42,ASAP1,MAPK3,RAL 23 B,ARPC5,KRAS,PTEN,MYL12A,RHOG,ITGA M (includesEG:16409),ACTR3,RHOT1,MYL12 B,ARF4,PPP1R12A,ARPC3,ZYX,ARPC1A,M AP2K1,VASP,CAPN3 Inositol Phosphate 1.78E+02 9.95E-02 9.95 MAPK1,MAPK3,GNAQ,LIMK2,INPP5A,PTE 19 Metabolism N,GNAI3,GNA15,CDK5,SYNJ1,PIM1,PRKC D,GRK6,PRKAA1,IMPA2,IRS2,MAP3K8,M AP2K1,MTMR3 Rac Signaling 1.78E+02 1.23E-01 12.3 ACTR2,MAPK1,CDC42,MAPK3,ARPC5,KRA 15 S,LIMK2,IQGAP1,ACTR3,NCF2,CD44 (includes EG:100330801),CYBB,ARPC3,ARPC1A,MA P2K1 Regulation of Actin-based 1.78E+02 1.46E-01 14.6 ACTR2,CDC42,MYL6,ARPC5,GSN,MYL12A, 13 Motility by Rho ACTR3,RHOG,RHOT1,MYL12B,PPP1R12A, ARPC3,ARPC1A HMGB1 Signaling 1.78E+02 1.41E-01 14.1 MAPK1,CDC42,HAT1,TNFRSF1A,MAPK3,I 14 FNGR2,IFNGR1,KRAS,TLR4,FOS,RHOG,MA 283

PK14,RHOT1,MAP2K1

Fructose and Mannose 1.70E+02 6.98E-02 6.98 NUDT5,PFKFB3,PGM2,HK2,PFKFB4,FBP1, 9 Metabolism ALDOA,HK3,TPI1 Role of Tissue Factor in 1.51E+02 1.35E-01 13.5 CDC42,MAPK1,MAPK3,GNAQ,PLAUR,KRA 15 Cancer S,LIMK2,JAK2,PTEN,ARRB2,MAPK14,HCK, IL1B,RPS6KA1 (includes EG:20111),FGR Acute Phase Response 1.23E+02 1.12E-01 11.2 ORM1/ORM2,MAPK1,TNFRSF1A,MYD88, 20 Signaling MAPK3,KRAS,JAK2,CEBPB (includes EG:1051),FOS,HMOX1,HP,SOD2,MAPK14, IL1RN,OSM,IL1B,SERPINA1,CHUK,IL1RAP, MAP2K1 Glioma Invasiveness 1.05E+02 1.67E-01 16.7 RHOG,MAPK1,RHOT1,TIMP1,MAPK3,CD4 10 Signaling 4 (includes EG:100330801),PLAUR,KRAS,MMP9,TIMP 2 (includes EG:21858) Ephrin Receptor Signaling 9.77E+01 1.01E-01 10.1 ACTR2,MAPK1,CDC42,MAPK3,ARPC5,GN 20 AQ,LIMK2,KRAS,JAK2,GNG5,GRINA,GNG1 0,GNAI3,ACTR3,SDCBP,GNA15,GNB2,ARP C3,ARPC1A,MAP2K1 Hepatic Cholestasis 8.91E+01 9.88E-02 9.88 IL18RAP,TNFRSF1A,MYD88,PRKAR2A,IRA 17 K3,IL1R2,TLR4,LY96 (includes EG:17087),IL1RN,RARA,PRKCD,IL1B,CD14 ,CHUK,RXRA,IL1RAP,IRAK2 Hepatic Fibrosis / Hepatic 8.71E+01 1.18E-01 11.8 IL18RAP,IL4R,MYL6,TNFRSF1A,IFNGR2,IF 17 Stellate Cell Activation NGR1,IL1R2,TLR4,LY96 (includes EG:17087),TIMP1,TGFA,IL1B,CD14,IL1RA P,MMP9,IFNAR1,TIMP2 (includes EG:21858) PPAR Signaling 8.71E+01 1.27E-01 12.7 IL18RAP,MAPK1,TNFRSF1A,MAPK3,KRAS, 13 IL1R2,FOS,IL1RN,IL1B,CHUK,RXRA,MAP2K 1,IL1RAP RhoGDI Signaling 8.71E+01 1.01E-01 10.1 ACTR2,MYL6,CDC42,ARPC5,GNAQ,LIMK2, 20 GNG5,GNG10,GNAI3,MYL12A,ACTR3,RH OG,GNA15,RHOT1,MYL12B,GNB2,CD44 (includes EG:100330801),PPP1R12A,ARPC3,ARPC1 A p38 MAPK Signaling 6.31E+01 1.32E-01 13.2 IL18RAP,DDIT3,MAPKAPK3,TNFRSF1A,IRA 14 K3,IL1R2,MAPK14,DUSP1,IL1RN,MKNK1,I L1B,MAPKAPK2,IL1RAP,IRAK2 PI3K Signaling in B 6.31E+01 1.13E-01 11.3 IL4R,MAPK1,MAPK3,CHP,GNAQ,KRAS,AT 16 Lymphocytes F6,PTEN,TLR4,FOS,GNA15,SYK,IRS2,PIK3A P1,CHUK,MAP2K1 LPS-stimulated MAPK 6.31E+01 1.34E-01 13.4 TLR4,FOS,MAPK14,CDC42,MAPK1,PRKCD 11 Signaling ,MAPK3,CD14,KRAS,CHUK,MAP2K1 Cardiac Hypertrophy 6.03E+01 9.54E-02 9.54 MYL6,MAPK1,MAPKAPK3,MAPK3,CHP,G 23 Signaling NAQ,PRKAR2A,KRAS,ATF6,GNG5,GNG10, GNAI3,MYL12A,MAPK14,RHOG,GNA15,R HOT1,MYL12B,GNB2,MAP3K8,RPS6KA1 (includes EG:20111),MAPKAPK2,MAP2K1 p70S6K Signaling 5.37E+01 1.15E-01 11.5 IL4R,YWHAG,MAPK1,YWHAH,PPP2CA,M 15 APK3,GNAQ,KRAS,PPP2R5A,GNAI3,GNA1 5,PRKCD,SYK,BCAP31,MAP2K1 α-Adrenergic Signaling 5.01E+01 1.14E-01 11.4 GNAI3,MAPK1,PRKCD,MAPK3,GNB2,PRK 12 AR2A,GNAQ,PYGL,KRAS,MAP2K1,GNG5, GNG10 G Beta Gamma Signaling 4.17E+01 1.06E-01 10.6 GNAI3,CDC42,MAPK1,GNA15,PRKCD,MA 12 PK3,GNB2,PRKAR2A,GNAQ,KRAS,GNG5,G NG10 TNFR2 Signaling 3.89E+01 1.82E-01 18.2 TANK,FOS,TNFAIP3,TBK1,CHUK,BIRC2 6

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TNFR1 Signaling 3.89E+01 1.54E-01 15.4 TANK,FOS,CDC42,TNFRSF1A,APAF1,TNFA 8 IP3,CHUK,BIRC2 Chemokine Signaling 3.89E+01 1.39E-01 13.9 GNAI3,FOS,MAPK14,MAPK1,MAPK3,GNA 10 Q,PPP1R12A,KRAS,LIMK2,MAP2K1 CXCR4 Signaling 3.89E+01 1.01E-01 10.1 MYL6,MAPK1,MAPK3,GNAQ,KRAS,GNG5, 17 GNG10,GNAI3,FOS,MYL12A,RHOG,GNA1 5,RHOT1,MYL12B,PRKCD,GNB2,MAP2K1 Dendritic Cell Maturation 3.89E+01 9.50E-02 9.5 MAPK1,TYROBP,MYD88,TNFRSF1A,MAPK 17 3,CD58,JAK2,TLR2,TLR4,MAPK14,IL1RN,F CER1G,IL1B,LTBR,CHUK,IFNAR1,FCGR1B NRF2-mediated Oxidative 3.89E+01 1.00E-01 10 RBX1,MGST1,MAPK1,NQO2,MAPK3,KRAS 19 Stress Response ,JUNB,GSTO1,TXNRD1,FOS,HMOX1,DNAJ C5,SOD2,MAPK14,PRKCD,DNAJB6,TXN (includes EG:116484),FKBP5,MAP2K1 Renal Cell Carcinoma 3.80E+01 1.37E-01 13.7 FOS,RBX1,CDC42,MAPK1,MAPK3,TGFA,K 10 Signaling RAS,HIF1A,MAP2K1,TCEB1 IL-17 Signaling 2.82E+01 1.35E-01 13.5 MAPK14,MAPK1,TIMP1,MAPK3,KRAS,CE 10 BPB (includes EG:1051),JAK2,MAPKAPK2,IL17RA,MAP2 K1 CD40 Signaling 2.63E+01 1.30E-01 13 TANK,FOS,MAPK14,MAPK1,MAPK3,TNFA 9 IP3,CHUK,MAPKAPK2,MAP2K1 GM-CSF Signaling 2.63E+01 1.34E-01 13.4 MAPK1,CSF2RA,PIM1,MAPK3,HCK,KRAS,J 9 AK2,BCL2A1,MAP2K1 Phospholipase C Signaling 2.63E+01 8.59E-02 8.59 HDAC4,MYL6,MAPK1,MAPK3,CHP,RALB, 22 GNAQ,KRAS,GNG5,GNG10,HMOX1,MYL1 2A,RHOG,RHOT1,MYL12B,PRKCD,SYK,GN B2,FCER1G,PPP1R12A,MARCKS,MAP2K1 Apoptosis Signaling 2.04E+01 1.17E-01 11.7 MAPK1,TNFRSF1A,MAPK3,APAF1,KRAS,C 11 HUK,RPS6KA1 (includes EG:20111),BCL2A1,MAP2K1,CAPN3,BIRC 2 Type II Diabetes Mellitus 2.00E+01 8.44E-02 8.44 PKM2,ACSL3,MAPK1,TNFRSF1A,MAPK3,C 13 Signaling D36,CEBPB (includes EG:1051),PRKCD,PRKAA1,ACSL4,IRS2,CH UK,ACSL1 Cholecystokinin/Gastrin- 2.00E+01 1.13E-01 11.3 FOS,RHOG,MAPK14,MAPK1,RHOT1,IL1R 12 mediated Signaling N,PRKCD,MAPK3,GNAQ,IL1B,KRAS,MAP2 K1 Acute Myeloid Leukemia 2.00E+01 1.22E-01 12.2 CSF3R,MAPK1,CSF2RA,PIM1,MAPK3,RAR 10 Signaling A,CEBPA,KRAS,MAP2K1,SPI1 (includes EG:20375) LPS/IL-1 Mediated 2.00E+01 8.92E-02 8.92 IL18RAP,MGST1,ACSL3,TNFRSF1A,MYD88 19 Inhibition of RXR Function ,ACOX1,GSTO1,ABCA1,IL1R2,TLR4,LY96 (includes EG:17087),RARA,CD14,ACSL4,IL1B,ALDH3 B1,RXRA,IL1RAP,ACSL1 Breast Cancer Regulation 2.00E+01 9.09E-02 9.09 MAPK1,CDC42,PPP2CA,MAPK3,GNAQ,PR 19 by Stathmin1 KAR2A,LIMK2,KRAS,E2F3,PPP2R5A,GNG5 ,GNG10,GNAI3,PPP1R3D,PRKCD,GNB2,PP P1R12A,MAP2K1,E2F2 Molecular Mechanisms of 2.00E+01 7.80E-02 7.8 MAPK1,APH1B,CDC42,MAPK3,CTNNA1,K 29 Cancer RAS,JAK2,HIF1A,E2F3,LAMTOR3,RHOG,G NA15,RHOT1,MAP2K1,E2F2,HAT1,RALB,C DKN2D,GNAQ,APAF1,PRKAR2A,NBN,GNA I3,FOS,MAPK14,CCND3,PRKCD,RBPJ,BIRC 2 Oncostatin M Signaling 2.00E+01 1.71E-01 17.1 MAPK1,MAPK3,OSM,KRAS,JAK2,MAP2K1 6 Thrombopoietin Signaling 2.00E+01 1.27E-01 12.7 FOS,MAPK1,PRKCD,MAPK3,IRS2,KRAS,JA 8 K2,MAP2K1

