THE EGYPTIAN JOURNAL OF IMMUNOLOGY Vol. 22 (1), 2015 Page: 29-40

Level of IL-16 and Reticulated Platelets Percentage during the Clinical Course of Immune Thrombocytopenic Purpura in Children

1Reem R. Abd El-Glil, 2Effat H. Assar Departments of 1Microbiology & Immunology and 2Pediatric, Faculty of Medicine, Benha University, Benha, Egypt.

Immune thrombocytopenic purpura (ITP) is an immune-mediated acquired disease with transient or persistent decrease of thrombocytes number in the blood. Cytokines play important roles in the immune regulation and are known to be deregulated in autoimmune diseases. This study aimed to investigate serum IL-16 levels in relation to reticulated platelets in children with ITP and platelet count. Twenty six children with ITP (11 with newly diagnosed ITP, 9 with persistent ITP and 6 with chronic ITP) and 12 age-matched healthy children controls were studied. Serum level of IL-16 and reticulated platelets count were assessed by Enzyme Linked Immunosorbent Assay (ELISA) and flow cytometry respectively. Serum IL-16 levels were significantly higher in patients as compared to controls (P<0.001).Within patients, the levels were higher in newly diagnosed compared to persistent and chronic ITP (P<0.01) and (P<0.001) respectively. IL-16 levels were also significantly higher in persistent ITP compared to chronic ITP (P<0.001). Reticulated platelets were also elevated in patients compared to controls and the increase was significant in newly diagnosed group (P<0.05). Negative correlation was found between IL-16 level and reticulated platelets and platelets counts (r=-0.284, P=0.028, r=0.274 P=0.25) respectively. It is concluded that IL-16 may be valuable in predicting the clinical course of pediatrics ITP. Measurement of reticulated platelets may provide significant information about thrombopoietic activity during the clinical course of ITP in children

mmune thrombocytopenic purpura (ITP), respectively. Chronic ITP was defined as also known as idiopathic persisting thrombocytopenia of less than I thrombocytopenic purpura, is an immune- 100x109/L lasting for more than 12 months mediated acquired disease of adults and (Rodeghiero et al., 2009). children characterized by transient or Interleukin 16 (IL-16), formerly known as persistent decrease of the platelet count and, lymphocyte chemo attractant factor or LCF, is depending upon the degree of a proinflammatory cytokine that is a major thrombocytopenia, increased risk of bleeding chemotactic signal for CD4+ T lymphocytes, (Cooper & Bussel, 2006). monocytes and eosinophils. It plays a role in Acute abrupt onset ITP is seen mainly in trafficking of several immune cells (Conti, childhood, and often follows a viral illness or 2002). IL-16 was originally identified as a immunization. The majority of children homotetramer consisting of individual 14 kDa require no treatment and in 80–85% of cases monomers of 130 amino acids each the disorder resolves within 6 months. Some (Cruikshank et al., 1994). Sources of IL-16 15–20% of children develop a chronic form of include epithelial cells, mast cells, ITP (Guideline – BCSH, 2003). lymphocytes, macrophages, synovial The new terms “newly diagnosed” and fibroblasts, and eosinophils (Laberge et al., “persistent” replaced the previous term 1999). The gene for human IL-16 maps to “acute” for children diagnosed with ITP chromosome 15 and the sequence displays > within the last 3 months and for cases lasting 90% homology to those of various nonhuman between 3 and 12 months from diagnosis, primates (Kim, 1999). 30 Level of IL-16 and Reticulated Platelets Percentage during the Clinical Course of ITP in Children

