Development of Novel Tools for Prevention and Diagnosis of Porphyromonas Gingivalis Infection and Periodontitis I Dedicate This Thesis to My Beloved Parents

Development of Novel Tools for Prevention And Diagnosis of Porphyromonas gingivalis Infection and Periodontitis I dedicate this thesis to my beloved parents

¨A small body of determined spirits fired by an unquenchable faith in their mission can alter the course of history¨ - Gandhi

Örebro Studies in Medicine 151

SRAVYA SOWDAMINI NAKKA

Development of Novel Tools for Prevention and Diagnosis of Porphyromonas gingivalis Infection and Periodontitis

Title: Development of Novel Tools for Prevention and Diagnosis of Porphyromonas gingivalis Infection and Periodontitis Publisher: Örebro University 2016 www.publications.oru.se

Print: Örebro University, Repro 09/2016

ISSN 1652-4063 ISBN 978-91-7668-162-8 Abstract

Sravya Sowdamini Nakka (2016): Development of Novel Tools for Prevention and Diagnosis of Porphyromonas gingivalis Infection and Periodontitis. Örebro studies in Medicine 151.

Periodontitis is a chronic inflammatory disease caused by exaggerated host immune responses to dysregulated microbiota in dental biofilms leading to degradation of tissues and alveolar bone loss. Porphyromonas gingivalis is a major periodontal pathogen and expresses several potent virulence factors. Among these factors, arginine and lysine gingipains are of special importance, both for the bacterial survival/proliferation and the pathological outcome. The major aim of this thesis was to develop and test novel methods for diagnosis and prevention of P. gingivalis infection and periodontitis. In study I, anti-P. gingivalis antibodies were developed in vitro for immunodetection of bacteria in clinical samples using a surface plasmon resonance (SPR)-based biosensor. Specific binding of the antibodies to P. gingivalis was demonstrated in samples of patients with periodontitis and the results were validated using real-time PCR and DNA-DNA checkerboard analysis. In study II, we elucidated the properties and antimicrobial effects of different lactobacillus species and the two-peptide bacteriocin PLNC8 on P. gingivalis. L. plantarum NC8 and 44048 effectively inhibited P. gingivalis growth and pure PLNC8 induced bacterial lysis by damaging P. gingivalisαβ membrane. In study III, we demonstrated that PLNC8 dose-dependently induces proliferation and release ofαβ growth factors in gingival epithelial cells (GECs). Furthermore, PLNC8 decreased P. gingivalis-induced αβcytotoxic effects in GECs but did not alter the effect of gingipains on cytokine expression. In study IV, we elucidated the effects ofαβ anti-P. gingivalis antibodies and PLNC8 in regulating cellular responses during P. gingivalis infection. Both antibodies and PLNC8 modulated P. gingivalis-induced expression of growth factorsαβ in GECs, however, their effects were diminished when used in combination. The results of thisαβ thesis demonstrate a possible role of anti-P. gingivalis antibodies and PLNC8 in prevention and treatment of P. gingivalis infection and periodontitis with no cytotoxic effects on human cells. αβ

Keywords: Periodontitis, Porphyromonas gingivalis, anti-P. gingivalis antibodies, surface plasmon resonance, PLNC8 , proliferation, growth factors.

Sravya Sowdamini Nakka, Örebro αβstudies in Medicine, Örebro University, SE-70182 Örebro, Sweden, Email: [email protected]

Table of Contents LIST OF PAPERS ...... 9 PUBLICATIONS NOT INCLUDED IN THIS STUDY ...... 10 LIST OF ABBREVIATIONS ...... 11 INTRODUCTION ...... 13 Periodontitis ...... 13 Porphyromonas gingivalis ...... 14 Oral Biofilms ...... 16 Periodontitis and P. gingivalis Associated Systemic Diseases ...... 16 Host Immune Responses ...... 17 Antibodies ...... 20 Diagnosis, Treatment and Prevention of Periodontitis ...... 21 Lactobacillus and Bacteriocins ...... 22 Aims ...... 25 METHODS ...... 26 Study Participants and Ethical Approval ...... 26 Bacterial Strains and Bacteriocins ...... 26 Cell Culturing ...... 27 Antibody Production ...... 27 Flow Cytometry ...... 28 Transmission Electron Microscopy ...... 28 Confocal Microscopy ...... 29 Surface Plasmon Resonance Analysis ...... 29 DNA-DNA Hybridization Technique ...... 30 Enzyme-linked Immunosorbent Assay ...... 31 Growth Factor Array ...... 31 Liposome Model ...... 32 Antibacterial Activity of PLNC8 ...... 32 Circular Dichroism Spectroscopy ...... 33 Western Blotting ...... αβ ...... 33 Statistics ...... 34 RESULTS AND DISCUSSION ...... 35 Activation of Leukocytes and Induction of Secondary Immune Responses (Study I) ...... 35

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 7

Detection and Attenuation of P. gingivalis by Anti-P. gingivalis antibodies (Study I) ...... 36 Antibacterial Activity of Lactobacillus spp. and PLNC8 αβ (Study II) .... 36 Effects of PLNC8 αβ per se on GECs and During P. gingivalis Infection (Studies III & IV) ...... 37 Effects of Anti-P. gingivalis Antibodies on GECs and During P. gingivalis Infection (Study IV) ...... 39 Combined Effects of Anti-P. gingivalis Antibodies and PLNC8 αβ on P. gingivalis-infected GECs (Study IV) ...... 40 CONCLUSIONS ...... 41 FUTURE PERSPECTIVES ...... 42 ACKNOWLEDGEMENTS ...... 43 REFERENCES ...... 46

8 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

List of Papers This thesis is based on the following original papers and manuscripts, which are referred to in the text by their roman numerals:

I. Sravya Sowdamini Nakka, Johanna Lönn, Carin Starkhammar Jo- hansson, Torbjörn Bengtsson and Fariba Nayeri. Antibodies produced in vitro in the detection of periodontal bacteria by using surface plasmon resonance analysis. Clinical and experimental dental research 2015, 1:32.

II. Hazem Khalaf, Sravya Sowdamini Nakka, Camilla Sandén, Anna Svärd, Kjell Hultenby, Nikolai Scherbak, Daniel Aili and Torbjörn Bengtsson. Antibacterial effects of Lactobacillus and bacteriocin PLNC8 αβ on the periodontal pathogen Porphyromonas gingivalis. BMC Microbiology 2016, 16:188.

III. Sravya Sowdamini Nakka, Eleonor Palm, Torbjörn Bengtsson, Hazem Khalaf. Bacteriocin plantaricin NC8 αβ antagonizes Por- phyromonas gingivalis infection and induces proliferation of gingival epithelial cells. (Manuscript).

IV. Sravya Sowdamini Nakka, Eleonor Palm, Fariba Nayeri, Torbjörn Bengtsson, Hazem Khalaf. Effects of plantaricin NC8 αβ and antibodies on gingival epithelial cells infected by Porphyromonas gingivalis. (Manuscript).

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 9

Publications not included in this study Chongcong Wu, Sravya Sowdamini Nakka, Sepahdar Mansouri, Tor- björn Bengtsson, Tayeb Nayeri and Fariba Nayeri. In vitro model of production of antibodies; a new approach to reveal the presence of key bacteria in polymicrobial environments. BMC Microbiology 2016 (in press).