285

Clathrin-mediated 2.00E+01 9.38E-02 9.38 ACTR2,ORM1/ORM2,RAB5A,CDC42,CHP, 18 Endocytosis Signaling CLTC,ARPC5,NUMB,SH3GLB1,ARRB2,ACT R3,LDLR,SYNJ1,RAB5C,ARPC3,S100A8,AR PC1A,SERPINA1 Synaptic Long Term 2.00E+01 1.06E-01 10.6 PPP1R3D,MAPK1,PRKCD,MAPK3,CHP,PRK 12 Potentiation AR2A,GNAQ,PPP1R12A,KRAS,RPS6KA1 (includes EG:20111),MAP2K1,GRINA CCR5 Signaling in 2.00E+01 1.02E-01 10.2 GNAI3,FOS,MAPK14,MAPK1,PRKCD,GNB 9 Macrophages 2,FCER1G,GNG5,GNG10 IL-17A Signaling in Gastric 2.00E+01 2.00E-01 20 FOS,MAPK14,MAPK1,MAPK3,IL17RA 5 Cells Role of JAK family kinases 2.00E+01 1.85E-01 18.5 MAPK14,MAPK1,MAPK3,OSM,JAK2 5 in IL-6-type Cytokine Signaling Germ Cell-Sertoli Cell 1.91E+01 9.70E-02 9.7 MAPK1,CDC42,TNFRSF1A,MAPK3,CTNNA 16 Junction Signaling 1,KRAS,LIMK2,IQGAP1,GSN,RHOG,MAPK 14,RHOT1,ZYX,MAP3K8,MAP2K1,RAB8B 14-3-3-mediated Signaling 1.91E+01 1.07E-01 10.7 YWHAG,MAPK1,YWHAH,TNFRSF1A,MAP 13 K3,GNAQ,VIM,KRAS,FOS,GNA15,PRKCD,R PS6KA1 (includes EG:20111),MAP2K1 ERK/MAPK Signaling 1.82E+01 8.82E-02 8.82 YWHAG,YWHAH,MAPK1,PPP2CA,MAPK3, 18 PRKAR2A,ETS2,KRAS,PPP2R5A,FOS,PPP1 R3D,LAMTOR3,DUSP1,PRKCD,MKNK1,PP P1R12A,RPS6KA1 (includes EG:20111),MAP2K1 Type I Diabetes Mellitus 1.62E+01 1.02E-01 10.2 MAPK14,MAPK1,MYD88,TNFRSF1A,APAF 12 Signaling 1,IFNGR2,FCER1G,IL1B,IFNGR1,CHUK,JAK 2,IL1RAP Aryl Hydrocarbon Receptor 1.62E+01 9.03E-02 9.03 MGST1,MAPK1,NQO2,MAPK3,APAF1,GST 14 Signaling O1,CYP1B1,CTSD,FOS,CCND3,RARA,IL1B, ALDH3B1,RXRA P2Y Purigenic Receptor 1.62E+01 9.35E-02 9.35 MAPK1,MAPK3,PRKAR2A,GNAQ,KRAS,GN 13 Signaling Pathway G5,GNG10,GNAI3,FOS,GNA15,PRKCD,GN B2,MAP2K1 Thrombin Signaling 1.58E+01 8.82E-02 8.82 MYL6,MAPK1,MAPK3,GNAQ,KRAS,GNG5, 18 GNG10,GNAI3,MYL12A,RHOG,MAPK14,G NA15,RHOT1,MYL12B,PRKCD,GNB2,PPP1 R12A,MAP2K1 Glucocorticoid Receptor 1.58E+01 7.88E-02 7.88 YWHAH,MAPK1,HSPA1A/HSPA1B,MAPK3 23 Signaling ,CHP,SLPI,KRAS,JAK2,CEBPB (includes EG:1051),TAF7,CD163,IL1R2,FOS,MAPK1 4,IL1RN,DUSP1,ANXA1,CEBPA,PRKAA1,IL 1B,CHUK,FKBP5,MAP2K1 Pyruvate Metabolism 1.48E+01 6.20E-02 6.2 PKM2,ACSL3,ACSS2,DLD,ME2,ACOT9,ACS 8 L1,LDHA Galactose Metabolism 1.45E+01 6.12E-02 6.12 PGM2,GLA,HK2,UGP2,PGM1,HK3 6 IGF-1 Signaling 1.45E+01 1.05E-01 10.5 FOS,YWHAG,MAPK1,YWHAH,MAPK3,PRK 11 AR2A,IRS2,KRAS,IGFBP7,JAK2,MAP2K1 Actin Cytoskeleton 1.45E+01 8.26E-02 8.26 ACTR2,MYL6,MAPK1,CDC42,MAPK3,ARP 19 Signaling C5,LIMK2,KRAS,IQGAP1,GSN,MYL12A,AC TR3,MYL12B,ARPC3,CD14,PPP1R12A,ARP C1A,SSH2,MAP2K1 Colorectal Cancer 1.45E+01 8.30E-02 8.3 TLR1,MAPK1,TNFRSF1A,MAPK3,PRKAR2A 21 Metastasis Signaling ,TLR8,KRAS,IFNGR1,JAK2,GNG5,GNG10,T LR2,TLR4,FOS,RHOG,TLR5,RHOT1,GNB2,P TGER2,MAP2K1,MMP9 Role of JAK1 and JAK3 in γc 1.41E+01 1.21E-01 12.1 IL4R,FES,MAPK1,MAPK3,SYK,IRS2,KRAS,J 8 Cytokine Signaling AK2 Amyloid Processing 1.38E+01 1.30E-01 13 MAPK14,APH1B,CDK5,MAPK1,MAPK3,PR 7 KAR2A,CAPN3

286

RANK Signaling in 1.38E+01 1.06E-01 10.6 FOS,MAPK14,MAPK1,MAPK3,CHP,MAP3 10 Osteoclasts K8,CHUK,GSN,MAP2K1,BIRC2 CDK5 Signaling 1.29E+01 1.06E-01 10.6 PPP1R3D,CDK5,MAPK1,PPP2CA,MAPK3,P 10 RKAR2A,PPP1R12A,KRAS,MAP2K1,PPP2R 5A CCR3 Signaling in 1.29E+01 9.60E-02 9.6 GNAI3,MAPK14,MAPK1,PRKCD,MAPK3,G 12 Eosinophils NB2,PPP1R12A,KRAS,LIMK2,MAP2K1,GN G5,GNG10 CD28 Signaling in T Helper 1.29E+01 9.30E-02 9.3 ACTR2,FOS,ACTR3,CDC42,SYK,ARPC5,CHP 12 Cells ,FCER1G,ARPC3,ARPC1A,CHUK,MAP2K1 Role of IL-17F in Allergic 1.29E+01 1.40E-01 14 MAPK1,MAPK3,IL1B,RPS6KA1 (includes 6 Inflammatory Airway EG:20111),IL17RA,MAP2K1 Diseases IL-17A Signaling in Airway 1.29E+01 1.11E-01 11.1 MAPK14,MAPK1,MAPK3,CHUK,JAK2,IL17 8 Cells RA,MAP2K1,PTEN Role of NFAT in Regulation 1.29E+01 8.21E-02 8.21 MAPK1,MAPK3,CHP,GNAQ,KRAS,GNG5,G 16 of the Immune Response NG10,GNAI3,FOS,GNA15,SYK,GNB2,FCER 1G,CHUK,MAP2K1,FCGR1B HIF1α Signaling 1.29E+01 1.04E-01 10.4 RBX1,MAPK14,MAPK1,MAPK3,COPS5,KR 11 AS,HIF1A,TCEB1,LDHA,MMP9,SLC2A3 Axonal Guidance Signaling 1.29E+01 7.21E-02 7.21 MAPK1,CDC42,MYL6,MAPK3,ARPC5,LIMK 31 2,KRAS,MICAL1,ACTR3,CDK5,GNA15,MK NK1,ARPC3,ARPC1A,SEMA4A,MAP2K1,V ASP,ACTR2,FES,CHP,GNAQ,PRKAR2A,GN G5,GNG10,GNAI3,MYL12A,SDCBP,MYL12 B,PRKCD,GNB2,RTN4 (includes EG:57142) Semaphorin Signaling in 1.26E+01 1.35E-01 13.5 RHOG,FES,CDK5,MAPK1,RHOT1,MAPK3,L 7 Neurons IMK2 PI3K/AKT Signaling 1.26E+01 8.82E-02 8.82 YWHAG,MAPK1,YWHAH,PPP2CA,MAPK3, 12 KRAS,MAP3K8,CHUK,JAK2,MAP2K1,PPP2 R5A,PTEN B Cell Receptor Signaling 1.26E+01 9.09E-02 9.09 CDC42,MAPK1,MAPK3,KRAS,BCL6,PTEN, 14 MAPK14,PAG1,SYK,PIK3AP1,MAP3K8,CH UK,BCL2A1,MAP2K1 MIF Regulation of Innate 1.26E+01 1.20E-01 12 TLR4,FOS,LY96 (includes 6 Immunity EG:17087),MAPK1,MAPK3,CD14 Hypoxia Signaling in the 1.17E+01 1.21E-01 12.1 UBE2H,UBE2A,COPS5,HIF1A,UBE2J1,LDH 8 Cardiovascular System A,UBE2F,PTEN

G Protein Signaling 1.17E+01 1.28E-01 12.8 GNB2,GNAQ,JAK2,GNG5,GNG10 5 Mediated by Tubby Leukocyte Extravasation 1.17E+01 8.72E-02 8.72 MYL6,MAPK1,CDC42,CTNNA1,NCF4,GNAI 17 Signaling 3,ITGAM (includes EG:16409),MAPK14,TIMP1,CYBA,PRKCD, NCF2,CYBB,CD44 (includes EG:100330801),MMP9,VASP,TIMP2 (includes EG:21858) VDR/RXR Activation 1.15E+01 1.11E-01 11.1 SERPINB1,CAMP (includes 9 EG:12796),GADD45A,PRKCD,MXD1,CEBP A,CD14,CEBPB (includes EG:1051),RXRA Role of IL-17A in Arthritis 1.12E+01 1.11E-01 11.1 MAPK14,MAPK1,MAPK3,RPS6KA1 7 (includes EG:20111),MAPKAPK2,IL17RA,MAP2K1 Inositol Metabolism 1.12E+01 1.18E-01 11.8 ALDOA,TPI1 2 Pancreatic 1.10E+01 9.32E-02 9.32 HMOX1,CDC42,MAPK1,MAPK3,TGFA,KRA 11 Adenocarcinoma Signaling S,JAK2,E2F3,MAP2K1,MMP9,E2F2 T Helper Cell 1.07E+01 1.14E-01 11.4 IL4R,TNFRSF1A,IL10RB,IFNGR2,FCER1G,IF 8 Differentiation NGR1,BCL6,IL18R1

287

Relaxin Signaling 1.07E+01 8.28E-02 8.28 MAPK1,MAPK3,PRKAR2A,GNAQ,PDE4D,G 13 NG5,GNG10,GNAI3,FOS,GNA15,GNB2,M AP2K1,MMP9 Polyamine Regulation in 1.07E+01 1.38E-01 13.8 AZIN1,MXD1,SAT1,KRAS 4 Colon Cancer Sphingolipid Metabolism 1.02E+01 8.18E-02 8.18 GLA,GALC,ARSD,VNN1,GBA,PIGF,VNN2,U 9 GCG,ASAH1 Altered T Cell and B Cell 1.02E+01 1.03E-01 10.3 TLR2,TLR4,TLR1,TLR5,IL1RN,TLR8,FCER1G 9 Signaling in Rheumatoid ,IL1B,CHUK Arthritis MIF-mediated 1.02E+01 1.19E-01 11.9 TLR4,LY96 (includes 5 Glucocorticoid Regulation EG:17087),MAPK1,MAPK3,CD14 Aminosugars Metabolism 9.79E+00 6.72E-02 6.72 POR,CYB5R4,HK2,CYB5R1,HK3,NANS,PDE 8 4D,NPL ILK Signaling 9.79E+00 8.60E-02 8.6 MYL6,MAPK1,CDC42,PPP2CA,TNFRSF1A, 16 MAPK3,VIM,HIF1A,PPP2R5A,PTEN,FOS,R HOG,RHOT1,PPP1R12A,IRS2,MMP9 Renin-Angiotensin 9.53E+00 8.73E-02 8.73 FOS,MAPK14,MAPK1,PRKCD,MAPK3,PRK 11 Signaling AR2A,GNAQ,KRAS,PTGER2,JAK2,MAP2K1 RhoA Signaling 9.53E+00 9.82E-02 9.82 ACTR2,MYL12A,ACTR3,MYL6,MYL12B,AR 11 PC5,PPP1R12A,ARPC3,ARPC1A,LIMK2,CD C42EP2 Melatonin Signaling 9.38E+00 1.03E-01 10.3 GNAI3,MAPK1,GNA15,PRKCD,MAPK3,PR 8 KAR2A,GNAQ,MAP2K1 GNRH Signaling 9.08E+00 8.45E-02 8.45 GNAI3,FOS,MAPK14,CDC42,MAPK1,PRKC 12 D,MAPK3,PRKAR2A,GNAQ,KRAS,MAP3K8 ,MAP2K1 Folate Biosynthesis 9.08E+00 4.17E-02 4.17 ALPP/ALPPL2,GGH,ALPL 3 IL-3 Signaling 8.95E+00 1.08E-01 10.8 FOS,MAPK1,PRKCD,MAPK3,CHP,KRAS,JA 8 K2,MAP2K1 IL-22 Signaling 8.95E+00 1.60E-01 16 MAPK14,MAPK1,MAPK3,IL10RB 4 Role of JAK1, JAK2 and 8.95E+00 1.48E-01 14.8 IFNGR2,IFNGR1,JAK2,IFNAR1 4 TYK2 in Interferon Signaling Protein Kinase A Signaling 8.75E+00 7.45E-02 7.45 HIST1H1C,YWHAG,YWHAH,MYL6,MAPK1, 24 MAPK3,CHP,GNAQ,PRKAR2A,PYGL,PDE4 D,GNG5,GNG10,GNAI3,MYL12A,PPP1R3D ,GNA15,MYL12B,PRKCD,GNB2,PPP1R12A ,CHUK,MAP2K1,VASP Huntington's Disease 8.09E+00 7.69E-02 7.69 HDAC4,MAPK1,HSPA1A/HSPA1B,MAPK3, 18 Signaling CLTC,GNAQ,APAF1,GNG5,GNG10,CTSD,D NAJC5,CDK5,CACNA1B,PRKCD,VAMP3,G NB2,GOSR2,CAPN3 Prolactin Signaling 8.09E+00 1.00E-01 10 FOS,MAPK1,PRKCD,MAPK3,KRAS,CEBPB 8 (includes EG:1051),JAK2,MAP2K1 IL-15 Signaling 7.74E+00 1.04E-01 10.4 MAPK14,MAPK1,MAPK3,SYK,KRAS,JAK2, 7 MAP2K1 ERK5 Signaling 7.74E+00 1.11E-01 11.1 FOS,YWHAG,YWHAH,GNAQ,KRAS,MAP3K 7 8,RPS6KA1 (includes EG:20111) CNTF Signaling 7.50E+00 1.15E-01 11.5 MAPK1,MAPK3,KRAS,JAK2,RPS6KA1 6 (includes EG:20111),MAP2K1 PAK Signaling 7.45E+00 8.49E-02 8.49 MYL12A,CDC42,MAPK1,MYL6,MYL12B,M 9 APK3,KRAS,LIMK2,MAP2K1 Antiproliferative Role of 7.36E+00 1.00E-01 10 MAPK1,MAPK3,GNB2,KRAS,MAP2K1,GN 7 Somatostatin Receptor 2 G5,GNG10 AMPK Signaling 7.05E+00 7.27E-02 7.27 CAB39,PFKFB3,MAPK14,MAPK1,PFKFB4,P 12 PP2CA,PPM1B,PRKAA1,PRKAR2A,IRS2,H MGCR,PPP2R5A