It is synthesized as a precursor molecule that ITP, or correlate with platelet count and is cleaved by caspase-3 to produce a pro-IL- assess their significance as markers in 16 molecule that functions as a regulator of T different stages of ITP. cell growth, and a secreted peptide that functions as a CD4 and/or CD9 ligand for Subjects and Methods induction of cell motility and activation. IL-16 Subjects has been predominantly studied as a This study was conducted on 26 children with ITP (14 contributing factor in the orchestration of an females and 12 males) attending Pediatric Department, immune response (Richmond et al., 2014). Benha University Hospital, during the period from June Many of studies have implicated IL-16 in 2014 to October 2014. Their age ranged from 7 months exacerbation of infectious, immune-mediated, to 12 years. 12 healthy children (Their age ranged and autoimmune inflammatory disorders, from, 9 months to 13 years) were tested as control group. The patients were classified into three groups. including asthma (Gantnere et al., 2002), atopic dermatitis (Lee et al., 2012), Newly diagnosed ITP group: Consist of 11 children rheumatoid arthritis (Franz et al., 1998) with ITP within the last 3 months. systemic lupus erythematosus, (Xue et al., Persistent ITP group: Consist of 9 children with ITP 2009), multiple sclerosis (Biddison et al., and their cases lasting between 3 to 12 months from 1997) and viral infections (Glass, 2006). diagnosis. However, little is known about how the IL-16 Chronic ITP group: Consist of 6 children who were levels in the ITP change during the course of defined as persisting thrombocytopenia of less than 100x109/L lasting for more than 12 months the disease. (Rodeghiero, 2009). Platelet membrane has a large number of glycoproteins that are essential for their History of preceding infection and vaccination, platelet count, bleeding manifestations and pervious normal functioning. Some glycoproteins are treatment were recorded. present in the resting state as well as after The study was approved by Research Ethics stimulation e.g. CD41. The CD41 antigen is Committee in Benha Faculty of Medicine. Written expressed on platelets and megakaryocytes. informed consent from children`s parents was obtained The expression of CD41 is associated with before participation in this study. early stages of hematopoiesis and is highly Methods regulated during hematopoietic development (Mitjavila et al., 2002). All participants in the current study were subjected to complete blood picture (CBC) and platelets count Reticulocytes are the youngest circulating (Sysmix K-21 automatic cell counter. Japan). platelet population and they contain abundant 5cc of venous blood was collected by sterile amounts of mRNA. It has been shown that the venipuncture. Each sample was divided into two parts: abundance of reticulated platelets mirrors the EDTA free part for ELISA, the other part was collected rate of megakaryopoiesis in the bone marrow, in EDTA containing blood collection tube and and conditions with high platelets turnover processed as whole blood within 24 h after can be discriminated from bone marrow venipuncture. Platelets count was done as part of the routine laboratory panel in the central laboratory of insufficiency by a significantly increased Benha University Hospital (Sysmix K-21 automatic percentage of reticulated platelets in the cell counter. Japan). Quantification of reticulated peripheral blood (Richards & Baglin, 1995). platelets in EDTA containing blood collection was This study evaluates serum level of IL-16 described later in Flow Cytometeric Analysis. Sera and reticulated platelets in children with ITP samples from patients and controls were liquated and kept frozen at-20oC. to determine whether these parameters differ in newly diagnosed, persistent and chronic IL-16 was measured by a commercial ELISA (Quantikine, R&D Systems, Inc. USA). The detection THE EGYPTIAN JOURNAL OF IMMUNOLOGY 31