Johanna Lönn, Carin Starkhammar Johansson, Sravya Sowdamini Nakka, Eleonor Palm, Torbjörn Bengtsson, Fariba Nayeri F, Nils Ravald. High concentration but low activity of hepatocyte growth factor in periodontitis. Journal of periodontology 2014, 85: 113.

10 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

List of Abbreviations Akt Serine/Threonine-specific protein kinase B AMP Antimicrobial peptides APC Allophycocyanin ATCC American type culture collection CD Cluster of differentiation CF Carboxyfluorescein CFU Colony forming unit CRP C-reactive protein DAPI 4, 6-diamidino-2-phenylindole DMEM Dulbecco’s modified eagle’s medium EGF Epidermal growth factor ELISA Enzyme-linked immunosorbent assay FBS Fetal bovine serum FGF Fibroblast growth factor Fim A Major fimbriae FITC Fluorescein isothiocyanate G-CSF Granulocyte colony stimulating factor GECs Gingival epithelial cells HRP Horse radish peroxidase Ig Immunoglobulin IGF Insulin-like growth factor IL Interleukin Kgp Lysine gingipain LCM Laser-capture microdissection LDL Low-density lipoprotein LPS Lipopolysaccharide Mfa 1 Minor fimbriae MMP Matrix metalloproteinase MOI Multiplicity of infection OMV Outer membrane vesicles PAGE Polyacrylamide gel electrophoresis PAR Protease activated receptor PDGF Platelet derived growth factor PE Phycoerythrin PerCP Peridinin chlorophyll protein PLGF Placental growth factor PLNC8 Plantaricin NC8

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 11

POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPS 1-palmitoyl-2-oleoyl-glycero-3-phosphoserine PPAD Peptidyl arginine deiminase PVDF Polyvinylidine fluoride qPCR Quantitative polymerase chain reaction RANK Receptor activator of nuclear factor kappa-B ligand Rgp Arginine gingipain ROS Reactive Oxygen species SCF Stem cell-like factor SDS Sodium dodecyl sulfate SPR Surface plasmon resonance TEM Transmission electron microscopy TGF Transforming growth factor TLR Toll-like receptor TNF Tumor necrosis factor VEGF Vascular endothelial growth factor

12 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

Introduction

Periodontitis Oral cavity harbours more than 700 bacterial species and a shift in microbial paradigm towards oral pathobionts is a major etiologic factor in periodontal disease [1]. Bacteria grow in polymicrobial biofilms where they reg- ulate nutrient supply, resist antibiotic treatment and clearance from host immune responses [2]. A microbial shift known as dysbiosis occurs when beneficial symbionts are gradually outnumbered by pathobionts and oral dysbiosis could lead to periodontitis [3].

Gingivitis and periodontitis are the two most commonly occurring periodontal diseases. During gingivitis an accumulation of dental plaque causes localized inflammation of gingival tissues without affecting periodontal ligaments, alveolar bone or teeth [4]. However, the disease if untreated, could progress into a chronic inflammatory condition affecting the gingiva, ligaments and tooth supporting tissues eventually leading to alveolar bone loss [5]. The progression of gingivitis to periodontitis fairly depends on host susceptibility and perturbations at the site of infection [6, 7].

Periodontitis is the most prevalent biofilm-induced chronic inflammatory disease caused by dysbiosis in the oral microbiota. Periodontitis is usually characterized by prolonged plaque-induced inflammation at the periodontal sites, >5mm deep pockets, degradation of periodontal tissues causing slow deterioration of alveolar bone and periodontal ligament and subsequently, tooth loss [7]. Several host factors such as lifestyle, genetics and oral hy- gienic conditions play an important role in periodontitis [8]. Furthermore, smoking, drug or alcohol abuse, stress, female hormonal changes, medica- tions (antidepressants) and genetic polymorphisms have been reported as potential risk factors for periodontal diseases. These factors have been shown to negatively affect the outcome of treatment [9-11].

Dental plaque is a key etiological agent in periodontitis and also plays a crucial role in initiation and progression of the disease [12]. Periodontal pockets are moist, low oxygenated and a suitable environment for anaerobic bacterial growth leading to the formation of bacterial plaque. The synergistic interactions between the oral microbiota and a balanced inflammatory condition determines the oral health, while imbalances in these interactions affect the severity of oral disease. The etiology of periodontal disease

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 13 is often associated with that the common oral microflora are outnumbered by pathobionts. Porphyromonas gingivalis (P. gingivalis), Treponema denticola and Tannerella forsythia are considered as the three most important periodontal pathogens and are referred to as ¨the red complex bacteria¨ [13].

Figure 1: Oral microbiota thrive in symbiosis within dental biofilms and under susceptible conditions healthy microflora are outnumbered by pathogenic bacteria. Dysregulated microbial community activates host immune responses. Periodontitis is a biofilm-induced chronic inflammatory disease characterized by deepened tooth pockets and elevated inflammatory mediators that causes destruction of tooth supporting tissues eventually leading to bone loss.

Porphyromonas gingivalis P. gingivalis is a widely studied species and has evolved sophisticated strategies to orchestrate the host immune responses for the growth and survival of the entire biofilm [12]. P. gingivalis is an anaerobic, gram-negative, asac- charolytic bacterium. Although found low in numbers along with other members of the oral microbiota, it has an ability to proliferate into high cell numbers to invade and destruct the oral epithelium through an arsenal of virulence factors [14].

14 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

The key virulence factors of P. gingivalis include gingipains, lipopolysaccharide (LPS), capsule, fimbriae, outer membrane vesicles (OMVs) and proteases. Gingipains are potent trypsin-like proteases secreted by P. gingivalis and are very essential virulence factors. P. gingivalis expresses arginine gingipains (Rgp), encoded by rgp A and rgp B genes, along with lysine gingipain (Kgp) encoded by the kgp gene. These gingipains contribute to about 85% of the total proteolytic activity and play a key role in adherence, colonization, manipulation of host immune responses and nutrition acquisition [15]. Rgps have previously been shown to be essential for Fim A maturation [16]. Gingipains per se act as adhesins for the bacterium to adhere to host and other bacterial cell components which facilitate biofilm formation [17]. P. gingivalis secretes several proteases that include collagenases, glycylprolyl enzyme, peptidyl arginine deiminase (PAD), sialidases, amino and carboxy- peptidases [18]. These enzymes have physiological functions and play an important role in the pathogenesis of periodontitis [18]. P. gingivalis LPS has been widely studied and is shown to be less endotoxic when compared to the classical gram-negative enterobacterial LPS due to the differences in the location, nature and phosphorylated groups of fatty acids in lipid A moiety [19]. Lipid A moiety of P. gingivalis LPS binds to Toll-like receptors (TLRs) 2 and 4 [20]. P. gingivalis is also able to alter the confirmation of lipid A moiety, depending upon the hemin availability in the environment [21, 22]. The capsule of P. gingivalis increases virulence and resistance to phagocytosis [19, 23, 24]. P. gingivalis LPS (O-antigenic part) in some strains of P. gingivalis is masked by a thick capsule, which prevents activation of the alternative complement pathway and subsequent phagocytosis [25]. There are six capsular antigenic serotypes (K1-6) identified in patients with periodontitis [26]. Fimbriae help P. gingivalis in adhesion to host cells during the invasion process. P. gingivalis expresses two types of fimbriae: major fimbriae (Fim A) encoded by fim A gene and minor fimbriae (Mfa 1) encoded by Mfa1 gene. Fim A is responsible for attachment and initiation of colonization, while Mfa 1 is involved in formation and maturation of microcolonies within the dental biofilms [27]. Fimbriae are also highly chemotactic and have haemagglutination activity that contributes to the chronic inflammatory condition [28]. P. gingivalis releases OMVs which are about 20-500 nm. These are cargoes of virulence factors, such as gingipains, LPS, fimbriae and capsule to favor the invasion process by modulating the immune responses from the distal sites of infection [29].