288

Mitochondrial Dysfunction 6.78E+00 6.98E-02 6.98 NDUFA4,ATP5C1,COX7B,COX7A2,NDUFB 12 3,COX17 (includes EG:10063),PRDX3,FURIN,SOD2,APH1B,PR DX5,GLRX2 NGF Signaling 6.75E+00 8.62E-02 8.62 RHOG,CDC42,MAPK1,PRKCD,MAPK3,KRA 10 S,MAP3K8,CHUK,RPS6KA1 (includes EG:20111),MAP2K1 Ceramide Signaling 6.28E+00 9.20E-02 9.2 CTSD,FOS,PPP2CA,TNFRSF1A,MAPK3,KRA 8 S,MAP2K1,PPP2R5A Endometrial Cancer 6.24E+00 1.05E-01 10.5 MAPK1,MAPK3,CTNNA1,KRAS,MAP2K1,P 6 Signaling TEN Glioblastoma Multiforme 6.24E+00 7.78E-02 7.78 CDC42,MAPK1,MAPK3,GNAQ,KRAS,E2F3, 13 Signaling PTEN,RHOG,GNA15,RHOT1,PRKCD,MAP2 K1,E2F2 Role of PKR in Interferon 6.17E+00 1.11E-01 11.1 MAPK14,TNFRSF1A,APAF1,CHUK,RNASEL 5 Induction and Antiviral Response Mechanisms of Viral Exit 6.17E+00 1.11E-01 11.1 CHMP2A,PRKCD,SH3GLB1,VPS25 5 from Host Cells (includes EG:28084),LMNB1 N-Glycan Biosynthesis 6.17E+00 6.41E-02 6.41 B4GALT4,MGAT4A,MAN1A1,MAN2A2,B4 5 GALT5 Role of NFAT in Cardiac 6.07E+00 7.39E-02 7.39 HDAC4,MAPK1,MAPK3,CHP,PRKAR2A,GN 15 Hypertrophy AQ,KRAS,GNG5,GNG10,GNAI3,MAPK14, GNA15,PRKCD,GNB2,MAP2K1 IL-2 Signaling 6.07E+00 1.03E-01 10.3 FOS,MAPK1,MAPK3,SYK,KRAS,MAP2K1 6 Glutathione Metabolism 6.07E+00 7.14E-02 7.14 MGST1,PGD,G6PD,GLRX,GSTO1,IDH1 6 Glioma Signaling 5.89E+00 8.11E-02 8.11 MAPK1,PRKCD,MAPK3,CDKN2D,KRAS,E2 9 F3,MAP2K1,E2F2,PTEN 4-1BB Signaling in T 5.85E+00 1.18E-01 11.8 MAPK1,MAPK3,CHUK,MAP2K1 4 Lymphocytes Erythropoietin Signaling 5.83E+00 8.97E-02 8.97 FOS,MAPK1,PRKCD,MAPK3,KRAS,JAK2,M 7 AP2K1 Lymphotoxin β Receptor 5.81E+00 1.00E-01 10 MAPK1,MAPK3,APAF1,CHUK,LTBR,BIRC2 6 Signaling Corticotropin Releasing 5.78E+00 7.69E-02 7.69 GNAI3,FOS,MAPK14,MAPK1,PRKCD,MAP 10 Hormone Signaling K3,ARPC5,PRKAR2A,GNAQ,MAP2K1

RAR Activation 5.73E+00 7.61E-02 7.61 MAPK1,PRKAR2A,JAK2,PTEN,FOS,MAPK1 14 4,DHRS9,DUSP1,RARA,PRKCD,RXRA,MAP KAPK2,ZBTB16,MAP2K1 Growth Hormone Signaling 5.66E+00 9.33E-02 9.33 FOS,MAPK1,PRKCD,MAPK3,CEBPA,JAK2,R 7 PS6KA1 (includes EG:20111) Neurotrophin/TRK 5.66E+00 9.21E-02 9.21 FOS,CDC42,MAPK1,MAPK3,KRAS,RPS6KA 7 Signaling 1 (includes EG:20111),MAP2K1 Melanoma Signaling 5.66E+00 1.09E-01 10.9 MAPK1,MAPK3,KRAS,MAP2K1,PTEN 5 Communication between 5.64E+00 8.08E-02 8.08 TLR2,TLR4,TLR1,TLR5,IL1RN,TLR8,FCER1G 8 Innate and Adaptive ,IL1B Immune Cells Androgen Signaling 5.47E+00 7.19E-02 7.19 GNAI3,MAPK1,GNA15,PRKCD,MAPK3,GN 10 B2,PRKAR2A,GNAQ,GNG5,GNG10 Thyroid Cancer Signaling 5.32E+00 1.11E-01 11.1 MAPK1,MAPK3,KRAS,RXRA,MAP2K1 5 HGF Signaling 4.94E+00 8.57E-02 8.57 FOS,CDC42,MAPK1,PRKCD,MAPK3,ETS2, 9 KRAS,MAP3K8,MAP2K1 Complement System 4.79E+00 1.18E-01 11.8 CR1,CD55,C5AR1,C3AR1 4 Death Receptor Signaling 4.79E+00 9.52E-02 9.52 TANK,TNFRSF1A,APAF1,TBK1,CHUK,BIRC 6 2 Eicosanoid Signaling 4.79E+00 7.79E-02 7.79 LTB4R,FPR2,ALOX5AP,PTGER2,ALOX5,TB 6 XAS1 289

Neuregulin Signaling 4.76E+00 8.00E-02 8 CDK5,MAPK1,PRKCD,MAPK3,TGFA,KRAS, 8 MAP2K1,PTEN Fatty Acid Metabolism 4.65E+00 5.52E-02 5.52 ACSL3,DHRS9,ACAA1,ACOX1,ACSL4,PECR, 9 ACSL1,CYP51A1,CYP1B1 Dopamine-DARPP32 4.61E+00 7.10E-02 7.1 KCNJ2,PPP2CA,CHP,PRKAR2A,GNAQ,PPP 13 Feedback in cAMP 2R5A,GRINA,GNAI3,PPP1R3D,CDK5,GNA1 Signaling 5,PRKCD,PPP1R12A Inhibition of Angiogenesis 4.52E+00 1.05E-01 10.5 MAPK14,MAPK1,CD36,MMP9 4 by TSP1 Xenobiotic Metabolism 4.46E+00 7.06E-02 7.06 RBX1,MGST1,MAPK1,PPP2CA,NQO2,MAP 19 Signaling K3,KRAS,PPP2R5A,CYP1B1,GSTO1,HMOX 1,MAPK14,PRKCD,IL1B,ALDH3B1,MAP3K 8,RXRA,PNPLA6,MAP2K1 Metabolism of Xenobiotics 4.45E+00 4.71E-02 4.71 MGST1,DHRS9,PECR,ALDH3B1,CSGALNA 8 by Cytochrome P450 CT1,GSTO1,CYP51A1,CYP1B1

Natural Killer Cell Signaling 4.38E+00 8.18E-02 8.18 LAIR1,MAPK1,TYROBP,PRKCD,MAPK3,SY 9 K,FCER1G,KRAS,MAP2K1 Interferon Signaling 4.26E+00 1.11E-01 11.1 IFNGR2,IFNGR1,JAK2,IFNAR1 4 Role of PI3K/AKT Signaling 4.22E+00 8.57E-02 8.57 GNAI3,MAPK1,MAPK3,MAP2K1,PLAC8,IF 6 in the Pathogenesis of NAR1 Influenza cAMP-mediated signaling 4.16E+00 7.34E-02 7.34 RGS18,MAPK1,MAPK3,PRKAR2A,FPR2,PD 16 E4D,OPRL1,FPR1,P2RY13,LAMTOR3,LTB4 R,DUSP1,PTGER2,RPS6KA1 (includes EG:20111),ADORA2A,MAP2K1 Lysine Biosynthesis 4.09E+00 3.08E-02 3.08 VNN1,VNN2 2 Cell Cycle Regulation by 4.03E+00 1.11E-01 11.1 PPP2CA,E2F3,E2F2,PPP2R5A 4 BTG Family Proteins

Cyclins and Cell Cycle 3.91E+00 8.05E-02 8.05 CCND3,HDAC4,PPP2CA,CDKN2D,E2F3,E2 7 Regulation F2,PPP2R5A Role of MAPK Signaling in 3.91E+00 9.09E-02 9.09 MAPK14,MAPK1,MAPK3,KRAS,MAP2K1,R 6 the Pathogenesis of ABGEF1 Influenza Chronic Myeloid Leukemia 3.91E+00 7.77E-02 7.77 HDAC4,MAPK1,MAPK3,KRAS,CHUK,E2F3, 8 Signaling MAP2K1,E2F2 Glycerolipid Metabolism 3.91E+00 5.56E-02 5.56 GLA,ABHD5,DHRS9,AGPAT2,DGAT2,PECR 8 ,AGPAT9,GK Leptin Signaling in Obesity 3.84E+00 8.05E-02 8.05 MAPK1,GNA15,MAPK3,PRKAR2A,GNAQ,J 7 AK2,MAP2K1 Regulation of IL-2 3.84E+00 7.95E-02 7.95 FOS,MAPK1,MAPK3,CHP,KRAS,CHUK,MA 7 Expression in Activated and P2K1 Anergic T Lymphocytes Glycerophospholipid 3.84E+00 5.41E-02 5.41 HMOX1,LPCAT2,GNA15,PIGF,AGPAT2,CH 10 Metabolism KA,GNAQ,CHPT1,ETNK1,AGPAT9 Insulin Receptor Signaling 3.84E+00 7.52E-02 7.52 PPP1R3D,MAPK1,MAPK3,PRKAR2A,PPP1 10 R12A,IRS2,KRAS,JAK2,MAP2K1,PTEN Role of Osteoblasts, 3.84E+00 6.75E-02 6.75 IL18RAP,MAPK1,TNFRSF1A,MAPK3,CHP, 16 Osteoclasts and GSN,IL18R1,IL1R2,FOS,MAPK14,IL1RN,IL1 Chondrocytes in B,CHUK,ALPL,IL1RAP,BIRC2 Rheumatoid Arthritis CREB Signaling in Neurons 3.80E+00 6.63E-02 6.63 MAPK1,MAPK3,PRKAR2A,GNAQ,KRAS,GN 13 G5,GNG10,GNAI3,GNA15,PRKCD,GNB2,R PS6KA1 (includes EG:20111),MAP2K1 Synaptic Long Term 3.76E+00 7.14E-02 7.14 GNAI3,MAPK1,GNA15,PPP2CA,PRKCD,M 10 Depression APK3,GNAQ,KRAS,MAP2K1,PPP2R5A Notch Signaling 3.71E+00 9.76E-02 9.76 FURIN,APH1B,NUMB,RBPJ 4 290

Non-Small Cell Lung Cancer 3.70E+00 7.59E-02 7.59 MAPK1,MAPK3,TGFA,KRAS,RXRA,MAP2K 6 Signaling 1 CD27 Signaling in 3.68E+00 8.93E-02 8.93 FOS,APAF1,MAP3K8,CHUK,MAP2K1 5 Lymphocytes April Mediated Signaling 3.52E+00 9.30E-02 9.3 FOS,MAPK14,MAPK1,CHUK 4