limits ranged from 2.7-13.4 pg/mL. ELISA procedures temperature to minimize nonspecific staining and were done according to instructions of the resuspended at 5×107/mL in phosphate-buffered saline manufacturers. (PBS). Fifty microliters of this suspension was mixed and incubated with 10 μl phycoerythrin (PE) -anti- In brief, 100 μL of Assay Diluent RD1W was added CD41-monoclonal antibody (CD-41 PE, DAKO, USA) to each well. 100 μL of standard, control, or sample per at room temperature for 30 min. Cells were then well were added then incubated for 2 hours at room washed twice in PBS. This suspension was then temperature. Aspiration and wash were repeated for incubated with 1 mL of To (Retic-COUNT, Becton three times. 200 μL of Human IL-16 Conjugate was Dickinson, San Jose, CA, USA) at room temperature in added to each well then incubated for 2 hours at room the dark for 1 hour. Labeled platelets were analyzed on temperature. Aspiration and wash were repeated. 200 FACS-caliber using cell quest software (Becton μL of Substrate Solution was added to each well then Dickinson San Jose, CA, USA). incubate for 30 minutes at room temperature. 50 μL of Stop Solution was added to each well. The optical Live gating on platelet-sized events was performed density of each well was determined within 30 minutes, using the distinctive forward light scatter (FSC) and vs using a microplate reader set to 450 nm. Obtained side light scatter (SSC) profile of platelets (figure 1). optical density values, were converted into pg/mL by Ten thousand events were measured for each sample. the Bio Rad ELISA data analysis software. All The second gating was performed using forward-scatter experiments were performed in duplicate and data vs FL2 fluorescence, and the CD41 PE-tagged platelet represent mean values. The cut-off value was population was selected. For phycoerythrin 580 nm calculated as the mean absorbance value of the negative filter is used. To exclude cell autofluorescence and controls plus three standard deviations. A sample was instrument background, an unstained control sample considered positive when the absorbance of the two was prepared simultaneously for each sample. TO- measurements was greater than 0.178. stained platelets with higher fluorescence than the 99% of the unstained control samples were considered to be Flow Cytometeric Analysis reticulated platelets. The histogram for these gated It was done at Benha Pediatrics Specialist Hospital. populations were then obtained and the mean fluorescence intensity (MFI) were calculated using Cell Whole-blood, dual-labelling flow cytometric Quest software which obtains the linear channel method was used. Direct, whole-blood double coverage numbers, determines the average values for 10000 was achieved using a monoclonal antiglycoprotein events, and then converts to mean log channel number. CD41 antibody (CD41-PE) for platelet identification and analysis of reticulated platelets (Youngest Statistical Analysis plateletes) by binding of thiazole orange (TO) (Retic- These data were tabulated, coded then analyzed using count(R) which is a fluorescent dye bind to mRNA the computer program SPSS (Statistical package for social content of platelets and allow their enumeration by science) version 16. Results were expressed as mean and Fluorescent activated cytometric (FACS )analysis. standard deviation (SD). parametric data were analysed 10 l of blood samples with EDTA as an with unpaired student`s t test were appropriate. anticoagulant, were centrifuged at 750 g for 5 minutes Correlation between variable were analyzed using to obtain platelet-rich plasma. The platelets were fixed Spearman rank correlation. P<0.05 was considered in 1% paraformaldehyde for 5 minutes at room significant and P<0.001 was considered highly significant.

32 Level of IL-16 and Reticulated Platelets Percentage during the Clinical Course of ITP in Children

a C b

b* c* a*

Figure 1. Flow cytometric dot-plot analysis of platelets. Platelets were identified by a combination of FSC/SSC gating (fig.1- a, a*), additional gating on the CD41 population by FSC versus PE-CD41 (FL2) staining (fig.1-b, b*). A third gating was set CD41 versus TO dot plot (fig.1-c, c*). 1a,b and c from an ITP patient. 1a*,b*and c* from a control subject.

Results was history of blood transfusion for 7 children (26.9%). There were no differences between This study was conducted on 26 children with patients with regards to mean platelet count, ITP and 12 healthy volunteers as control age and sex. Platelets counts were group. Patients’ and controls’ data were significantly different between patients and summarized in Table (1).The most frequent controls (P<0.001) but no significant symptoms in ITP patients were petechia in 19 difference between patients and controls patients (73.1 %). Ten children (38.5%) were regarding the age. admitted with epistaxis with petechia. There THE EGYPTIAN JOURNAL OF IMMUNOLOGY 33

Table 1. Demographic and clinical data of ITP patients and control groups. P Variable Newly diagnosed ITP Persistent ITP Chronic ITP Control Value

Age (years)(mean ±SD) 5.55 ± 2.278 5.45±1.383 5.65±2.224 6.79±2.65 NS

Sex F/M 6/5 4/5 4/2 5/7

Platelets count (mm3) 10430 23560 25370 240000 <0.001 (mean, range) (2500-50000) (4200-53000) (4600-5900) (150000-440000)

Bleeding manifestations

Petechia 11 5 3 -

Epistaxis 3 2 - -

Petechia and epistaxis 3 2 - -

Melena 1 - - -

Hematuria 1 - - -

History of blood - 3 4 - transfusion.