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 15

Oral Biofilms The tooth-associated biofilms play a crucial role in initiation and progression of periodontitis. Bacteria are well protected within biofilms and have been shown to be resistant to antibiotics and clearance by the host immune system [30, 31]. The development of periodontopathic biofilms is proposed to occur in three stages [32]. Early colonizers, including Actinomyces and Streptococci, initiate the biofilm formation by adhering to surfaces in the oral cavity. Adhesins, such as fimbriae and polysaccharides, are used to adhere to the host. The biofilm is then colonized by intermediate colonizers, such as Fusobacterium nucleatum, and serve to enhance co-aggregation with late colonizers that include red complex bacteria. P. gingivalis, which is considered as a keystone pathogen in periodontitis is a late colonizer that facilitates growth and survival of the entire biofilm community [12]. How- ever, it still remains unclear how the bacterial species compete, coexist and synergize their interactions within dental biofilms leading to disruption of the healthy symbiotic state and in causing the chronic inflammatory condition.

Periodontitis and P. gingivalis Associated Systemic Diseases Chronic inflammatory conditions during periodontal disease have been associated with systemic diseases, such as atherosclerosis, diabetes and rheumatoid arthritis [33]. Several studies have reported an association of different clinical parameters of periodontitis, such as the number of teeth, bleeding on probing and pocket depth, with atherosclerotic plaque formation, thickening of the intima, myocardial infarction, angina pectoris and other cardiovascular diseases [34, 35]. Oral pathobionts may gain access to the circulation through bleeding gums at the site of infection and injury [34].

P. gingivalis along with other periodontal pathogens has been identified within atherosclerotic plaque and is widely associated as a potential risk factor in several systemic diseases [36]. In mice studies, P. gingivalis was shown to spread to systemic organs, like heart, liver, spleen and kidney, within 12-24 weeks of infection [37]. Gingipains are released into the circulation and promote platelet aggregation leading to intravascular clotting [38]. Endothelial-derived factors, such as adenosine, prostaglandin and nitric oxide, are released continuously into the circulation protecting from thrombus formation, leukocyte infiltration and adhesion. P. gingivalis dis-

16 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection rupts these mediators in the vessel walls and also modifies low-density lip- oproteins (LDL) to an atherogenic form [39]. Elevated levels of cytokines and chemokines induced by P. gingivalis, such as IL-6, TNF- , TGF- , CXCL-8, prostaglandin E2, CRP and IL-1 , and other inflammatory mediators have also been associated with the progression of atherosclerosisα andβ adverse cardiovascular diseases [40]. β

Periodontitis is also a risk factor for diabetes where P. gingivalis LPS is able to modify glucose, fructose and glycolytic adducts and accumulation of these modified proteins play a central role in chronic diabetes [41]. The elevated levels of TNF- and IL-6 during severe periodontitis were shown to impair intracellular insulin signaling and possibly induce insulin resistance [42-44]. Furthermore,α periodontitis increases the risk for renal diseases, hy- perglycemia, retinopathy and cardiovascular diseases in diabetic patients [42].

Furthermore, there are several studies suggesting an association of periodontitis and P. gingivalis infection with rheumatoid arthritis. The PAD enzymes of P. gingivalis have been shown to citrullinate host peptides leading to the production of antibodies [45]. In mice studies, these antibodies were proven to be pathogenic and induce arthritis [46]. In addition, antibodies against periodontal bacterial heat shock proteins (HSP) cross-react with human HSPs leading to autoimmune responses [47].

Host Immune Responses The host immune system is conferred to protect from continuous microbial challenges in the periodontal biofilms, however, these responses may be destructive and dysregulated during the course of eliminating the invading pathogens. P. gingivalis has evolved effective strategies to evade recognition by antibacterial host effectors, phagocytosis, cytokine, chemokine and antibody activity and thereby, manipulate both the innate and adaptive immune responses [48].

Innate Immune Responses The innate immune cells comprising gingival epithelial cells (GECs), fibroblasts, neutrophils, monocytes/macrophages and dendritic cells (DCs) help in retrieving the host from infection with the involvement of humoral responses from lymphocytes [49]. P. gingivalis has also been shown to invade fibroblasts, DCs, endothelial and smooth muscle cells [50, 51].

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 17

GECs are the first line of the physical barrier and secrete several cytokines and chemokines on encountering a pathogen. Zhao et al., [52] have shown that the expression of IL-6, CXCL-8 and IL-1 in GECs is altered upon interaction with bacteria. Inflammatory mediators such as TNF- , IL-1 , IL-6 and CXCL-8, elicit signaling pathways andβ transcription of genes regulating key pathways during host-pathogen interaction [49, 53]. CXCLα -8 βis a chemokine and functions as a chemoattractant for neutrophils, macrophages and lymphocytes and promotes osteoclast differentiation [54, 55]. Accumulation of neutrophils during dysbiosis at the gingival crevice is a hallmark of periodontitis. Changes in normal neutrophil activity, such as enhanced recruitment and hyperactivity, lead to destruction of periodontal tissues [56]. Neutrophils secrete matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) that degrades host tissues and also membrane- bound RANKL that induces osteoclastic bone resorption [56, 57].

TLRs and protease-activated receptors (PARs) are pattern recognition receptors found on various cell types and are essential in activating several inflammatory processes in response to pathogen and danger associated signals. P. gingivalis LPS and fimbriae are able to activate TLR2 and TLR4 to induce expression of inflammatory mediators [58]. Animal studies have shown that TLR2-/- mice were able to clear recurrent P. gingivalis infection and also resist alveolar bone loss [59]. An altered expression of PAR2 associated with increase in cytokine levels in patients with periodontitis when compared to healthy controls suggests modulation of host responses during a periodontal disease [60, 61].