Telomerase Signaling 3.52E+00 8.08E-02 8.08 HDAC4,MAPK1,PPP2CA,MAPK3,ETS2,KRA 8 S,MAP2K1,PPP2R5A Role of IL-17A in Psoriasis 3.43E+00 1.54E-01 15.4 S100A8,IL17RA 2 Sertoli Cell-Sertoli Cell 3.41E+00 6.70E-02 6.7 CDC42,MAPK1,TNFRSF1A,MAPK3,CTNNA 13 Junction Signaling 1,PRKAR2A,KRAS,PTEN,MAPK14,ZAK,MA P3K8,MAP2K1,RAB8B TGF-β Signaling 3.27E+00 7.87E-02 7.87 FOS,MAPK14,MAPK1,MAPK3,KRAS,MAP2 7 K1,ACVR1B mTOR Signaling 3.25E+00 6.67E-02 6.67 MAPK1,PPP2CA,MAPK3,FKBP1A,KRAS,HI 13 F1A,PPP2R5A,HMOX1,RHOG,RHOT1,PRK CD,PRKAA1,RPS6KA1 (includes EG:20111) B Cell Activating Factor 3.18E+00 8.89E-02 8.89 FOS,MAPK14,MAPK1,CHUK 4 Signaling FLT3 Signaling in 3.18E+00 8.45E-02 8.45 MAPK14,MAPK1,MAPK3,KRAS,RPS6KA1 6 Hematopoietic Progenitor (includes EG:20111),MAP2K1 Cells Role of RIG1-like Receptors 3.02E+00 8.70E-02 8.7 TANK,TBK1,CHUK,TRIM25 4 in Antiviral Innate Immunity FAK Signaling 2.96E+00 6.93E-02 6.93 MAPK1,ASAP1,MAPK3,KRAS,MAP2K1,PT 7 EN,CAPN3 PDGF Signaling 2.95E+00 7.59E-02 7.59 FOS,MAPK1,MAPK3,KRAS,JAK2,MAP2K1 6 Gα12/13 Signaling 2.90E+00 7.09E-02 7.09 MYL12A,CDC42,MAPK1,MYL6,MYL12B,M 9 APK3,KRAS,CHUK,MAP2K1 Caveolar-mediated 2.89E+00 7.23E-02 7.23 CD55,FLOT2,ITGAM (includes 6 Endocytosis Signaling EG:16409),RAB5A,RAB5C,FLOT1 BMP signaling pathway 2.89E+00 7.59E-02 7.59 MAPK14,MAPK1,MAPK3,PRKAR2A,KRAS, 6 MAP2K1 Fc Epsilon RI Signaling 2.89E+00 7.21E-02 7.21 MAPK14,MAPK1,PRKCD,MAPK3,SYK,FCER 8 1G,KRAS,MAP2K1 Neuropathic Pain Signaling 2.89E+00 7.21E-02 7.21 FOS,MAPK1,GNA15,PRKCD,MAPK3,PRKA 8 In Dorsal Horn Neurons R2A,GNAQ,GRINA Cell Cycle: G2/M DNA 2.81E+00 8.33E-02 8.33 YWHAG,GADD45A,YWHAH,RPS6KA1 4 Damage Checkpoint (includes EG:20111) Regulation Tight Junction Signaling 2.81E+00 6.88E-02 6.88 FOS,CDC42,MYL6,PPP2CA,TNFRSF1A,CEB 11 PA,CTNNA1,PRKAR2A,VASP,PPP2R5A,PTE N Granzyme B Signaling 2.72E+00 1.25E-01 12.5 APAF1,LMNB1 2 Sulfur Metabolism 2.72E+00 3.64E-02 3.64 PAPSS1,SUOX 2 MSP-RON Signaling 2.72E+00 8.16E-02 8.16 TLR2,TLR4,ITGAM (includes 4 Pathway EG:16409),JAK2 Retinoic acid Mediated 2.72E+00 7.81E-02 7.81 RARA,APAF1,RXRA,TIPARP,IFNAR1 5 Apoptosis Signaling CTLA4 Signaling in 2.72E+00 7.29E-02 7.29 PPP2CA,SYK,CLTC,FCER1G,JAK2,PTPN22,P 7 Cytotoxic T Lymphocytes PP2R5A PTEN Signaling 2.62E+00 6.50E-02 6.5 CDC42,MAPK1,YWHAH,MAPK3,KRAS,CH 8 UK,MAP2K1,PTEN Aldosterone Signaling in 2.61E+00 6.36E-02 6.36 DNAJC5,MAPK1,GNA15,HSPA1A/HSPA1B 11 Epithelial Cells ,DUSP1,PRKCD,MAPK3,GNAQ,KRAS,DNAJ B6,MAP2K1 291

Granzyme A Signaling 2.54E+00 1.11E-01 11.1 HIST1H1C,HMGB2 2 JAK/Stat Signaling 2.54E+00 7.81E-02 7.81 MAPK1,MAPK3,KRAS,JAK2,MAP2K1 5 EGF Signaling 2.52E+00 7.69E-02 7.69 FOS,MAPK1,MAPK3,MAP2K1 4 Protein Ubiquitination 2.44E+00 6.23E-02 6.23 RBX1,PSMA6,UBE2H,USP15,UBE2A,HSPA 17 Pathway 1A/HSPA1B,PSMD6,PSMA1,USP6,TCEB1, UBE2F,DNAJC5,PSMD12,DNAJB6,PSMD4, UBE2J1,BIRC2 Fatty Acid Elongation in 2.40E+00 4.35E-02 4.35 ACAA1,PECR 2 Mitochondria Parkinson's Signaling 2.40E+00 1.11E-01 11.1 MAPK14,MAPK1 2 Stilbene, Coumarine and 2.40E+00 2.74E-02 2.74 GBA,PRDX5 2 Lignin Biosynthesis Prostate Cancer Signaling 2.39E+00 6.32E-02 6.32 MAPK1,MAPK3,KRAS,CHUK,MAP2K1,PTE 6 N Sphingosine-1-phosphate 2.38E+00 6.50E-02 6.5 GNAI3,RHOG,MAPK1,GNA15,RHOT1,MA 8 Signaling PK3,GNAQ,ASAH1 Role of CHK Proteins in Cell 2.38E+00 8.57E-02 8.57 E2F3,E2F2,NBN 3 Cycle Checkpoint Control TWEAK Signaling 2.38E+00 7.89E-02 7.89 APAF1,CHUK,BIRC2 3 Role of JAK2 in Hormone- 2.38E+00 8.57E-02 8.57 IRS2,JAK2,SIRPA 3 like Cytokine Signaling Pentose and Glucuronate 2.38E+00 2.29E-02 2.29 UGP2,HPSE,CSGALNACT1 3 Interconversions T Cell Receptor Signaling 2.38E+00 6.48E-02 6.48 FOS,MAPK1,PAG1,MAPK3,KRAS,CHUK,M 7 AP2K1 PKCθ Signaling in T 2.35E+00 5.88E-02 5.88 FOS,MAPK1,MAPK3,CHP,FCER1G,KRAS,M 8 Lymphocytes AP3K8,CHUK Endothelin-1 Signaling 2.24E+00 6.01E-02 6.01 HMOX1,GNAI3,FOS,MAPK14,MAPK1,GN 11 A15,PRKCD,MAPK3,GNAQ,KRAS,PTGER2 Cardiac β-adrenergic 2.22E+00 5.88E-02 5.88 PPP1R3D,PPP2CA,GNB2,PRKAR2A,PPP1R 9 Signaling 12A,PDE4D,GNG5,PPP2R5A,GNG10 G-Protein Coupled 2.21E+00 5.85E-02 5.85 RGS18,MAPK1,MAPK3,KRAS,PDE4D,OPRL 31 Receptor Signaling 1,LTB4R,LAMTOR3,C5AR1,CHUK,MAP2K1 ,CCR1,GPR97,GNAQ,FPR2,PRKAR2A,PCNX ,FPR1,P2RY13,GPR137B,DUSP1,GPR65,G PR160,MAP3K8,EMR1,PTGER2,RPS6KA1 (includes EG:20111),PTAFR,EMR2,C3AR1,ADORA2 A Atherosclerosis Signaling 2.21E+00 6.20E-02 6.2 ORM1/ORM2,IL1RN,CD36,IL1B,SERPINA1 8 ,S100A8,ALOX5,MMP9 Nitrogen Metabolism 2.21E+00 2.50E-02 2.5 VNN1,VNN2,CA4 3 DNA Methylation and 2.21E+00 8.70E-02 8.7 MTA1,SAP30 (includes EG:60406) 2 Transcriptional Repression Signaling One Carbon Pool by Folate 2.21E+00 5.56E-02 5.56 MTHFS,MTHFD2 2 Nur77 Signaling in T 2.20E+00 6.78E-02 6.78 CHP,APAF1,FCER1G,RXRA 4 Lymphocytes Macropinocytosis Signaling 2.14E+00 6.58E-02 6.58 RAB5A,CDC42,PRKCD,CD14,KRAS 5 TR/RXR Activation 2.12E+00 6.25E-02 6.25 SLC16A3,HP,LDLR,BCL3,HIF1A,RXRA 6 Melanocyte Development 2.12E+00 6.52E-02 6.52 MAPK1,MAPK3,PRKAR2A,KRAS,RPS6KA1 6 and Pigmentation Signaling (includes EG:20111),MAP2K1 Biosynthesis of Steroids 2.11E+00 1.65E-02 1.65 IDI1,HMGCR 2 Arginine and Proline 2.03E+00 2.87E-02 2.87 P4HA1,VNN1,VNN2,OAT,SAT1 5 Metabolism

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Cell Cycle: G1/S Checkpoint 2.00E+00 6.78E-02 6.78 CCND3,HDAC4,E2F3,E2F2 4 Regulation Docosahexaenoic Acid 2.00E+00 6.25E-02 6.25 APAF1,IL1B,BCL2A1 3 (DHA) Signaling Small Cell Lung Cancer 2.00E+00 5.68E-02 5.68 APAF1,CHUK,RXRA,PTEN,BIRC2 5 Signaling Differential Regulation of 1.92E+00 8.70E-02 8.7 LCN2,IL1B 2 Cytokine Production in Intestinal Epithelial Cells by IL-17A and IL-17F Nucleotide Sugars 1.92E+00 1.54E-02 1.54 UGP2 1 Metabolism Airway Pathology in 1.92E+00 1.11E-01 11.1 MMP9 1 Chronic Obstructive Pulmonary Disease Role of BRCA1 in DNA 1.92E+00 6.56E-02 6.56 GADD45A,E2F3,E2F2,NBN 4 Damage Response Propanoate Metabolism 1.92E+00 3.31E-02 3.31 ACSL3,ACSS2,ACSL1,LDHA 4 NF-κB Activation by Viruses 1.92E+00 6.10E-02 6.1 MAPK1,PRKCD,MAPK3,KRAS,CHUK 5 Regulation of eIF4 and 1.92E+00 5.36E-02 5.36 EIF2C4,MAPK14,MAPK1,PPP2CA,MAPK3, 9 p70S6K Signaling MKNK1,KRAS,MAP2K1,PPP2R5A Aminophosphonate 1.87E+00 3.51E-02 3.51 PIGF,CHPT1 2 Metabolism Tumoricidal Function of 1.87E+00 8.33E-02 8.33 SRGN,APAF1 2 Hepatic Natural Killer Cells Role of 1.85E+00 7.32E-02 7.32 CCR1,IL1RN,IL1B 3 Hypercytokinemia/hyperch emokinemia in the Pathogenesis of Influenza Mitotic Roles of Polo-Like 1.85E+00 6.67E-02 6.67 SLK,PPP2CA,PRC1 (includes 4 Kinase EG:233406),PPP2R5A Myc Mediated Apoptosis 1.85E+00 6.67E-02 6.67 YWHAG,YWHAH,APAF1,KRAS 4 Signaling SAPK/JNK Signaling 1.84E+00 5.94E-02 5.94 CDC42,GADD45A,ZAK,FCER1G,KRAS,GNG 6 5 Pathogenesis of Multiple 1.84E+00 1.11E-01 11.1 CCR1 1 Sclerosis Dopamine Receptor 1.84E+00 5.26E-02 5.26 PPP1R3D,PPP2CA,PRKAR2A,PPP1R12A,PP 5 Signaling P2R5A Glycosaminoglycan 1.84E+00 3.64E-02 3.64 HPSE,GNS 2 Degradation N-Glycan Degradation 1.84E+00 8.00E-02 8 MAN1A1,MAN2A2 2 Induction of Apoptosis by 1.83E+00 6.25E-02 6.25 TNFRSF1A,APAF1,CHUK,BIRC2 4 HIV1 Cellular Effects of Sildenafil 1.80E+00 5.37E-02 5.37 MYL12A,MYL6,GNA15,MYL12B,PRKAR2A, 8 (Viagra) GNAQ,PPP1R12A,PDE4D Activation of IRF by 1.80E+00 5.80E-02 5.8 TANK,TBK1,CHUK,IFNAR1 4 Cytosolic Pattern Recognition Receptors Estrogen-Dependent 1.80E+00 5.71E-02 5.71 FOS,MAPK1,MAPK3,KRAS 4 Breast Cancer Signaling Antiproliferative Role of 1.79E+00 7.69E-02 7.69 MAPK1,RPS6KA1 (includes EG:20111) 2 TOB in T Cell Signaling Ovarian Cancer Signaling 1.79E+00 5.67E-02 5.67 MAPK1,MAPK3,CD44 (includes 8 EG:100330801),PRKAR2A,KRAS,MAP2K1, MMP9,PTEN Fatty Acid Biosynthesis 1.75E+00 2.04E-02 2.04 ACSL3 1