Comparisons between each of the four groups were analysed by unpaired t test; comparisons among the four groups were also compared using one-way analysis of variance with adjustment for multiple comparisons. P<0.05 was considered statistically significant. NS: not significant.

The study revealed that all patients with ITP ITP respectively that differed significantly had markedly increased serum IL-16 from those of the healthy subjects (24.3±6.7) (782.5±730.8, 284.2±309.5 and 159.2±142.6) (P<0.001) (Figure 2). in newly diagnosed, persistent and chronic

Figure 2. Comparison between serum IL-16 levels in different groups of patients with ITP and controls. 34 Level of IL-16 and Reticulated Platelets Percentage during the Clinical Course of ITP in Children

Our study also demonstrated that serum IL-16 groups (11.6±0.3 and 11.5±0.8) compared to levels were significantly elevated in newly the control group. However, these values were diagnosed ITP compared to its levels in statistically significant in newly diagnosed persistent ITP and chronic ITP (P<0.01), group (P<0.05) and insignificant in persistent (P<0.001) respectively. IL-16 levels were also and chronic group (P>0.05) Table 2. significantly higher in persistent ITP Percentage of positive CD41 platelets was compared to chronic ITP (P<0.001) Table 2 & 20.1±0.7 in newly diagnosed, ITP, 11.6±0.3 in 3. persistent ITP and 11.5±0.8 in chronic ITP. Patients with newly diagnosed ITP had These values were significantly different in markedly increased positive CD41 platelets newly diagnosed ITP compared to its levels in percent (20.1 ± 0.7) that differed significantly other two ITP groups (P<0.05) each but no from those of the healthy subjects (9.4±2.4). It significant difference in persistent ITP was also elevated in persistent and chronic compared to chronic ITP (P>0.05) Table 3.

Table 2. Comparison of serum IL-16 levels and platelet’s count and CD41expression between ITP patients and control groups. Newly diagnosed ITP Persistent ITP Chronic ITP Control P value

P1<0.001 IL-16 (pg/ml) 782.5±730.8 284.2±309.5 159.2±142.6 24.3±6.7 P2<0.001 (mean ±SD) P3<0.001

P1< 0.05 CD 41% 20.1±0.7 11.6±0.3 11.5±0.8 9.4±2.4 P2 = NS (mean ±SD) P3 =NS

P1= newly diagnosed ITP vs control, P2= chronic ITP vs control, P3= chronic ITP vs control. P> 0.05 is not significant (NS)

Table 3. Comparison of serum level of IL-16 and percent CD 41 positive platelets among pediatric groups with ITP. Newly diagnosed ITP Persistent ITP Chronic ITP IL-16(pg/ml)(mean ±SD) 782.5±730.8 284.2±309.5 159.2±142.6 P value P1<0.01 P2<0.001 P3<0.001 CD 41% 20.1±0.7 11.6±0.3 11.5±0.8 P value P1<0.05 P2<0.05 P3=NS P1= newly diagnosed ITP vs persistent ITP, P2= newly diagnosed ITP vs chronic ITP, P3= persistent ITP vs chronic ITP. P>0.05 is not significant (NS)

Histogram of TO labeled platelets from persistent and newly diagnosed ITP, representative normal control and a respectively. representative ITP patient is presented in Negative correlation was found between figure 3. Labeled platelets from ITP patient platelets count and IL-16 level and CD41% in groups showed more fluorescence intensity patients with ITP (r=-0.284, P=0.028, than those of the normal control group. MFI r=0.274, P=0.025) respectively figure 4 & 5. of the platelets were 57.59±12.22, 77±15.22, 92±16.11 and 105±25.83 in arbitrary fluorescence units for control, chronic, THE EGYPTIAN JOURNAL OF IMMUNOLOGY 35

Figure 3. Flow cytometric histogram of TO-labeled platelets. Platelets were stained with TO dye and their intensity was measured as MFI using a flow cytometer. M markers were set at the inflection points on the curve. The M1 indicates platelets that stained positive for TO in a representative healthy control and M2 in a representative ITP patient. MFI values were (23 and 89) respectively.