Inflammatory mediators, such as IL-6, CXCL-8 and TGF- , are widely studied during P. gingivalis infection and periodontitis. IL-6 is an important inflammatory mediator and induces angiogenesis, B cell maturationβ and T cell differentiation [62] and promotes osteoclastogenesis and alveolar bone loss and thereby is a key component of periodontitis [54]. [63, 64]. TGF- , a pleiotropic anti-inflammatory cytokine is involved in several cellular processes and wound healing [62]. TGF- suppresses elevated responses of im-β mune cells, such as B cells, T cells, neutrophils, macrophages and also MMPs, cytokines and chemokines, suchβ as IL-1 , CXCL-8 and TNF- [62, 65, 66]. β α

18 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

P. gingivalis has been shown to tolerate oxidative stress, manipulate host immune cells leading to tissue destruction and can paralyze the host complement system, cytokines, chemokines and phagocytic activity [67-70]. Previous studies have shown that P. gingivalis can utilize the inflammatory exudate as a source of nutrient for survival [14]. P. gingivalis abrogates IL- 6 and CXCL-8 responses which were reverted when using heat-killed P. gingivalis, suggesting a role of gingipains (heat sensitive proteases) in cleaving these major inflammatory mediators.

Adaptive Immune Responses Adaptive immune responses include recognition, memory, antigen presentation and binding to effector molecules to eliminate the pathogens and their toxins. Plasma and B cells are dominant in periodontal lesions which are affected by the severity of the disease [71]. Both B and T cells are essential for the regulation of humoral and cell-mediated responses during host-pathogen interaction [72]. B cell maturation into memory and antibody secreting plasma cells could either be T cell dependent or T cell independent. Further- more, increase in RANKL secretion was also shown to induce T cell-mediated antigen-specific responses [73]. However, the secreted antibodies act as effector molecules in activating cell-mediated immunity (major histocom- patibility complexes). T cell extracts from patients with periodontitis showed decreased response to stimuli [74], while lymphocyte reactivity was restored after periodontal therapy [75]. B cells have effector functions and secrete reactive oxygen metabolites, cytokines, lysosomal components and nitric oxide and also aid in antigen presentation to professional phagocytes, such as neutrophils, monocytes and macrophages. B cell-deficient mice were shown to have increased susceptibility to bone loss and abscess formation during P. gingivalis infection [76]. B cells mature into plasma cells that secrete immunoglobulins, which are key components of adaptive immune responses and with their unique and versatile structure they recognize pathogens and their antigens leading to elimination.

An imbalance in Th1 and Th2 responses observed in patients with periodontitis is correlated with severity of the disease and is probably due to proteolytic degradation of several host inflammatory mediators [77]. Thereby, an impaired immune response with an altered cytokine activity associates periodontitis to several systemic inflammatory diseases. However, P. gingivalis is able to inactivate IL-4 and IL-5, thereby hinder B cell maturation

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 19 and antibody secretion [77]. P. gingivalis alters T cell responses by disrupt- ing IL-2 secretion [78] and impairs leukocyte function by degradation of TNF- [79].

Figure 2: A schematic representation of immune cell activation by P. gingivalis. The microbial invasion into resident epithelial cells and fibroblasts induces the release of inflammatory mediators and chemoattracts immune cells, such as neutrophils, lymphocytes and monocytes. Prolonged chronic inflammatory state at the gingival crevice leads to degradation of tooth supporting tissues and alveolar bone loss.

Antibodies Antibodies are glycoproteins secreted by plasma cells in response to an antigen that is considered as a threat to the host and antibodies either neutral- ize the target antigen or elicit an immune response to recruit effector molecules to eliminate the invaded pathogen [80]. There are five classes of antibodies in higher mammals comprising IgA, IgD, IgE, IgM and IgG, each having a distinct structure, size, amino acid composition, charge, target and mode of action. Briefly, IgA responses are critical in mucosal immunity, IgD activates basophils and mast cells [81], IgE responses are associated with allergies and parasitic infections, IgM is secreted as a primary response to

20 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection an antigen followed by IgG secretion during prolonged or subsequent responses to pathogens or their toxins [82].

The exquisite specificity and diversity of antibodies have made them valua- ble tools in diagnosis, therapy and research. The advent of hybridoma technology has revolutionized antibody production technology and production of monoclonal antibodies became feasible. Monoclonal antibodies developed from single B cell clones are widely used for their specificity, whereas polyclonal antibodies are developed from B cells from different lineages, thereby expressing varying specificity [83, 84]. Recombinant antibody technology is more popular in recent times, where specific antibody fragments could be engineered. Synthetic antibodies are now synthesized and designed creating diverse antibody libraries [85].

Diagnosis, Treatment and Prevention of Periodontitis Oral examinations including a complete patient history (determine the source, hygiene, associated systemic diseases, severity of pain), oral casts (to determine the margins of gingiva), radiographs (determine tooth anomalies, pathologic lesions, severity of bone destruction), lymph node examination (determine signs of infection, inflammation or malignancies), bleeding on probing (determine signs of inflammation) are currently practiced during initial diagnosis of periodontal diseases [86].

Treatment of periodontitis focuses on elimination of plaque, calculus or tartar deposits to restore the establishment of a healthy, well-functioning periodontium. The treatment phase includes scaling and root planning (removing dental biofilms and excavation of caries), antibiotic therapy, flap surgery (removing the tartar deposits by lifting back the gums) and bone or tissue grafts (bone and connective tissue regeneration) [86]. It has been reported that almost 20 different antimicrobial treatments have been utilized to treat recurrent periodontitis, however, it is extremely difficult to control the huge bacterial load in biofilms with acceptable doses of antibiotics [87, 88]. Antibiotics, such as penicillin, amoxicillin, tetracycline and metronida- zole, are currently prescribed to combat oral infections in dental practice [89]. Antibiotic prescriptions account for careful consideration of age, systemic diseases and ailment and the impact of prolonged periodontal therapy in patients. Studies have not only reported the emergence of antibiotic resistant strains but also have shown the transmission of resistance to other bacterial species within the dental plaque [90, 91]. However, it is speculated

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 21 that total plaque removal proposed in different experimental studies is not feasible in clinical practice [86, 87]. This together with an increase in antibiotic resistance suggests a need for alternatives for treating P. gingivalis infection and periodontitis and prevent an inflammatory burden leading to several associated systemic diseases.

Lactobacillus and Bacteriocins Lactobacillus spp. are rod-shaped, gram-positive bacteria capable of fer- menting carbohydrates and produce lactic acid as major metabolite. Several strains of Lactobacillus are a part of urogenital, gut and salivary microflora. They are widely known for their beneficial activities and are used as probiotics. Probiotics are live microorganisms that have favorable effects on the host when ingested in adequate amounts [92]. The major probiotic mechanisms include increased adhesion to mucosal layer, competitive exclusion of pathogens, enhancement, and maintenance of epithelial barrier functions, secretion of antimicrobial substances and modulation of host immune responses [92]. Lactobacillus spp. are also known for their beneficial activities on host homeostasis, however, they may be outnumbered by gram-negative pathogenic bacteria, such as P. gingivalis, in the oral cavity during disease progression. The use of lactobacillus for its anti-pathogen properties has been reported in previous studies and it was shown that L. rhamnosus and L. salivarius suppress the growth of P. gingivalis [93]. It was also demonstrated that Lactobacillus spp. alter the adhesive ability of oral pathogens and also aid in preventing cariogenic bacteria in the oral cavity [94].