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Graft-versus-Host Disease 1.75E+00 6.25E-02 6.25 IL1RN,FCER1G,IL1B 3 Signaling Amyotrophic Lateral 1.75E+00 5.17E-02 5.17 RAB5A,RAB5C,APAF1,GRINA,CAPN3,BIRC 6 Sclerosis Signaling 2 Role of Oct4 in Mammalian 1.71E+00 6.67E-02 6.67 RARA,SH3GLB1,HOXB1 3 Embryonic Stem Cell Pluripotency Paxillin Signaling 1.69E+00 5.45E-02 5.45 ITGAM (includes 6 EG:16409),MAPK14,CDC42,MAPK1,KRAS, PTPN12 Citrate Cycle 1.68E+00 3.57E-02 3.57 DLD,IDH1 2 Sonic Hedgehog Signaling 1.62E+00 6.06E-02 6.06 ARRB2,PRKAR2A 2 FXR/RXR Activation 1.60E+00 5.05E-02 5.05 IL1RN,RARA,FBP1,IL1B,RXRA 5 FGF Signaling 1.60E+00 5.56E-02 5.56 MAPK14,MAPK1,MAPK3,MAPKAPK2,MA 5 P2K1 VEGF Signaling 1.57E+00 5.15E-02 5.15 MAPK1,MAPK3,KRAS,HIF1A,MAP2K1 5 Agrin Interactions at 1.57E+00 5.88E-02 5.88 CDC42,MAPK1,MAPK3,KRAS 4 Neuromuscular Junction Gap Junction Signaling 1.57E+00 5.00E-02 5 GNAI3,MAPK1,GNA15,PRKCD,MAPK3,PR 9 KAR2A,GNAQ,KRAS,MAP2K1 Bile Acid Biosynthesis 1.56E+00 2.97E-02 2.97 DHRS9,ACAA1,PECR 3 Bladder Cancer Signaling 1.53E+00 5.56E-02 5.56 MAPK1,MAPK3,KRAS,MAP2K1,MMP9 5 Chondroitin Sulfate 1.53E+00 4.62E-02 4.62 CHSY1,CSGALNACT1,DSE 3 Biosynthesis Virus Entry via Endocytic 1.48E+00 5.05E-02 5.05 CD55,CDC42,PRKCD,CLTC,KRAS 5 Pathways ATM Signaling 1.48E+00 5.56E-02 5.56 MAPK14,GADD45A,NBN 3 DNA Double-Strand Break 1.48E+00 5.88E-02 5.88 NBN 1 Repair by Homologous Recombination DNA Double-Strand Break 1.48E+00 5.26E-02 5.26 NBN 1 Repair by Non-Homologous End Joining Glycine, Serine and 1.48E+00 2.74E-02 2.74 GNA15,CHKA,DLD,GNAQ 4 Threonine Metabolism IL-9 Signaling 1.45E+00 5.00E-02 5 IRS2,BCL3 2 Telomere Extension by 1.43E+00 5.88E-02 5.88 NBN 1 Telomerase Methane Metabolism 1.43E+00 1.54E-02 1.54 PRDX5 1 Coagulation System 1.42E+00 5.26E-02 5.26 PLAUR,SERPINA1 2 Phenylalanine Metabolism 1.39E+00 1.83E-02 1.83 PRDX5,ALDH3B1 2 RAN Signaling 1.39E+00 4.35E-02 4.35 KPNA4 1 Glyoxylate and 1.35E+00 9.26E-03 0.926 MTHFD2 1 Dicarboxylate Metabolism Ascorbate and Aldarate 1.35E+00 1.30E-02 1.3 GSTO1 1 Metabolism Differential Regulation of 1.31E+00 5.56E-02 5.56 IL1B 1 Cytokine Production in Macrophages and T Helper Cells by IL-17A and IL-17F Endoplasmic Reticulum 1.31E+00 5.56E-02 5.56 ATF6 1 Stress Pathway

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Appendix 8.4: Signalling pathways from 492 SDE downregulated gene transcripts

Ingenuity Canonical B H p Ratio % Molecules No. of Pathways value molecules Calcium-induced T 2.00E+14 3.03E-01 30.3 CD247,HLA-DMA,HLA- 20 Lymphocyte Apoptosis DOA,CD3E,CD4,HLA- DQA1,PLCG1,HLA-DMB,PPP3CC,HLA- DQB1,CD3D,CD3G,LCK,MEF2D,HLA- DRA,ZAP70,ITPR3,HLA- DOB,PRKCH,PRKCA iCOS-iCOSL Signaling in T 2.51E+12 1.93E-01 19.3 CD247,HLA-DMA,HLA- 23 Helper Cells DOA,CD3E,CD4,HLA- DQA1,PLCG1,HLA-DMB,PPP3CC,HLA- DQB1,CD3D,IL2RB,CD3G,ICOS,LCK,LA T,HLA-DRA,ZAP70,ITPR3,HLA- DOB,PLEKHA1,ATM,ITK Cytotoxic T Lymphocyte- 2.51E+12 3.21E-01 32.1 CD247,HLA-DMA,HLA- 17 mediated Apoptosis of DOA,CD3E,HLA-DQA1,HLA-DMB,HLA- Target Cells DRB3 (includes others),HLA- DQB1,CD3D,HLA- DPA1,BCL2,CD3G,PRF1,HLA- DRA,HLA-DOB,HLA-DPB1,GZMB CD28 Signaling in T 6.31E+11 1.78E-01 17.8 CD247,HLA-DMA,FYN,HLA- 23 Helper Cells DOA,CD3E,CD4,HLA- DQA1,PLCG1,HLA-DMB,PPP3CC,HLA- DQB1,CD3D,CD3G,LCK,LAT,HLA- DRA,ZAP70,ITPR3,CD86,HLA- DOB,CARD11,ATM,ITK OX40 Signaling Pathway 6.31E+11 2.70E-01 27 CD247,HLA-DMA,HLA- 17 DOA,CD3E,CD4,HLA-DQA1,HLA- DMB,HLA-DRB3 (includes others),HLA-DQB1,CD3D,HLA- DPA1,BCL2,CD3G,HLA-DRA,HLA- DOB,TRAF5,HLA-DPB1 Role of NFAT in 5.01E+10 1.33E-01 13.3 CD247,FYN,HLA-DOA,CD3E,CD4,HLA- 26 Regulation of the DQA1,HLA-DMB,HLA- Immune Response DQB1,CD79A,LCK,HLA- DRA,ITK,ATM,HLA- DMA,CD79B,PLCG1,PPP3CC,CD3D,CD 3G,MEF2D,LAT,ZAP70,ITPR3,CD86,HL A-DOB,MEF2C PKCθ Signaling in T 1.26E+10 1.54E-01 15.4 CD247,HLA-DMA,FYN,HLA- 21 Lymphocytes DOA,CD3E,CD4,HLA- DQA1,PLCG1,HLA-DMB,PPP3CC,HLA- DQB1,CD3D,CD3G,LCK,LAT,HLA- DRA,ZAP70,CD86,HLA- DOB,CARD11,ATM CTLA4 Signaling in 1.26E+10 1.98E-01 19.8 CD247,HLA-DMA,FYN,HLA- 19 Cytotoxic T Lymphocytes DOA,CD3E,CD4,HLA- DQA1,PLCG1,HLA-DMB,HLA- DQB1,CD3D,CD3G,LCK,LAT,HLA- DRA,ZAP70,HLA-DOB,CD86,ATM Nur77 Signaling in T 1.00E+10 2.54E-01 25.4 CD247,HLA-DMA,HLA- 15 Lymphocytes DOA,CD3E,HLA-DQA1,HLA- DMB,PPP3CC,HLA- DQB1,CD3D,BCL2,CD3G,MEF2D,HLA- DRA,HLA-DOB,CD86 B Cell Development 8.13E+09 4.00E-01 40 IL7R,HLA-DMA,CD19,HLA- 12 DOA,CD79B,HLA-DRA,HLA- DQA1,CD86,HLA-DOB,HLA-DMB,HLA- DQB1,CD79A

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Primary 4.17E+09 2.55E-01 25.5 CD19,CD3E,CD4,RFX5,CD8A,CD3D,CD 14 Immunodeficiency 79A,IL7R,ICOS,LCK,ZAP70,ADA,TAP2, Signaling UNG

Allograft Rejection 1.55E+08 2.17E-01 21.7 HLA-DMA,HLA-DOA,HLA-DQA1,HLA- 13 Signaling DRB3 (includes others),HLA- DMB,HLA-DQB1,HLA- DPA1,PRF1,HLA-DRA,CD86,HLA- DOB,HLA-DPB1,GZMB Antigen Presentation 1.29E+08 2.79E-01 27.9 PSMB9,HLA-DMA,HLA-DOA,HLA- 12 Pathway DRA,HLA-DQA1,HLA-DOB,HLA-DRB3 (includes others),HLA- DMB,CD74,HLA-DPB1,TAP2,HLA- DPA1 Type I Diabetes Mellitus 1.02E+08 1.53E-01 15.3 CD247,HLA-DMA,HLA- 18 Signaling DOA,CD3E,SOCS2,HLA-DQA1,HLA- DMB,HLA- DQB1,CD3D,BCL2,CD3G,PRF1,LTA,HL A-DRA,HLA-DOB,CD86,STAT1,GZMB T Cell Receptor Signaling 8.13E+07 1.57E-01 15.7 CD247,FYN,CD3E,CD4,PLCG1,PPP3CC, 17 CD8A,CD3D,CD3G,LCK,TXK,RASGRP1, ZAP70,LAT,CARD11,ATM,ITK

T Helper Cell 2.45E+07 2.00E-01 20 HLA-DMA,HLA- 14 Differentiation DOA,IL12RB1,IL21R,HLA-DQA1,HLA- DMB,HLA-DQB1,STAT4,ICOS,HLA- DRA,CD86,HLA-DOB,CXCR5,STAT1 Phospholipase C 1.20E+06 8.98E-02 8.98 CD247,PEBP1,FYN,MPRIP,CD3E,CD79 23 Signaling B,PLCG1,PPP3CC,CD3D,CD79A,CD3G, LCK,AHNAK,MEF2D,LAT,JMJD7- PLA2G4B,ZAP70,ITPR3,MEF2C,PRKCH ,RALGDS,PRKCA,ITK Autoimmune Thyroid 4.90E+05 1.82E-01 18.2 HLA-DMA,PRF1,HLA-DOA,HLA- 10 Disease Signaling DRA,HLA-DQA1,CD86,HLA-DOB,HLA- DMB,HLA-DQB1,GZMB

Graft-versus-Host 3.24E+05 2.08E-01 20.8 HLA-DMA,PRF1,HLA-DOA,HLA- 10 Disease Signaling DRA,HLA-DQA1,CD86,HLA-DOB,HLA- DMB,HLA-DQB1,GZMB

Altered T Cell and B Cell 2.95E+05 1.49E-01 14.9 HLA-DMA,HLA-DOA,CD79B,HLA- 13 Signaling in Rheumatoid DQA1,LTB,HLA-DMB,HLA- Arthritis DQB1,CD79A,LTA,HLA- DRA,TLR7,CD86,HLA-DOB Cdc42 Signaling 1.95E+05 1.10E-01 11 CD247,HLA-DMA,HLA- 16 DOA,MPRIP,CD3E,HLA-DQA1,HLA- DRB3 (includes others),HLA- DMB,HLA-DQB1,CD3D,HLA- DPA1,CD3G,HLA-DRA,HLA-DOB,HLA- DPB1,ITK Natural Killer Cell 1.15E+05 1.27E-01 12.7 CD247,FYN,KLRD1,PLCG1,NCR3,LCK,S 14 Signaling H2D1A,KLRB1,LAT,ZAP70,PRKCH,HCS T,ATM,PRKCA

Regulation of IL-2 1.07E+04 1.25E-01 12.5 CD247,FYN,CD3G,CD3E,SMAD3,LAT,Z 11 Expression in Activated AP70,PLCG1,PPP3CC,CD3D,CARD11 and Anergic T Lymphocytes