Figure 4. Correlation between platelets counts and serum IL-16 level in patients with ITP. Negative correlation was found between platelets counts and IL-16 level in patients with ITP (r=-0.284, P=0.028).

36 Level of IL-16 and Reticulated Platelets Percentage during the Clinical Course of ITP in Children

Figure 5. Correlation between platelets counts and platelets CD41 expression in patients with ITP. Negative correlation was found between platelets count and CD41% in patients’ groups (r=0.274, P=0.025).

Discussion in a second Chinese cohort of newly diagnosed ITP children and children with ITP is a common human autoimmune disease. chronic ITP. The study also revealed that Il- It characterized by destructive thrombo- 16 was elevated in all patients groups cytopenia due to the presence of auto compared to the control group. Richmond et antibodies with a normal or an increased al., 2014 explained that IL-16 has been a number of bone marrow megakaryocytes contributing factor in the orchestration of an (Kaplan et al., 2004). These autoantibodies immune response during auto immune opsonize platelets for splenic clearance. disease. Therefore, the life spans of circulating To the best of our knowledge no other platelets in ITP are reduced. relevant work regarding to the role of Il-16 in The disease is characterized by recurring ITP has been done to compare our work with. episodes of severe thrombocytopenia and is Gu et al., 2012, showed that serum IL-16 associated with a bleeding diathesis. levels were elevated both in Hashiomoto Treatment for ITP includes splenectomy, thyroiditis (HT) and higher in untreated high-dose corticosteroids and intravenous Graves disease (GD) patients when compared immunoglobulin's (IVIg) (Stasi et al., 2008). healthy individuals, which were decreased The present study demonstrated significant after therapy in untreated GD patients. They differences in serum level of IL-16 between concluded that IL-16 might be involved in the newly diagnosed pediatric patients with ITP, pathogenesis of GD and HT, and serum IL-16 persistent and chronic pediatric patients with levels in GD maybe a potential marker of ITP. This was in agreement with Jernås et al., disease activity and severity. 2013, who found that plasma IL-16 was In a study done by Wu et al., 2011, they significantly higher in newly diagnosed than found that serum levels of total IgE, IL-16 in chronic ITP. They also verify this finding THE EGYPTIAN JOURNAL OF IMMUNOLOGY 37