L. plantarum is active at pH below 3.2 and is a natural inhabitant of the human gastrointestinal tract [95]. The bacterium is able to ferment sugars homolactically or heterolactically through Embden-Meyerhof-Parnas pathway (EMP) or the phosphoketolase pathway [96]. They have sodium proton pumps, alkaline shock proteins, catalases, reductases and peroxidases, which help in maintaining intracellular pH, tolerance to low pH and meet- ing oxidative stress and thereby are more flexible to hostile environmental niches [96]. Their ability to produce peroxide radicals, peptidases and bacteriocins render them beneficial against pathogens. Furthermore, bacteriocin producing L. plantarum strains have been reported to modulate host immune responses [97].

22 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

Bacteriocins are antimicrobial peptides that could be easily synthesized and are cationic substances that enable them to bind to negatively charged microbial membranes. They are secreted by several bacterial species and have gained much attention in recent years for their antimicrobial properties. Bacteriocin production gives a selective advantage for the producer strain to survive within the competitive niche of a polymicrobial environment. Several studies suggest a narrow spectrum activity of bacteriocins secreted by the producer strains against closely related bacteria. However, some bacteriocins have broad-spectrum antimicrobial activities, such as Plantaricin from L. plantarum was has been shown to be effective against the food- borne pathogen, such as Listeria monocytogens [98]. They are broadly clas- sified into three classes depending on size, structure, mode and mechanism of action [99]. Class I bacteriocins comprise lantibiotics that contain post- translationally modified peptides with unusual amino acids, such as -me- thyl-lanthionine and lanthionine. Class II bacteriocins are usually composed of two peptides that dimerize to induce pore formation and can resistβ pro- teolysis, heat and pH changes. Class III bacteriocins are comprised of larger heat-sensitive peptides.

Bacteriocins are able to form pores upon binding to bacterial membranes and could also inhibit cell wall synthesis [100]. The interaction of bacteriocins with the bacterial membrane is widely studied and two distinct mechanisms are proposed using three possible models – transmembrane pore formation (barrel-stave model and toroidal model) and disruption of membrane (carpet model) [101]. Some bacteriocins, such as nisin (class I lantibiotics), target lipid II, thereby interfering with peptidoglycan synthesis in bacteria. Other bacteriocins bind to lipid II to initiate pore formation leading to loss of membrane potential and ultimately cause bacterial lysis [102]. Class II bacteriocins, such as Lactococcin A, have been shown to interact with mannose phosphotransferase system (Man-PTS) to induce pore formation [103].

Plantaricin (PLNC8 ) is a class IIb bacteriocin secreted by L. plantarum NC8 and has been recently genetically characterized [104, 105]. The authors of αβthese studiesαβ suggest that both peptides are required for full plantaricin activity. They also reported that an immunity protein in plantaricin protects the producer strain from the toxicity of the secreted bacteriocin [105].

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 23

Figure 3: Three different models are proposed for the possible interaction of bacteriocins with microbes [106]. In the barrel-stave model, peptides insert themselves perpendicularly into the microbial membranes and form barrel-like bundles. In the carpet model, peptides cover the microbial membrane in a carpet-like cluster and an abrupt lysis of the microbe is induced. Lastly, in the toroidal pore model, peptides insert into the membrane and cluster into bundles that span through the membrane to act on the intracellular targets to exhibit killing activities. The figure shown is modified and used with permission from [101].

24 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

Aims This thesis focuses on developing and testing novel tools for diagnosis and prevention of P. gingivalis infection and periodontitis. The specific aims of the thesis are to:

• develop anti-P. gingivalis antibodies and use these antibodies in immunodetection in clinical samples

• elucidate the antibacterial properties of lactobacillus species and the two peptide bacteriocin PLNC8 on P. gingivalis

• study the effects of PLNC8 on GECsαβ and to demonstrate their possible role in modulating the innate and inflammatory responses against P. gingivalis αβ

• study the effects of anti-P. gingivalis antibodies on GECs and to demonstrate their ability in combination with PLNC8 in suppressing P. gingivalis-mediated innate and inflammatory responses αβ

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 25

Methods

Study Participants and Ethical Approval Patients with severe periodontitis (n=30, mean age 55 yrs, 13 females and 17 males) were recruited at the Center of Oral Rehabilitation, Public Dental Health Care, County Council of Östergötland, Linköping, Sweden. Gingi- val crevicular fluid (GCF) samples were collected from the four deepest periodontal pockets. Supragingival plaque was removed and the tooth was allowed to air dry. Periopaper paper strips were inserted subgingivally to about 1–2 mm depth. GCF was collected and the volume absorbed by the strips were determined using a Periotron 8000 (Oraflow Inc, New York, USA). GCF from all the four sites were then pooled into diluent buffer (Quantikine Human HGF immunoassay, R&D Systems, Minneapolis, MN, USA) for each of the patient and were frozen at -20°C until analysis. GCF samples from age and sex matched periodontally healthy controls (n=30) were included in the study.

Recruitment of patients and the protocol were in agreement with Helsinki declaration, the regional ethical committee in Linköping approved the study (2010/307-31) and all participating patients gave written informed consent.

Bacterial Strains and Bacteriocins P. gingivalis is an anaerobic rod-shaped bacterium that forms black-pig- mented colonies on blood agar plates due to hemin accumulation on cell surfaces [107]. P. gingivalis wild-types (WT) ATCC 33277, W50 and 381 and their mutants - E8 (W50 derived arginine gingipain mutant) and K1A (W50 derived lysine gingipain mutant), DPG-3 (381-derived major fimbriae mutant) and KRX-178 (381-derived minor fimbriae mutant) strains were used. W50, E8 and K1A strains were a kind gift from Dr. M. Curtis, Queen Mary’s School of medicine and dentistry, London, UK, while 381, DPG-3 and KRX-178 strains were provided by Prof. Genco RJ and Prof. Sharma A, School of dental medicine, University of Buffalo, State University of New York, USA.

L. plantarum and L. brevis are widely studied probiotic strains of Lactoba- cillus sp. L. plantarum NC8, L. plantarum 44048 and L. brevis 30670 strains (Culture collection, University of Gothenberg, Sweden) were used in

26 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection study II. L. plantarum NC8 is a model strain used widely in metabolic en- gineering, fermentation processes and for the production of bacteriocins, such as PLNC8 .

PLNC8 is aαβ novel class IIb, two-peptide bacteriocin secreted by L. plantarum NC8, was characterized by Maldonado et al., [97]. The amino acid sequenceαβ s of the two peptides used in our studies are given below:

PLNC8 - DLTTKLWSSWGYYLGKKARWNLKHPYVQF PLNC8 - SVPTSVYTLGIKILWSAYKHRKTIEKSFNKGFYH [97] α Cell Culturingβ Leukocytes were isolated using gradient centrifugation method from a do- nor who previously has been treated for severe periodontitis. Cells were cultured overnight in L-15 medium (ATCC, Borås, Sweden), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, Sweden). Cells were then stimulated with heat-killed P. gingivalis ATCC 33277 to develop anti-P. gingivalis antibodies in study I.

Human GECs (Ca9-22 cell line, JCRB0625) were cultured in Dulbecco’s modified minimum essential medium (DMEM, Thermo Fischer Scientific, Sweden), supplemented with 10% FBS (Thermo Fischer Scientific, Sweden). GECs are a part of the innate immune system and actively secrete cytokines upon encountering a pathogen and are thereby able to recruit immune cells to the site of infection.