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Dendritic Cell Maturation 5.37E+03 8.38E-02 8.38 HLA-DMA,HLA-DOA,HLA- 15 DQA1,LTB,HLA-DRB3 (includes others),HLA-DMB,HLA- DQB1,STAT4,LTA,HLA- DRA,CD86,HLA- DOB,STAT1,CCR7,ATM Crosstalk between 3.24E+03 1.16E-01 11.6 PRF1,KLRD1,LTA,HLA- 11 Dendritic Cells and DRA,TLR7,LTB,CD86,NCR3,HLA-DRB3 Natural Killer Cells (includes others),IL2RB,CCR7 CCR5 Signaling in 1.23E+03 1.02E-01 10.2 CD247,CD3G,CD3E,CD4,PLCG1,PRKC 9 Macrophages H,CCL5,CD3D,PRKCA IL-4 Signaling 1.23E+03 1.25E-01 12.5 HLA-DMA,HLA-DOA,HLA-DRA,HLA- 9 DQA1,HLA-DOB,HLA-DMB,HLA- DQB1,ATM,FCER2 Prolactin Signaling 6.46E+02 1.12E-01 11.2 MYC,FYN,SOCS2,PLCG1,PRKCH,STAT1 9 ,TCF7,PRKCA,ATM Systemic Lupus 1.70E+02 6.54E-02 6.54 CD247,PRPF19,CD79B,CD3E,PLCG1,C 14 Erythematosus Signaling D3D,CD79A,SNRPN,CD3G,LCK,LAT,TL R7,CD86,ATM Cell Cycle: G1/S 1.70E+02 1.19E-01 11.9 MYC,CCND2,CDK4,SMAD3,CDK6,ATR, 7 Checkpoint Regulation ATM Communication between 6.31E+01 8.08E-02 8.08 CD4,HLA-DRA,TLR7,CD86,HLA-DRB3 8 Innate and Adaptive (includes others),CCL5,CD8A,CCR7 Immune Cells Growth Hormone 4.57E+01 9.33E-02 9.33 SOCS2,PLCG1,RPS6KA5,PRKCH,STAT1 7 Signaling ,PRKCA,ATM Small Cell Lung Cancer 3.63E+01 7.95E-02 7.95 PTK2 (includes 7 Signaling EG:14083),MYC,CDK4,CDK6,TRAF5,AT M,BCL2 Aryl Hydrocarbon 3.47E+01 6.45E-02 6.45 MYC,GSTM1,GSTM2,ALDH1A1,CCND 10 Receptor Signaling 2,CDK4,CDK6,ATR,ATM,MCM7 Thrombopoietin 3.24E+01 9.52E-02 9.52 MYC,PLCG1,PRKCH,STAT1,PRKCA,AT 6 Signaling M HER-2 Signaling in Breast 2.88E+01 8.86E-02 8.86 FOXO1,CDK6,PLCG1,PRKCH,ITGB7,PR 7 Cancer KCA,ATM Molecular Mechanisms 2.88E+01 4.84E-02 4.84 FYN,SMAD3,CDK6,BCL2,MYC,CDC25B 18 of Cancer ,PTK2 (includes EG:14083),CCND2,FOXO1,RASGRP1,C DK4,PRKCH,LEF1,BIRC3,ATR,RALGDS, ATM,PRKCA Activation of IRF by 2.29E+01 8.70E-02 8.7 IFIH1,LTA,ZBP1,STAT1,IFIT2,ISG15 6 Cytosolic Pattern Recognition Receptors Cell Cycle Control of 2.24E+01 1.29E-01 12.9 MCM3,CDK4,CDK6,MCM7 4 Chromosomal Replication IL-15 Signaling 2.19E+01 8.96E-02 8.96 PTK2 (includes 6 EG:14083),LCK,PLCG1,IL2RB,ATM,BCL 2 EGF Signaling 1.74E+01 9.62E-02 9.62 ITPR3,PLCG1,STAT1,PRKCA,ATM 5 VEGF Signaling 1.74E+01 7.22E-02 7.22 PTK2 (includes 7 EG:14083),VEGFB,FOXO1,PLCG1,PRK CA,ATM,BCL2 Non-Small Cell Lung 1.74E+01 7.59E-02 7.59 CDK4,ITPR3,CDK6,PLCG1,PRKCA,ATM 6 Cancer Signaling Granzyme B Signaling 1.66E+01 1.88E-01 18.8 PRF1,GZMB,PARP1 3 Renin-Angiotensin 1.58E+01 6.35E-02 6.35 PTK2 (includes 8 Signaling EG:14083),ITPR3,PLCG1,PRKCH,CCL5, STAT1,PRKCA,ATM

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Chemokine Signaling 1.51E+01 8.33E-02 8.33 PTK2 (includes 6 EG:14083),CCR3,MPRIP,PLCG1,CCL5, PRKCA ERK/MAPK Signaling 1.38E+01 5.39E-02 5.39 PTK2 (includes 11 EG:14083),ETS1,MYC,FYN,ELF2,JMJD 7- PLA2G4B,PLCG1,RPS6KA5,STAT1,PRK CA,ATM Role of Macrophages, 1.32E+01 4.69E-02 4.69 VEGFB,IL32,PLCG1,LTB,PPP3CC,CCL5, 15 Fibroblasts and TCF7,MYC,LTA,TLR7,PRKCH,LEF1,TRA Endothelial Cells in F5,ATM,PRKCA Rheumatoid Arthritis IL-3 Signaling 1.32E+01 8.11E-02 8.11 FOXO1,PRKCH,PPP3CC,STAT1,PRKCA, 6 ATM Interferon Signaling 1.26E+01 1.11E-01 11.1 IFIT3,MX1,STAT1,BCL2 4 NF-κB Activation by 1.20E+01 7.32E-02 7.32 LCK,CD4,PRKCH,CXCR5,PRKCA,ATM 6 Viruses HGF Signaling 9.62E+00 6.67E-02 6.67 PTK2 (includes 7 EG:14083),ETS1,ELF2,PLCG1,PRKCH,P RKCA,ATM Fc Epsilon RI Signaling 8.05E+00 6.31E-02 6.31 FYN,LAT,JMJD7- 7 PLA2G4B,PLCG1,PRKCH,PRKCA,ATM Pancreatic 7.53E+00 5.93E-02 5.93 VEGFB,CDK4,SMAD3,STAT1,RALGDS, 7 Adenocarcinoma ATM,BCL2 Signaling Role of JAK1 and JAK3 in 7.53E+00 7.58E-02 7.58 IL7R,IL21R,STAT1,IL2RB,ATM 5 γc Cytokine Signaling PI3K Signaling in B 7.53E+00 5.63E-02 5.63 FYN,CD19,CD79B,ITPR3,PLCG1,PPP3C 8 Lymphocytes C,PLEKHA1,CD79A p38 MAPK Signaling 7.53E+00 6.60E-02 6.6 MYC,MEF2D,JMJD7- 7 PLA2G4B,MAP4K1,MEF2C,RPS6KA5,S TAT1 Factors Promoting 6.22E+00 6.38E-02 6.38 TGFBR3,LEF1,MEF2C,PRKCH,TCF7,PR 6 Cardiogenesis in KCA Vertebrates CCR3 Signaling in 5.85E+00 5.60E-02 5.6 CCR3,MPRIP,JMJD7- 7 Eosinophils PLA2G4B,ITPR3,PRKCH,PRKCA,ATM Virus Entry via Endocytic 5.85E+00 6.06E-02 6.06 FYN,PLCG1,PRKCH,ITGB7,PRKCA,ATM 6 Pathways IL-15 Production 5.47E+00 9.68E-02 9.68 PTK2 (includes EG:14083),TXK,STAT1 3 Macropinocytosis 5.47E+00 6.58E-02 6.58 PLCG1,PRKCH,ITGB7,PRKCA,ATM 5 Signaling FLT3 Signaling in 5.27E+00 7.04E-02 7.04 STAT4,RPS6KA5,STAT1,FLT3LG,ATM 5 Hematopoietic Progenitor Cells p53 Signaling 5.19E+00 6.25E-02 6.25 CCND2,CDK4,ATR,ATM,BCL2,SERPINE 6 2 Glioma Signaling 5.04E+00 5.41E-02 5.41 CDK4,CDK6,PLCG1,PRKCH,PRKCA,AT 6 M PDGF Signaling 4.97E+00 6.33E-02 6.33 MYC,PLCG1,STAT1,PRKCA,ATM 5 p70S6K Signaling 4.76E+00 5.34E-02 5.34 CD19,CD79B,PLCG1,PRKCH,CD79A,PR 7 KCA,ATM Role of Pattern 4.63E+00 6.10E-02 6.1 IFIH1,OAS2,TLR7,CCL5,ATM 5 Recognition Receptors in Recognition of Bacteria and Viruses Glioblastoma Multiforme 4.58E+00 4.79E-02 4.79 MYC,FOXO1,CDK4,ITPR3,CDK6,PLCG1 8 Signaling ,LEF1,ATM DNA Double-Strand 4.12E+00 1.05E-01 10.5 PARP1,ATM 2 Break Repair by Non- Homologous End Joining 298

Cholecystokinin/Gastrin- 4.11E+00 5.66E-02 5.66 PTK2 (includes 6 mediated Signaling EG:14083),MEF2D,ITPR3,MEF2C,PRK CH,PRKCA Acute Myeloid Leukemia 4.11E+00 6.10E-02 6.1 MYC,LEF1,FLT3LG,TCF7,ATM 5 Signaling Cyclins and Cell Cycle 4.11E+00 5.75E-02 5.75 CCND2,CDK4,CDK6,ATR,ATM 5 Regulation Lymphotoxin β Receptor 4.10E+00 6.67E-02 6.67 LTA,LTB,TRAF5,ATM 4 Signaling Prostate Cancer Signaling 3.88E+00 5.26E-02 5.26 FOXO1,NKX3-1,LEF1,ATM,BCL2 5 IL-9 Signaling 3.88E+00 7.50E-02 7.5 SOCS2,STAT1,ATM 3 Role of BRCA1 in DNA 3.88E+00 6.56E-02 6.56 C17orf70,STAT1,ATR,ATM 4 Damage Response Propanoate Metabolism 3.88E+00 3.31E-02 3.31 ACAD11,ALDH1A1,ACSS1,LDHB 4 Role of Tissue Factor in 3.79E+00 5.41E-02 5.41 FYN,LCK,RPS6KA5,PDXP,PRKCA,ATM 6 Cancer NF-κB Signaling 3.53E+00 4.68E-02 4.68 LCK,LTA,TGFBR3,ZAP70,TLR7,TRAF5,C 8 ARD11,ATM Corticotropin Releasing 3.53E+00 4.62E-02 4.62 MEF2D,ITPR3,PLCG1,MEF2C,PRKCH,P 6 Hormone Signaling RKCA RhoA Signaling 3.53E+00 5.36E-02 5.36 PTK2 (includes 6 EG:14083),MPRIP,EPHA1,LPAR5,SEPT 9,SEPT6 Phospholipid 3.49E+00 4.95E-02 4.95 LPIN1,DGKQ,CLC,JMJD7- 5 Degradation PLA2G4B,PLCG1 Granzyme A Signaling 3.49E+00 1.11E-01 11.1 GZMA,PRF1 2 Pyruvate Metabolism 3.45E+00 3.10E-02 3.1 ALDH1A1,ACSS1,AKR1B1,LDHB 4 ERK5 Signaling 3.42E+00 6.35E-02 6.35 MYC,MEF2D,MEF2C,RPS6KA5 4 JAK/Stat Signaling 3.42E+00 6.25E-02 6.25 STAT4,SOCS2,STAT1,ATM 4 Neuregulin Signaling 3.42E+00 5.00E-02 5 MYC,PLCG1,PRKCH,PRKCA,MATK 5 Docosahexaenoic Acid 3.42E+00 6.25E-02 6.25 FOXO1,ATM,BCL2 3 (DHA) Signaling CD40 Signaling 3.37E+00 5.80E-02 5.8 LTA,TRAF5,ATM,FCER2 4 GM-CSF Signaling 3.37E+00 5.97E-02 5.97 ETS1,PPP3CC,STAT1,ATM 4 Apoptosis Signaling 3.32E+00 5.32E-02 5.32 PLCG1,BIRC3,PRKCA,PARP1,BCL2 5 PXR/RXR Activation 3.32E+00 4.82E-02 4.82 GSTM1,GSTM2,ALDH1A1,FOXO1 4 Role of MAPK Signaling in 3.32E+00 6.06E-02 6.06 JMJD7-PLA2G4B,CCL5,PRKCA,BCL2 4 the Pathogenesis of Influenza FcγRIIB Signaling in B 3.06E+00 5.45E-02 5.45 CD79B,CD79A,ATM 3 Lymphocytes Chronic Myeloid 2.97E+00 4.85E-02 4.85 MYC,CDK4,SMAD3,CDK6,ATM 5 Leukemia Signaling Erythropoietin Signaling 2.88E+00 5.13E-02 5.13 PLCG1,PRKCH,PRKCA,ATM 4 Thyroid Cancer Signaling 2.86E+00 6.67E-02 6.67 MYC,LEF1,TCF7 3 Cell Cycle: G2/M DNA 2.86E+00 6.25E-02 6.25 CDC25B,ATR,ATM 3 Damage Checkpoint Regulation Role of NFAT in Cardiac 2.82E+00 3.94E-02 3.94 MEF2D,ITPR3,PLCG1,MEF2C,PRKCH,P 8 Hypertrophy PP3CC,PRKCA,ATM Telomerase Signaling 2.74E+00 5.05E-02 5.05 ETS1,MYC,ELF2,IL2RB,ATM 5 Melatonin Signaling 2.69E+00 5.13E-02 5.13 RORA,PLCG1,PRKCH,PRKCA 4 Aminophosphonate 2.40E+00 3.51E-02 3.51 MGMT,PRMT6 2 Metabolism Tumoricidal Function of 2.40E+00 8.33E-02 8.33 PRF1,GZMB 2 Hepatic Natural Killer Cells