correlated with the SCORAD index in ITP (equivalent to persistent ITP in our study) pediatric patients with atopic dermatitis. than in healthy subjects (P<0.01) and in These serum parameters declined significantly contrast with them as regard chronic ITP after conventional treatment with clinical (P<0.001).They stated that their finding improvement. Thus, they postulated that they probably mirror ongoing massive platelet can serve as serum markers for monitoring destruction, do not, therefore, seem to have a disease activity in childhood atopic dermatitis. prognostic meaning with regards to ITP Zak et al., 2010, reported that elevated progression. levels of IL-16 with the lower blood content Our results confirmed by observation of of the cytokine IL-4 were long before the Rajantie et al., 2004, as regard of increase development of DM type1 in children with reticulated platelets count in persistent ITP normal blood glucose level in the presence of who demonstrated that after 6 months of ITP Langerhans islet autoantibodies (LIAA), (persistent stage) reticulated platelet counts which should be borne in mind while increased in children with disease developing the immune mechanisms progression. specifically directed against block, which The present work was in contrast with participate by means of cytokines in beta-cell Yildirmak et al., 2005, who found that platelet destruction, as well as methods for preventing surface antigens were significantly decreased the development of T1DM in subjects with in both acute and chronic ITP when compared LIAA. Meagher et al., 2010, also to the control group (P=0.001). Zhongguo et demonstrated that IL-16 production by al., 2009, found that the expression of CD41 leukocytes in islets of Langerhans augments in acute ITP and the chronic ITP groups were the severity of insulitis during the onset of significantly lower than those in the control type 1 diabetes. Neutralization of IL-16 may group (P<0.01). The acute ITP group had represent a novel therapy for the prevention of lower expression of CD41 than the chronic type 1 diabetes. ITP group (P<0.01). Jimin et al., 2008, Kimura et al., 2011, reported that IL-16- explained that the numbers of antibody- deficiency reduced graft inflammatory cell binding sites of CD41 in the ITP patients were recruitment, and allograft inflammatory less than in the normal controls (P<0.05) by cytokine and chemokine production. that the antibody-binding sites of CD41 may Therefore, IL-16 neutralization may provide a be occupied by autoantibodies and that the potential target for novel therapeutic treatment available binding sites may be decreased in for cardiac allograft rejection. ITP. This study revealed that percentage of In study done by Yoshiyuki et al., 2001, reticulated platelets was elevated in all they found that the percentage of reticulated patients groups compared to the control platelets was markedly elevated in patients group, however, these values were statistically with ITP but normal or slightly elevated in significant in newly diagnosed group and patients with aplastic anemia or insignificant in persistent and chronic groups chemotherapy-induced thrombocytopenia. compared to control. This was in accordance They reported the measurement of reticulated with Giovanni et al., 2012, as regard newly platelets percentage was an excellent method diagnosed and persistent ITP as they found for discriminating between hyper destructive that the percentage of reticulated platelets and hypo plastic thrombocytopenia were significantly higher in children with Thomas et al., 2007, showed that patients acute and those with chronic progression of with thrombocytopenic myelodysplastic 38 Level of IL-16 and Reticulated Platelets Percentage during the Clinical Course of ITP in Children