Antibody Production Leukocytes stimulated with P. gingivalis were grown in L-15 medium for 3 weeks after which antibodies were recovered from culture supernatants ap- plying series of sterile filtrations, centrifugation and enrichment steps using 150 KDa amicon cut-off filters (Millipore, France) in study I. Anti-P. gingivalis antibodies were of IgG class as the donor previously has sustained the P. gingivalis infection. This was confirmed by obtaining bands at 50 and 25KDa that correspond to the heavy and light chains, respectively of an IgG molecule.

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 27

Flow Cytometry Flow cytometry is used to study the composition of cell suspensions and allows multiparametric analysis. This technique is utilized in the fields of biomedicine to study surface antigens, intracellular antigens, apoptosis, cell adherence, pigmentation, protein expression and localization. It has a wide range of clinical applications, such as monitoring cell counts in patients with leukemia, lymphoma or HIV, and properties of erythrocytes, leukocytes and platelets [108]. Flow cytometry measures the optical and fluorescence characteristics of a single cell. The forward light scatter measures the size or morphology of the cell, while the side scatter measures the integrity or internal complexity of the cell. Several fluorescent dyes are available to inter- calate or bind to different cellular components, including DNA and RNA.

Lymphocytes isolated from a donor for antibody production were analyzed using flow cytometry before and after stimulation with P. gingivalis (study I). An antibody cocktail with markers for CD19 (B cells), CD3 (T cells), CD16/56 NK cells, CD45 (lymphocytes) tagged with fluorochromes APC, FITC, PE, and Per CP, respectively was used. BD FACS Canto TM (BD Biosciences, Stockholm, Sweden) instrument was utilized to identify the cell composition and the results were plotted on a logarithmic scale by gating CD45+ lymphocytes for analysis.

Transmission Electron Microscopy Transmission electron microscopy (TEM) is a technique to visualize minute details of the specimens and has been widely used to study cellular and microbial structures in medical and biological research. Electrons are trans- mitted through a vacuum and are focused into electromagnetic lenses into a very thin beam that passes through the specimen. The unscattered electrons hit a fluorescent screen giving rise to an image of the sample displaying different parts in varied darkness depending on the density. TEM produces 2D images of the specimen in very high resolution and requires a very thin section of the sample that is mounted on a grid. Furthermore, the heavy metal staining (lead, uranium) of samples can help to intensify the electron deposits on the sample in order to visualize cell or protein regions [109, 110].

In study I, TEM (JEOL 1230, Jeol Ltd, Tokyo, Japan) was used to visualize the binding interaction of anti-P. gingivalis antibodies to P. gingivalis ATCC

28 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

33277. Osmium tetroxide and nano-gold secondary antibody staining were included during sample preparation. The effect of PLNC8 in distorting the membrane and causing lysis of P. gingivalis ATCC 33277 was visualized using TEM (Hitachi HT 7700, Tokyo, Japan). In study II, theαβ pelleted sections of the bacterium were counter stained with uranyl acetate and lead citrate.

Confocal Microscopy Confocal microscopy facilitates fluorescence imaging with great sensitivity, contrast and resolution in the examination of cell and tissue structures. It is advantageous over light microscopy in minimizing the out of focus blur effect due to illumination of light through thick sections of the sample. This is overcome by having the same focus for the light source, specimen and detector in confocal microscopy [111]. In study I, antibody secreting CD 38+ plasma cells were visualized using confocal microscopy. Plasma cells were below the detection limits in flow cytometry and therefore we used FITC-CD38 antibodies and DAPI to visualize plasma cells with LCM Zeiss confocal microscope (Department of Pathology, Linköping University, Swe- den).

Surface Plasmon Resonance Analysis Surface plasmon resonance (SPR) is a label-free, real-time biosensor detection system and is used in the food and pharmaceutical industries for detection of food-borne pathogens and their toxins, insecticides, antibiotics, hor- mones, vitamins, additives and adulterants in food samples [112, 113]. The method is widely used to determine kinetic parameters, specificity and affinity of an immobilized ligand (antibodies, enzymes, peptides, receptors) to a sample analyte (cell or microbial proteins, peptides, antigens, polysaccharides, substrates, drugs). Ligands, such as antibodies that are immobilized on biosensor chip interact with the analyte, such as antigens in the sample. A specific interaction between ligand and analyte gives rise to a change in the angular position of an optical signal due to the difference in the refractive index which can be measured in response units (RU). These interactions could be monitored in real-time and for most proteins, 1 RU corresponds to a surface concentration of 1 pg/mm2 of the immobilized protein [114].

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 29

In paper I, the binding interaction of anti-P. gingivalis antibodies with P. gingivalis in GCF samples of patients with periodontitis and healthy controls was studied. Anti-P. gingivalis antibodies were immobilized to CM-5 sensor chip. The interaction of PLNC8 and anti-P. gingivalis antibodies with P. gingivalis wild-types (ATCC 33277 and W50), was verified using SPR in study II. Bia-evaluation softwareαβ (GE healthcare, Sweden) was used to obtain the response units (RU).

Figure 4: Kretschmann prism model of light refraction obtained from ligand interaction with immobilized proteins. The sensor chip has a gold film adhered to a dex- tran matrix and is immobilized with a ligand, such as an antibody of interest. A specific interaction of immobilized ligand to sample analyte causes a change in refractive index due to the change in mass bound, which displaces the angular position of an optical signal from I to II as shown in the figure. A typical response curve during these interactions could be visualized in real-time.

DNA-DNA Hybridization Technique DNA-DNA hybridization technique is a molecular tool and is used to study genetic similarities between bacterial species. Labeled DNA of a bacterial

30 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection species is mixed with an unlabeled bacterial DNA pool and compared. La- beled and unlabeled DNA are allowed to form hybrid double-stranded DNA, where the DNA of closely related species bind more firmly and are hard to be separated using melting procedure compared to hybrid DNA formed between distantly related DNA samples. The results are then combined to determine the degree of genetic similarity between the organisms. In paper I, DNA-DNA hybridization technique was used in the detection of P. gingivalis in GCF samples for validating the results obtained in SPR analysis.

Enzyme-linked Immunosorbent Assay Enzyme-linked immunosorbent assay (ELISA) is a popular technique widely used to detect proteins, peptides or antigens of interest. In a sandwich ELISA type, the capture antibody is coated on a 96-well microtiter plate and nonspecific binding sites are blocked upon which samples are added. A specific primary antibody that binds to the agent of interest is added. An enzyme-linked secondary antibody which can bind to the primary antibody is added. A suitable chromogenic substrate is used to generate a detection signal in proportion to the amount of the specific agent that binds to the capture antibody and the optical density is measured at 450nm.