299

IL-12 Signaling and 2.40E+00 3.95E-02 3.95 STAT4,IL12RB1,PRKCH,STAT1,PRKCA, 6 Production in ATM Macrophages Ovarian Cancer Signaling 2.37E+00 4.26E-02 4.26 VEGFB,CDK4,LEF1,TCF7,ATM,BCL2 6 Neuropathic Pain 2.37E+00 4.50E-02 4.5 ITPR3,PLCG1,PRKCH,PRKCA,ATM 5 Signaling In Dorsal Horn Neurons CNTF Signaling 2.37E+00 5.77E-02 5.77 RPS6KA5,STAT1,ATM 3 Histidine Metabolism 2.37E+00 2.59E-02 2.59 ALDH1A1,MGMT,PRMT6 3 Endothelin-1 Signaling 2.24E+00 3.83E-02 3.83 MYC,JMJD7- 7 PLA2G4B,ITPR3,PLCG1,PRKCH,PRKCA, ATM TREM1 Signaling 2.24E+00 4.84E-02 4.84 TLR7,PLCG1,CD86 3 VDR/RXR Activation 2.24E+00 4.94E-02 4.94 FOXO1,PRKCH,CCL5,PRKCA 4 Glucocorticoid Receptor 2.24E+00 3.42E-02 3.42 CD247,CD3G,CD3E,SMAD3,PPP3CC,C 10 Signaling CL5,STAT1,CD3D,ATM,BCL2 fMLP Signaling in 2.24E+00 3.94E-02 3.94 ITPR3,PRKCH,PPP3CC,PRKCA,ATM 5 Neutrophils Hepatic Fibrosis / Hepatic 2.24E+00 4.17E-02 4.17 VEGFB,SMAD3,CCL5,STAT1,CCR7,BCL 6 Stellate Cell Activation 2 Reelin Signaling in 2.24E+00 4.88E-02 4.88 FYN,LCK,MAP4K1,ATM 4 Neurons Endometrial Cancer 2.24E+00 5.26E-02 5.26 MYC,LEF1,ATM 3 Signaling Pyrimidine Metabolism 2.22E+00 2.80E-02 2.8 POLR1C,POLR3H,POLR1E,PUS1,POLR 6 MT,UNG IL-2 Signaling 2.19E+00 5.17E-02 5.17 LCK,IL2RB,ATM 3 Purine Metabolism 2.18E+00 2.56E-02 2.56 CECR1,POLR1C,POLR3H,IMPDH2,PDE 10 7A,POLR1E,ADA,POLRMT,ATIC,APRT TNFR2 Signaling 2.18E+00 6.06E-02 6.06 LTA,BIRC3 2 IL-8 Signaling 2.17E+00 3.66E-02 3.66 PTK2 (includes 7 EG:14083),VEGFB,CCND2,PRKCH,PRK CA,ATM,BCL2 Colorectal Cancer 2.17E+00 3.56E-02 3.56 MYC,VEGFB,SMAD3,TLR7,LEF1,STAT1 9 Metastasis Signaling ,RALGDS,TCF7,ATM B Cell Receptor Signaling 2.13E+00 3.90E-02 3.9 ETS1,CD19,CD79B,PPP3CC,CD79A,AT 6 M TGF-β Signaling 2.13E+00 4.49E-02 4.49 RUNX3,SMAD3,MAP4K1,BCL2 4 EIF2 Signaling 2.13E+00 3.68E-02 3.68 RPS4X,RPL22,RPL14,RPS23,RPL13A,R 7 PL13,ATM Hereditary Breast Cancer 2.13E+00 4.03E-02 4.03 C17orf70,CDK4,CDK6,ATR,ATM 5 Signaling Protein Kinase A 2.13E+00 3.42E-02 3.42 PTK2 (includes 11 Signaling EG:14083),ANAPC1/LOC100286979,P DE7A,SMAD3,ITPR3,PLCG1,LEF1,PRK CH,PPP3CC,TCF7,PRKCA FGF Signaling 2.13E+00 4.44E-02 4.44 PLCG1,RPS6KA5,PRKCA,ATM 4 Melanocyte 2.13E+00 4.35E-02 4.35 PLCG1,RPS6KA5,ATM,BCL2 4 Development and Pigmentation Signaling mTOR Signaling 2.13E+00 3.59E-02 3.59 RPS4X,VEGFB,RPS6KA5,PRKCH,RPS23 7 ,PRKCA,ATM FAK Signaling 2.13E+00 3.96E-02 3.96 PTK2 (includes 4 EG:14083),FYN,PLCG1,ATM RANK Signaling in 2.13E+00 4.26E-02 4.26 PPP3CC,TRAF5,BIRC3,ATM 4 Osteoclasts ILK Signaling 2.13E+00 3.76E-02 3.76 PTK2 (includes 7 EG:14083),MYC,VEGFB,LEF1,RPS6KA5 ,ITGB7,ATM

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G Protein Signaling 2.13E+00 5.13E-02 5.13 LCK,PLCG1 2 Mediated by Tubby Myc Mediated Apoptosis 2.13E+00 5.00E-02 5 MYC,ATM,BCL2 3 Signaling Death Receptor Signaling 2.13E+00 4.76E-02 4.76 TNFRSF25,BIRC3,BCL2 3 α-Adrenergic Signaling 2.13E+00 3.81E-02 3.81 ITPR3,PLCG1,PRKCH,PRKCA 4 Bladder Cancer Signaling 2.13E+00 4.44E-02 4.44 MYC,VEGFB,CDK4,RPS6KA5 4 14-3-3-mediated 2.13E+00 4.13E-02 4.13 FOXO1,PLCG1,PRKCH,PRKCA,ATM 5 Signaling Gα12/13 Signaling 2.12E+00 3.94E-02 3.94 PTK2 (includes 5 EG:14083),MEF2D,LPAR5,MEF2C,AT M Retinoic acid Mediated 2.12E+00 4.69E-02 4.69 PARP12,PARP14,PARP1 3 Apoptosis Signaling Valine, Leucine and 2.12E+00 2.94E-02 2.94 ACAD11,ALDH1A1,HADH 3 Isoleucine Degradation Pathogenesis of Multiple 2.12E+00 1.11E-01 11.1 CCL5 1 Sclerosis Assembly of RNA 2.12E+00 7.69E-02 7.69 POLR1C 1 Polymerase I Complex P2Y Purigenic Receptor 2.12E+00 3.60E-02 3.6 MYC,PLCG1,PRKCH,PRKCA,ATM 5 Signaling Pathway Role of CHK Proteins in 2.12E+00 5.71E-02 5.71 ATR,ATM 2 Cell Cycle Checkpoint Control TWEAK Signaling 2.12E+00 5.26E-02 5.26 TNFRSF25,BIRC3 2 MIF-mediated 2.12E+00 4.76E-02 4.76 JMJD7-PLA2G4B,CD74 2 Glucocorticoid Regulation Role of JAK2 in Hormone- 2.12E+00 5.71E-02 5.71 SOCS2,STAT1 2 like Cytokine Signaling Aminoacyl-tRNA 2.12E+00 2.63E-02 2.63 FARS2,VARS 2 Biosynthesis CXCR4 Signaling 2.12E+00 3.57E-02 3.57 PTK2 (includes 6 EG:14083),CD4,ITPR3,PRKCH,PRKCA, ATM Role of Osteoblasts, 2.10E+00 3.38E-02 3.38 FOXO1,LEF1,PPP3CC,TRAF5,BIRC3,TC 8 Osteoclasts and F7,ATM,BCL2 Chondrocytes in Rheumatoid Arthritis Leukocyte Extravasation 2.09E+00 3.59E-02 3.59 PTK2 (includes 7 Signaling EG:14083),TXK,PLCG1,PRKCH,PRKCA, ATM,ITK Fcγ Receptor-mediated 2.09E+00 3.96E-02 3.96 FYN,PLCG1,PRKCH,PRKCA 4 Phagocytosis in Macrophages and Monocytes Glycerolipid Metabolism 2.09E+00 2.78E-02 2.78 LPIN1,ALDH1A1,DGKQ,AKR1B1 4 Glycerophospholipid 2.09E+00 2.70E-02 2.7 LPIN1,DGKQ,CLC,JMJD7- 5 Metabolism PLA2G4B,PLCG1 Thrombin Signaling 2.09E+00 3.43E-02 3.43 PTK2 (includes 7 EG:14083),MPRIP,ITPR3,PLCG1,PRKC H,PRKCA,ATM Valine, Leucine and 2.09E+00 2.44E-02 2.44 VARS 1 Isoleucine Biosynthesis Fatty Acid Biosynthesis 2.09E+00 2.04E-02 2.04 FASN 1 Aldosterone Signaling in 2.09E+00 3.47E-02 3.47 ITPR3,PLCG1,PRKCH,DNAJC30,PRKCA, 6 Epithelial Cells ATM Nitrogen Metabolism 2.09E+00 1.67E-02 1.67 CA5B,CCDC92 2 Selenoamino Acid 2.09E+00 2.74E-02 2.74 MGMT,PRMT6 2 301

Metabolism Angiopoietin Signaling 2.02E+00 4.05E-02 4.05 PTK2 (includes 3 EG:14083),FOXO1,ATM Gap Junction Signaling 2.02E+00 3.33E-02 3.33 ITPR3,PLCG1,PRKCH,PPP3CC,PRKCA,A 6 TM Amyotrophic Lateral 1.98E+00 3.45E-02 3.45 VEGFB,BIRC3,ATM,BCL2 4 Sclerosis Signaling IGF-1 Signaling 1.98E+00 3.81E-02 3.81 PTK2 (includes 4 EG:14083),FOXO1,SOCS2,ATM Human Embryonic Stem 1.94E+00 3.31E-02 3.31 S1PR5,SMAD3,LEF1,TCF7,ATM 5 Cell Pluripotency Cleavage and 1.87E+00 8.33E-02 8.33 WDR33 1 Polyadenylation of Pre- mRNA Fatty Acid Metabolism 1.85E+00 2.45E-02 2.45 ACAD11,ALDH1A1,ECI2,HADH 4 Wnt/β-catenin Signaling 1.84E+00 3.51E-02 3.51 MYC,AXIN2,TGFBR3,MAP4K1,LEF1,TC 6 F7 Role of PKR in Interferon 1.84E+00 4.44E-02 4.44 TRAF5,STAT1 2 Induction and Antiviral Response Mechanisms of Viral Exit 1.84E+00 4.44E-02 4.44 PRKCH,PRKCA 2 from Host Cells Synaptic Long Term 1.82E+00 3.54E-02 3.54 ITPR3,PRKCH,PPP3CC,PRKCA 4 Potentiation G-Protein Coupled 1.82E+00 3.02E-02 3.02 CCR3,FYN,S1PR5,PDE7A,GPR18,CX3C 16 Receptor Signaling R1,EMR3,GPR183,GPR56,P2RY10,RAS GRP1,GPR114,LPAR5,CCR7,ATM,PRK CA MIF Regulation of Innate 1.82E+00 4.00E-02 4 JMJD7-PLA2G4B,CD74 2 Immunity Caveolar-mediated 1.82E+00 3.61E-02 3.61 FYN,ITGB7,PRKCA 3 Endocytosis Signaling Androgen and Estrogen 1.82E+00 2.42E-02 2.42 MGMT,PRMT6,SULF2 3 Metabolism LPS-stimulated MAPK 1.79E+00 3.66E-02 3.66 PRKCH,PRKCA,ATM 3 Signaling Melanoma Signaling 1.79E+00 4.35E-02 4.35 CDK4,ATM 2 DNA Double-Strand 1.77E+00 5.88E-02 5.88 ATM 1 Break Repair by Homologous Recombination Nitric Oxide Signaling in 1.77E+00 3.19E-02 3.19 VEGFB,ITPR3,ATM 3 the Cardiovascular System PTEN Signaling 1.76E+00 3.25E-02 3.25 PTK2 (includes 4 EG:14083),FOXO1,TGFBR3,BCL2 Phenylalanine, Tyrosine 1.70E+00 1.54E-02 1.54 FARS2 1 and Tryptophan Biosynthesis Tryptophan Metabolism 1.70E+00 1.86E-02 1.86 ALDH1A1,MGMT,PRMT6,HADH 4 β-alanine Metabolism 1.70E+00 2.20E-02 2.2 ACAD11,ALDH1A1 2 NRF2-mediated Oxidative 1.70E+00 3.16E-02 3.16 GSTM1,GSTM2,DNAJA3,PRKCH,PRKC 6 Stress Response A,ATM Ceramide Signaling 1.69E+00 3.45E-02 3.45 S1PR5,ATM,BCL2 3 Leptin Signaling in 1.69E+00 3.45E-02 3.45 FOXO1,PLCG1,ATM 3 Obesity Sphingosine-1-phosphate 1.67E+00 3.25E-02 3.25 PTK2 (includes 4 Signaling EG:14083),S1PR5,PLCG1,ATM Clathrin-mediated 1.67E+00 3.12E-02 3.12 VEGFB,LDLRAP1,PPP3CC,SH3KBP1,IT 6 Endocytosis Signaling GB7,ATM