syndrome had normal reticulated platelets References counts compared to healthy subjects. ITP 1. Biddison WE, Taub DD, Cruikshank WW, Center patients had elevated reticulated platelets DM, Connor EW, Honma K. (1997). Chemokine counts. They concluded that reticulated and matrix metalloproteinase secretion by myelin platelets counts had a diagnostic and proteolipid protein-specific CD8+ T cells: potential prognostic value in patients with ITP and help roles in inflammation. J. Immunol.158:3046-53. to identify the complex causes of 2. Conti P, Kempuraj D, Kandere K, Di Gioacchino thrombocytopenia. M, Reale M, Barbacane RC, Castellani ML, Mortari The present study demonstrated that serum U, Boucher W, Letourneau R, Theoharides TC. (2002). Interleukin-16 network in inflammation and level of IL-16 and reticulated platelets counts allergy. Allergy Asthma Proc. 23(2):103-8. correlated inversely with platelets counts. Our results represent a remarkable contrast with 3. Cooper N, Bussel J. (2006). The pathogenesis of immune thrombocytopaenic purpura. Br J those of Giovanni et al., 2012 who did not Haematol. 133:364-374. find a correlation between platelet count and 4. Cruikshank WW, Center DM, Nisar N, Wu M, reticulated platelets. They speculated that may Natke B, Theodore AC, Kornfeld H. (1994). be related to the fact that even if the number Molecular and functional analysis of a lymphocyte of circulating platelets is predominantly chemoattractant factor: association of biologic determined by platelet destruction, it can also function with CD4 expression. Proc Natl Acad Sci. be affected by platelet production. 24;91(11):5109-13. These results indicated that IL-16 may play 5. Franz JK, Kolb SA, Hummel KM, Lahrtz F, an important role in activation of the immune Neidhart M, Aicher WK, Pap T, Gay RE, Fontana response in ITP and suggested that the two A, Gay S. (1998). Interleukin-16, produced by synovial fibroblasts, mediates chemoattraction for markers (IL-16 and CD41 percentage) are CD4+ T lymphocytes in rheumatoid arthritis. Eur J altered in the different stages of pediatrics Immunol. 28(9):2661-71. ITP. 6. Gantner F, Sakai K, Tusche MW, Cruikshank WW, We conclude, that increased IL-16 and Center DM, Bacon KB. (2002). Histamine h(4) and elevated reticulated platelets are observed in h(2) receptors control histamine-induced all forms of ITP. They seem to predict the interleukin-16 release from human CD8(+) T cells.J clinical course of ITP, as IL-16 is Pharmacol Exp Ther. 303(1):300-7. significantly higher and reticulated platelets 7. Giovanni Carlo Del Vecchio, Paola Giordano, significantly elevated in the newly diagnosed Riccardina Tesse, Laura Piacente, Maria Altomare, ITP. Furthermore, IL-16 should be considered and Domenico De Mattia. (2012). Clinical significance of serum cytokine levels and as a marker for discrimination between thrombopoietic markers in childhood idiopathic healthy children and those with ITP. thrombocytopenic purpura Blood Transfus. 10(2): Further studies as well as new 194–199. experimental designs are recommended to 8. Glass WG, Sarisky RT, Vecchio AM. (2006).The elucidate the role of IL-16 in pathogenesis, role of IL-16 in infectious and immune-mediated disease progression, diagnosis, and prognosis inflammatory diseases. J Interferon Cytokine Res. of ITP. IL -16 correlate with clinical 26(8):511-20. manifestations of ITP, and may enable better 9. Gu X, Zheng L, Chen X, Ruan L, Zhang H, Ge S, prediction and/or directed therapy once Zhu H, Lin X, Shen F.(2012). Elevated serum IL-16 confirmed in future studies. and RANTES levels in patients with autoimmune thyroid diseases and modulation by methimazole therapy. Horm Metab Res. 44(6):482-7. 10. Guidelines for the investigation and management of idiopathic thrombocytopenic purpura in adults, THE EGYPTIAN JOURNAL OF IMMUNOLOGY 39