In paper I, we elucidated the IgG subclass responses to heat killed P. gingivalis ATCC 33277 by using an IgG subclass assay kit (Invitrogen, Stock- holm, Sweden). The concentrations of IgG 1-4 among the anti-P. gingivalis antibodies were measured and compared to human serum control provided by the manufacturer. In paper III and IV, we measured IL-6, CXCL-8 and

TGF- 1 levels in culture supernatants of GECs challenged with P. gingivalis, with and without PLNC8 or anti-P. gingivalis antibodies or in their com- β bination. Human IL-6, CXCL-8 and TGF- 1 ELISA kits (Biolegend, San Di- ego, USA) were used. αβ β Growth Factor Array Membrane-based antibody arrays are gaining popularity in recent times for their wide use in screening for the expression of several target samples, such as cytokines, growth factors, proteases, chemokines, receptors and other proteins. The technique is based on a sandwich immunoassay, where a panel of capture antibodies is printed on a nitrocellulose membrane which then is processed as a chemiluminescent readout. Detection signals are analyzed in

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 31 a digital image processor and fold-changes for each detected protein is semi- quantified.

In studies III and IV, growth factors involved in cell proliferation and wound repair were semi-quantified in culture supernatants of GECs challenged with P. gingivalis with and without PLNC8 or anti-P. gingivalis antibodies or in their combination. We used a growth factor array kit (Raybiotech, Ga, USA) and membranes were semi-quantifiedαβ with ImageJ software (National Institute of health, MD, USA).

Liposome Model A Liposome is a spherical vesicle artificially constructed with one or more phospholipid bilayers and is used to administer nutrients or pharmaceutical drugs in biomedical research [115]. The phospholipids POPS and POPC were used to construct a liposome model in study II to elucidate the effects of PLNC8 and PLNC8 peptides. The ratio of these phospholipids which resemble the membrane composition of P. gingivalis was analyzed by measuring the hydrodynamicα radiusβ and zeta potential using dynamic light scat- tering. Hydrodynamic radius measures the size of a macromolecule in solution through the diffusional properties of the sample of interest, while zeta potential is the minimum electrokinetic potential to attract the oppositely charged particle.

Carboxyfluorescein (CF) is a fluorescent dye which is used as a tracer agent in labeling and sequencing nucleic acids. In paper II, liposomes were loaded with CF and the leakage of the dye was studied upon treatment of liposomes with PLNC8 and peptides.

Antibacterialα Activityβ of PLNC8 αβ Sytox green is a high-affinity nucleic acid stain and can penetrate only through the cells with damaged plasma membranes. It could be used for both gram-positive and gram-negative bacteria. When bacteria is incubated with sytox green dye, the nucleic acids of the dead cells fluoresce in bright green. The staining is a simple and quantitative dead-cell indicator and fluorescence microscopes, fluorometers, fluorescence microplate readers or flow cytometers can be used for detection. The dye can also be used as a DNA counterstain for chromosome labeling in fixed cells and tissues.

32 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

P. gingivalis ATCC 33277 or W50 treated with or without PLNC8 or anti-P. gingivalis antibodies or their combination were visualized using sytox green dye (Invitrogen, Stockholm, Sweden) in studies III andαβ IV. Olympus BX41 instrument (Core facility, Örebro University, Sweden) was used to visualize the samples.

Circular Dichroism Spectroscopy Circular dichroism is used in evaluating the secondary structure and binding and folding properties of proteins [116]. Briefly, when a polarized light passes through suitable prisms and filters, an electromagnetic radiation con- sisting of an electric and magnetic field oscillate perpendicular to one an- other generating a sinusoidal wave in a specific direction. The wave traces out in circles that rotates clockwise/right side and the other in anticlock- wise/left side. When asymmetric molecules interact with light, they absorb right and left-handed circularly polarized light to a different extent and have different refractive indices for the two waves.

In study II, the secondary structure of PLNC8 and peptides was predicted using circular dichroism spectroscopy. The changes in the structure of bacteriocin peptides when in contact with liposomesα β membranes were predicted using Chirascan spectropolarimeter (Applied Photophysics, UK) and Savitzky-Golay algorithm was used for processing the obtained curves.

Western Blotting Western blotting is widely used to detect specific proteins in samples. The proteins are first separated using gel electrophoresis and then transferred to a PVDF or nitrocellulose membrane. The membrane is incubated with a primary antibody that is specific to the target protein followed by a suitable HRP-conjugated secondary antibody against the species-specific portion of the primary antibody. The membrane is then incubated with a suitable substrate to visualize the protein bands.

In paper III, the p-akt expression in GECs treated with or without PLNC8 during P. gingivalis infection was studied. Rabbit anti-p-akt primary antibodies (Cell signalling technology, Sweden) and rabbit anti-GAPDH pri- maryαβ antibodies were used, HRP-anti-rabbit secondary antibodies (San- tacruz biotechnology Ltd, USA) and Luminata forte western blot substrate solution were used to visualize the bands for p-akt and GAPDH at 60 and 37 KDa, respectively, in chemidoc MP imager (Bio-rad, Sweden).

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 33

Statistics Chi square test was used to compare the results obtained in SPR, DNA- DNA checkerboard analysis and real-time PCR in study I. The chi square distribution table for degree of freedom 1 was used to reject the null hypothesis corresponding to no statistical differences in results obtained in these methods. One way ANOVA test (Tukey’s multiple comparison test) was used for the comparisons between the different treatments in studies II, III and IV and * p<0.05; ** p<0.01; *** p<0.001 was used to determine statistical significance. All the data were analyzed using Graph Pad prism 5.0 (GraphPad Software, California, USA).

34 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

Results and Discussion

Activation of Leukocytes and Induction of Secondary Immune Responses (Study I) In study I, we aimed to develop anti-P. gingivalis antibodies in vitro and to investigate their role in the detection of bacteria in clinical samples using SPR-based biosensor. Furthermore, we elucidated the specificity of antibodies and their role in attenuating wild-type and mutant strains of P. gingivalis.

Leukocytes isolated from a donor were challenged with heat-killed P. gingivalis ATCC 33277 to trigger the secondary immune responses in vitro. Previous studies have suggested a role of T cell functions in activation of B cells and that cytokines secreted by T cells promote B cell differentiation into antibody secreting cells [117]. We have shown that there is an increase in T cell population (CD3+) in leukocytes after bacterial stimulation. Anti- body secreting plasma cells (CD 38+) were visualized after 2 and 3 weeks of infection by P. gingivalis indicating a T cell-mediated B cell maturation.

Re-infection by a specific pathogen activates secondary immune responses leading to IgG secretion in a simplified antibody secretion process [118]. We showed that anti-P. gingivalis antibodies yielded bands at 50 KDa and 25 KDa in SDS-PAGE analysis, which correspond to heavy and light chains, respectively, of an IgG molecule [119]. Studies have reported the importance of IgG subclass responses within the host, where IgG 1 and IgG 3 activate the complement system, IgG 2 recognizes glycolipid antigens and encapsu- lated bacterial strains, while IgG 4 is elicited upon persistent exposure to an antigen [119]. The different IgG subclasses induced by various virulence factors of P. gingivalis determines the biological effects, such as P. gingivalis whole cells elicit IgG 1 and IgG 3 [120], purified P. gingivalis fimbrial protein elicits IgG 3 or IgG 4 responses [121, 122], while P. gingivalis LPS induces IgG 2 dominant responses [122, 123]. Anti-P. gingivalis antibodies developed in our study are of IgG class and the increased levels of IgG 1 and IgG 2 shown in our study correspond to responses to P. gingivalis or it’s LPS.