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Type II Diabetes Mellitus 1.66E+00 2.60E-02 2.6 SOCS2,PRKCH,PRKCA,ATM 4 Signaling Glycolysis/Gluconeogene 1.66E+00 2.42E-02 2.42 ALDH1A1,ACSS1,LDHB 3 sis Inositol Phosphate 1.66E+00 2.62E-02 2.62 CDK4,CDK6,PLCG1,PRKCH,ATM 5 Metabolism Sphingolipid Metabolism 1.64E+00 2.73E-02 2.73 NEU1,LPIN1,SULF2 3 Ascorbate and Aldarate 1.64E+00 1.30E-02 1.3 ALDH1A1 1 Metabolism Fatty Acid Elongation in 1.58E+00 2.17E-02 2.17 HADH 1 Mitochondria Differential Regulation of 1.58E+00 5.56E-02 5.56 CCL5 1 Cytokine Production in Macrophages and T Helper Cells by IL-17A and IL-17F CD27 Signaling in 1.55E+00 3.57E-02 3.57 TRAF5,CD27 2 Lymphocytes Lipid Antigen 1.53E+00 4.55E-02 4.55 CD3E 1 Presentation by CD1 Semaphorin Signaling in 1.53E+00 3.85E-02 3.85 PTK2 (includes EG:14083),FYN 2 Neurons Integrin Signaling 1.53E+00 2.93E-02 2.93 PTK2 (includes 6 EG:14083),FYN,MPRIP,PLCG1,ITGB7, ATM Glutathione Metabolism 1.53E+00 2.38E-02 2.38 GSTM1,GSTM2 2 DNA Methylation and 1.53E+00 4.35E-02 4.35 DNMT1 1 Transcriptional Repression Signaling Role of Lipids/Lipid Rafts 1.53E+00 4.17E-02 4.17 RSAD2 1 in the Pathogenesis of Influenza One Carbon Pool by 1.53E+00 2.78E-02 2.78 ATIC 1 Folate Synaptic Long Term 1.53E+00 2.86E-02 2.86 JMJD7-PLA2G4B,ITPR3,PRKCH,PRKCA 4 Depression GNRH Signaling 1.53E+00 2.82E-02 2.82 PTK2 (includes 4 EG:14083),ITPR3,PRKCH,PRKCA PPARα/RXRα Activation 1.53E+00 2.82E-02 2.82 FASN,SMAD3,TGFBR3,PLCG1,PRKCA 5 Role of IL-17A in Arthritis 1.53E+00 3.17E-02 3.17 CCL5,ATM 2 G Beta Gamma Signaling 1.53E+00 2.65E-02 2.65 PLCG1,PRKCH,PRKCA 3 Dopamine-DARPP32 1.52E+00 2.73E-02 2.73 ITPR3,PLCG1,PRKCH,PPP3CC,PRKCA 5 Feedback in cAMP Signaling Biosynthesis of Steroids 1.51E+00 8.26E-03 0.82 COX10 (includes EG:1352) 1 6 Polyamine Regulation in 1.48E+00 3.45E-02 3.45 MYC 1 Colon Cancer Glioma Invasiveness 1.48E+00 3.33E-02 3.33 PTK2 (includes EG:14083),ATM 2 Signaling Butanoate Metabolism 1.48E+00 1.59E-02 1.59 ALDH1A1,HADH 2 SAPK/JNK Signaling 1.48E+00 2.97E-02 2.97 LCK,MAP4K1,ATM 3 Mitotic Roles of Polo-Like 1.46E+00 3.33E-02 3.33 CDC25B,ANAPC1/LOC100286979 2 Kinase Glycosphingolipid 1.46E+00 1.56E-02 1.56 ST3GAL5 1 Biosynthesis - Neolactoseries Differential Regulation of 1.46E+00 4.35E-02 4.35 CCL5 1 Cytokine Production in Intestinal Epithelial Cells

303 by IL-17A and IL-17F RAR Activation 1.46E+00 2.72E-02 2.72 ALDH1A1,SMAD3,PRKCH,PRKCA,PAR 5 P1 Induction of Apoptosis by 1.46E+00 3.12E-02 3.12 BIRC3,BCL2 2 HIV1 Lysine Degradation 1.46E+00 1.47E-02 1.47 ALDH1A1,HADH 2 CREB Signaling in 1.46E+00 2.55E-02 2.55 ITPR3,PLCG1,PRKCH,PRKCA,ATM 5 Neurons Glycosphingolipid 1.46E+00 1.82E-02 1.82 ST3GAL5 1 Biosynthesis - Ganglioseries IL-22 Signaling 1.46E+00 4.00E-02 4 STAT1 1 Role of JAK1, JAK2 and 1.46E+00 3.70E-02 3.7 STAT1 1 TYK2 in Interferon Signaling Role of PI3K/AKT 1.46E+00 2.86E-02 2.86 CCL5,ATM 2 Signaling in the Pathogenesis of Influenza Nicotinate and 1.46E+00 2.24E-02 2.24 CDK4,CDK6,PRKCH 3 Nicotinamide Metabolism Glycosaminoglycan 1.46E+00 1.82E-02 1.82 SULF2 1 Degradation N-Glycan Degradation 1.46E+00 4.00E-02 4 NEU1 1 IL-17A Signaling in 1.46E+00 4.00E-02 4 CCL5 1 Gastric Cells Role of JAK family kinases 1.46E+00 3.70E-02 3.7 STAT1 1 in IL-6-type Cytokine Signaling Paxillin Signaling 1.46E+00 2.73E-02 2.73 PTK2 (includes EG:14083),ITGB7,ATM 3 Antiproliferative Role of 1.44E+00 3.85E-02 3.85 SMAD3 1 TOB in T Cell Signaling Methionine Metabolism 1.44E+00 1.33E-02 1.33 DNMT1 1 HIF1α Signaling 1.44E+00 2.83E-02 2.83 VEGFB,LDHB,ATM 3 Production of Nitric 1.42E+00 2.38E-02 2.38 PLCG1,PRKCH,STAT1,PRKCA,ATM 5 Oxide and Reactive Oxygen Species in Macrophages Hypoxia Signaling in the 1.41E+00 3.03E-02 3.03 UBE2L6,ATM 2 Cardiovascular System Xenobiotic Metabolism 1.41E+00 2.60E-02 2.6 GSTM1,GSTM2,ALDH1A1,MGMT,PRK 7 Signaling CH,PRKCA,ATM NGF Signaling 1.41E+00 2.59E-02 2.59 PLCG1,RPS6KA5,ATM 3 Ubiquinone Biosynthesis 1.41E+00 1.82E-02 1.82 MGMT,PRMT6 2 Citrate Cycle 1.41E+00 1.79E-02 1.79 CLYBL 1 Tyrosine Metabolism 1.40E+00 1.04E-02 1.04 MGMT,PRMT6 2 Neurotrophin/TRK 1.38E+00 2.63E-02 2.63 PLCG1,ATM 2 Signaling Renal Cell Carcinoma 1.37E+00 2.74E-02 2.74 ETS1,ATM 2 Signaling O-Glycan Biosynthesis 1.37E+00 2.27E-02 2.27 ST6GALNAC6 1 Basal Cell Carcinoma 1.36E+00 2.74E-02 2.74 LEF1,TCF7 2 Signaling Androgen Signaling 1.34E+00 2.16E-02 2.16 SMAD3,PRKCH,PRKCA 3 Complement System 1.33E+00 2.94E-02 2.94 CFD 1 Inhibition of 1.32E+00 2.63E-02 2.63 FYN 1 Angiogenesis by TSP1 Pentose and Glucuronate 1.32E+00 7.63E-03 0.76 AKR1B1 1 304

Interconversions 3 Role of Wnt/GSK-3β 1.32E+00 2.60E-02 2.6 LEF1,TCF7 2 Signaling in the Pathogenesis of Influenza Oncostatin M Signaling 1.30E+00 2.86E-02 2.86 STAT1 1 Cell Cycle Regulation by 1.28E+00 2.78E-02 2.78 CDK4 1 BTG Family Proteins Notch Signaling 1.25E+00 2.44E-02 2.44 MFNG 1 Glutamate Metabolism 1.25E+00 1.33E-02 1.33 CCDC92 1 Cellular Effects of 1.80E+00 5.37E-02 5.37 MYL12A,MYL6,GNA15,MYL12B,PRKA 8 Sildenafil (Viagra) R2A,GNAQ,PPP1R12A,PDE4D Activation of IRF by 1.80E+00 5.80E-02 5.8 TANK,TBK1,CHUK,IFNAR1 4 Cytosolic Pattern Recognition Receptors Estrogen-Dependent 1.80E+00 5.71E-02 5.71 FOS,MAPK1,MAPK3,KRAS 4 Breast Cancer Signaling Antiproliferative Role of 1.79E+00 7.69E-02 7.69 MAPK1,RPS6KA1 (includes EG:20111) 2 TOB in T Cell Signaling Ovarian Cancer Signaling 1.79E+00 5.67E-02 5.67 MAPK1,MAPK3,CD44 (includes 8 EG:100330801),PRKAR2A,KRAS,MAP 2K1,MMP9,PTEN Fatty Acid Biosynthesis 1.75E+00 2.04E-02 2.04 ACSL3 1 Graft-versus-Host 1.75E+00 6.25E-02 6.25 IL1RN,FCER1G,IL1B 3 Disease Signaling Amyotrophic Lateral 1.75E+00 5.17E-02 5.17 RAB5A,RAB5C,APAF1,GRINA,CAPN3,B 6 Sclerosis Signaling IRC2 Role of Oct4 in 1.71E+00 6.67E-02 6.67 RARA,SH3GLB1,HOXB1 3 Mammalian Embryonic Stem Cell Pluripotency Paxillin Signaling 1.69E+00 5.45E-02 5.45 ITGAM (includes 6 EG:16409),MAPK14,CDC42,MAPK1,K RAS,PTPN12 Citrate Cycle 1.68E+00 3.57E-02 3.57 DLD,IDH1 2 Sonic Hedgehog Signaling 1.62E+00 6.06E-02 6.06 ARRB2,PRKAR2A 2 FXR/RXR Activation 1.60E+00 5.05E-02 5.05 IL1RN,RARA,FBP1,IL1B,RXRA 5 FGF Signaling 1.60E+00 5.56E-02 5.56 MAPK14,MAPK1,MAPK3,MAPKAPK2, 5 MAP2K1 VEGF Signaling 1.57E+00 5.15E-02 5.15 MAPK1,MAPK3,KRAS,HIF1A,MAP2K1 5 Agrin Interactions at 1.57E+00 5.88E-02 5.88 CDC42,MAPK1,MAPK3,KRAS 4 Neuromuscular Junction Gap Junction Signaling 1.57E+00 5.00E-02 5 GNAI3,MAPK1,GNA15,PRKCD,MAPK3 9 ,PRKAR2A,GNAQ,KRAS,MAP2K1 Bile Acid Biosynthesis 1.56E+00 2.97E-02 2.97 DHRS9,ACAA1,PECR 3 Bladder Cancer Signaling 1.53E+00 5.56E-02 5.56 MAPK1,MAPK3,KRAS,MAP2K1,MMP9 5 Chondroitin Sulfate 1.53E+00 4.62E-02 4.62 CHSY1,CSGALNACT1,DSE 3 Biosynthesis Virus Entry via Endocytic 1.48E+00 5.05E-02 5.05 CD55,CDC42,PRKCD,CLTC,KRAS 5 Pathways ATM Signaling 1.48E+00 5.56E-02 5.56 MAPK14,GADD45A,NBN 3 DNA Double-Strand 1.48E+00 5.88E-02 5.88 NBN 1 Break Repair by Homologous Recombination DNA Double-Strand 1.48E+00 5.26E-02 5.26 NBN 1 Break Repair by Non- Homologous End Joining Glycine, Serine and 1.48E+00 2.74E-02 2.74 GNA15,CHKA,DLD,GNAQ 4 Threonine Metabolism

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IL-9 Signaling 1.45E+00 5.00E-02 5 IRS2,BCL3 2 Telomere Extension by 1.43E+00 5.88E-02 5.88 NBN 1 Telomerase Methane Metabolism 1.43E+00 1.54E-02 1.54 PRDX5 1 Coagulation System 1.42E+00 5.26E-02 5.26 PLAUR,SERPINA1 2 Phenylalanine 1.39E+00 1.83E-02 1.83 PRDX5,ALDH3B1 2 Metabolism RAN Signaling 1.39E+00 4.35E-02 4.35 KPNA4 1 Glyoxylate and 1.35E+00 9.26E-03 0.92 MTHFD2 1 Dicarboxylate 6 Metabolism Ascorbate and Aldarate 1.35E+00 1.30E-02 1.3 GSTO1 1 Metabolism Differential Regulation of 1.31E+00 5.56E-02 5.56 IL1B 1 Cytokine Production in Macrophages and T Helper Cells by IL-17A and IL-17F Endoplasmic Reticulum 1.31E+00 5.56E-02 5.56 ATF6 1 Stress Pathway

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