children and in pregnancy. (2003). Brit J Haematol. progenitors derived from embryonic hematopoietic 120, 574–596. cells. Development. 129(8):2003-13. 11. Jernås M, Hou Y, Strömberg Célind F, Shao L, 20. Rajantie J, Javela K, Joutsi-Korhonen L, Kekomäki Nookaew I, WangQ, Ju X, Mellgren K, Wadenvik R. (2004). Chronic thrombocytopenia of childhood: H, Hou M, Olsson B. (2013).Differences in gene use of non-invasive methods in clinical evaluation. expression and cytokine levels between newly Eur J Haematol.72:268–72. diagnosed and chronic pediatric ITP. 5; 21. Richards EM, Baglin TP. (1995). Quantitation of 122(10):1789-92. reticulated platelets: Methodology and clinical 12. Jimin Kahng,Hun Hee Park, Kyungja Han, Bo application. Br J Haematol.91:445–451. Yeon Choi and Wonbae Lee. (2008). Quantitative 22. Richmond J, Tuzova M, Cruikshank W, Center D. Comparisons of Antibody-Binding Sites of Platelet (2014). J Cell Physiol. Regulation of cellular Glycoprotein IIb/IIIa in Aplastic Anemia and processes by interleukin-16 in homeostasis and Idiopathic Thrombocytopenic Purpura. Ann Clin cancer. 229(2):139-47. Lab Sc. 38(1):6-11. 23. Rodeghiero F, Stasi R, Gernsheimer T, Michel M 13. Kaplan RN, Bussel JB. (2004). Differential Provan D, Arnold DM, Bussel JB, Cines DB, diagnosis and management of thrombocytopenia in Chong BH, Cooper N, Godeau B, Lechner K, childhood. Pediatr Clin North Am.51:1109–1140. Mazzucconi MG, McMillan R, Sanz MA, Imbach 14. Kim HS. (1999). Assignment of human interleukin P, Blanchette V, Kühne T, Ruggeri M, George JN. 16 (IL16) to chromosome 15q26.3 by radiation (2009). Standardization of terminology, definitions hybrid mapping. Cytogenet Cell Genet.84(1-2):93. and outcome criteria in immune international working group. Blood. 113:2386–2393. 15. Kimura N, Itoh S, Nakae S, Axtell RC, Velotta JB, Bos EJ, Merk DR, Gong Y, Okamura H, Nagamine 24. Stasi, R., M. L. Evangelista, E. Stipa, F. Buccisano, CM, Adachi H, Kornfeld H, Robbins RC, Fischbein A. Venditti, S. Amadori. (2008). Idiopathic MP (2011).Interleukin-16 deficiency suppresses the thrombocytopenic purpura: current concepts in development of chronic rejection in murine cardiac pathophysiology and management. Thromb. transplantation model. J Heart Lung Transplant. Haemost. 99: 4-13. 30(12):1409-1417. 25. Thomas-Kaskel AK, Mattern D, Köhler G, Finke J, 16. Laberge S, Pinsonneault S, Ernst P, Olivenstein R, Behringer D. (2007). Reticulated platelet counts Ghaffar O, Center DM, Hamid Q. (1999). correlate with treatment response in patients with Phenotype of IL-16-producing cells in bronchial idiopathic thrombocytopenic purpura and help mucosa: evidence for the human eosinophil and identify the complex causes of thrombocytopenia in mast cell as cellular sources of IL-16 in asthma. Int patients after allogeneic hematopoietic stem cell Arch Allergy Immunol. 119(2):120-5. transplantation. Cytometry B Clin Cytom. 72(4):241-8. 17. Lee J, Noh G, Lee S, Youn Y, Rhim J. (2012).Atopic dermatitis and cytokines: recent 26. Wu KG, Li TH, Chen CJ, Cheng HI, Wang TY. patents in immunoregulatory and therapeutic (2011).Correlations of serum Interleukin-16, total implications of cytokines in atopic dermatitis--part IgE, eosinophil cationic protein and total eosinophil I: cytokines in atopic dermatitis.Recent Pat Inflamm counts with disease activity in children with atopic Allergy Drug Discov. 6(3):222-47. dermatitis. Int J Immunopathol Pharmacol. 24(1):15-23. 18. Meagher C, Beilke J, Arreaza G, Mi QS, Chen W, Salojin K, Horst N, Cruikshank WW, Delovitch TL. 27. Xue H, Gao L, Wu Y. Wang L, Li C, Li Y, Liang (2010).Neutralization of interleukin-16 protects W, Zhang L. (2009). The IL-16 gene nonobese diabetic mice from autoimmune type 1 polymorphisms and the risk of the systemic lupus diabetes by a CCL4-dependent mechanism. erythematosus.Clin Chim Acta. 403(1-2):223-5. Diabetes. 59(11):2862-2871. 28. Yildirmak Y, Yanikkaya-Demirel G, Palanduz A, 19. Mitjavila-Garcia MT, Cailleret M, Godin I, Kayaalp N. (2005). Antiplatelet antibodies and their Nogueira MM, Cohen-Solal K, Schiavon V, correlation with clinical findings in childhood Lecluse Y, Le Pesteur F, Lagrue AH, Vainchenker immune thrombocytopenic purpura. Acta Haematol W.(2002). Expression of CD41 on hematopoietic 113(2):109-12. 40 Level of IL-16 and Reticulated Platelets Percentage during the Clinical Course of ITP in Children

29. Yoshiyuki Kurata, Satoru Hayashi, Teruo Kiyoi, circulating cytokines and chemokines in the Satoru Kosugi, Hirokazu Kashiwagi, Shigenori preclinical and early clinical stages of type IA Honda, Yoshiaki Tomiyama. (2001). Diagnostic diabetes mellitus development. Ter Arkh.82 Value of Tests for Reticulated Platelets, Plasma (10):10-5. Glycocalicin, and Thrombopoietin Levels for 31. Zhongguo Dang Dai Er Ke Za Zhi. (2009).Levels of Discriminating Between Hyperdestructive and Toll-like receptors-2,-4 on platelets in children with Hypoplastic Thrombocytopenia Am J Clin Pathol. idiopathic thrombocytopenic purpura. Zhongguo 115:656-664. Dang Dai Er Ke Za Zhi. Chinese J of contemp. 30. Zak KP, Popova VV, Mel'nichenko SV, Tron'ko Pediat. 11(10):797-801. EN, Man'kovskiĭ BN. (2010). The level of