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 35

Detection and Attenuation of P. gingivalis by Anti-P. gingivalis antibodies (Study I) In study I, the specificity of anti-P. gingivalis antibodies was investigated by immobilizing the antibodies in a SPR-based biosensor. Several studies have reported the use of SPR-based biosensors in the detection of food-borne pathogens, such as Salmonella typhimurium [124], Escherichia coli and enterotoxin B [125, 126], Yersinia enterocolitica [127] and Listeria monocytogenes [128]. P. gingivalis in GCF samples of patients with periodontitis was detected by using anti-P. gingivalis antibodies and the results obtained were validated using DNA-DNA-checkerboard analysis and real-time PCR. Furthermore, the interaction of antibodies with P. gingivalis was observed using ELISA and TEM. Our observations suggest a specific interaction of in vitro developed antibodies with P. gingivalis.

We have shown that antibodies were able to bind to wild-type and mutant strains of P. gingivalis in plasma samples indicating that the antibodies are directed against an epitope common to these tested strains. Furthermore, pre-incubation of these plasma samples with antibodies decreased the binding responses to immobilized antibodies indicating a role of antibodies in attenuating P. gingivalis infection. We have recently shown that antibodies developed in vitro could detect specific bacteria in polymicrobial prepara- tions which suggest their role in the real-time detection of specific bacterial species of interest [129]. This approach may possibly decrease the antibiotic consumption and thus, prevents resistance and harmful effects on host microbiota. Our study suggests a role of anti-P. gingivalis antibodies in iden- tifying the infection to ensure timely onset of periodontal treatment and a decrease in the use of broad-spectrum antibiotics

Antibacterial Activity of Lactobacillus spp. and PLNC8 αβ (Study II) Several Lactobacillus spp. are a part of healthy microflora and have been widely studied for their antimicrobial properties. Lactobacillus spp. including L. casei, L. rhamnosus, L. salivarus, L. gasseri, L. plantarum and L. brevis have previously been reported in the oral cavity [130]. The interaction between Lactobacillus spp. and periodontal pathogens are sparsely studied. In study II, we have elucidated the antibacterial effects of L. brevis 30670, L. plantarum NC8 and L. plantarum 44048 on P. gingivalis. We show that L. brevis and L. plantarum have different effects on P. gingivalis. Both the strains of L. plantarum significantly inhibited P. gingivalis growth,

36 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection while L. brevis had no effects in our co-culture experiments. It was interesting to observe that K1A (Kgp mutant) was more susceptible to growth in- hibition by L. plantarum compared to the parent wild-type P. gingivalis W50 and E8 (Rgp mutant), suggesting the importance of Kgp in P. gingivalis survival. Similar inhibiting effects were obtained when using cell-free culture supernatants of L. plantarum strains, proposing that of secretory AMPs that are effective in suppressing P. gingivalis growth.

Maldonado et al., [104] have characterized a novel class IIb bacteriocin, PLNC8 , from L. plantarum NC8 and we hypothesized that PLNC8 could possibly be involved in the L. plantarum-mediated suppression of P. gingivalisαβ. Hence, further experiments were carried out to test the antibac-αβ terial effects of PLNC8 . Pore formation is one of the key mechanisms of bacteriocins [131] and we show that PLNC8 is able to rapidly damage the outer membrane of P.αβ gingivalis using the fluorescence DNA stain, sytox green. Furthermore, the distortion of P. gingivalisαβ outer membrane and leakage of cellular contents was clearly demonstrated using TEM. A liposome model was used to investigate the interaction of PLNC8 and with lipid membranes. We show that PLNC8 rapidly interacts with lipid membrane and is able to damage the membrane integrity. Thisα couldβ possibly be due to an initial electrostatic interactionαβ between cationic peptides and anionic membranes, which is a common mechanism reported in several AMPs [132]. We showed that both the peptides were able to interact with lipid membranes, however, the effects of PLNC8 were more pronounced. Moreover, PLNC8 and lack a definite secondary structure in suspen- sion, however, they were shown to undergo a structuralβ transition on interaction with liposomesα formβing a -helix and a -sheet, respectively.

Effects of PLNC8 αβ per se on GECsα and Duringβ P. gingivalis Infection (Studies III & IV) The possible risk factors associated with infection by P. gingivalis, a key stone periodontopathogen, makes it an immediate concern in looking for novel strategies in treating periodontal disease and we suggest that this could be achieved by using PLNC8 . The antimicrobial properties of bacteriocins have gained much attention as promising alternatives to antibiotics. The ability of bacteriocins in αβtargeting bacterial membranes renders them hard to develop resistance against as it is quite an expensive process for microbes to modify their basic structural integrity [131]. Furthermore, the beneficial effects of bacteriocins, such as their potency, low cytotoxicity

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 37 and both broad and narrow spectrum activity are used in food preservation industry to inhibit the growth of pathogens, as well as, in prevention and treatment of diseases in veterinary healthcare [133, 134].

In study III, we demonstrated the effects of PLNC8 per se on GECs and in the regulation of cellular responses during P. gingivalis infection. Growth factors are used as therapeutical agents and have beenαβ shown to be effective in the reconstruction of cementum, restoration of periodontal ligaments and tissue regeneration [135-137]. We show that PLNC8 per se dose-dependently promotes cell proliferation and induces several growth factors, such as platelet-derived growth factor (PDGF), insulin-αβlike growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), EGF receptor (EGFR), TGF- , TGF- 3, vascular endothelial growth factor D (VEGF-D), placental growth factor (PLGF), IGF-binding protein 4 (IGFBP- 4) and G-CSF in GECs andα severalβ of these growth factors have previously been reported to be involved in repair and regeneration of periodontal tissues [135, 137]. Furthermore, PLNC8 increases the expression of TGF-

1, which is an anti-inflammatory cytokine and plays an important role in cell proliferation [138]. Importantly, PLNC8αβ showed no cytotoxic ef- βfects on GECs in all the tested concentrations of the peptides, which along with other mentioned factors, suggests their possibleαβ role in prevention and treatment of P. gingivalis infection.

We studied the effects of PLNC8 on P. gingivalis infected GECs and show that PLNC8 antagonizes the cytotoxic effects of the bacterium. P. gingivalis cleaves host immune mediatorsαβ to paralyze immune signalling, and thereby successfullyαβ colonizes and invades the host cells. Gingipains have previously been reported to cleave IL-6 and CXCL-8 [58] and furthermore P. gingivalis induces TGF- 1 that downregulates CXCL-8 levels [136]. However, these effects of P. gingivalis on immune mediators were not reverted by PLNC8 and this mayβ be due to the potent activity of P. gingivalis gingipains. It is possible that gingipains proteolytically cleave and thus inactivate PLNC8 αβ . However, these effects are yet to be investigated.

Interestingly, PLNC8αβ extended immunomodulatory and antibacterial effects on GECs and P. gingivalis. However, PLNC8 suppressed the release of several growth factorsαβ triggered by P. gingivalis in GECs. A previous report has indicated high concentration but a lowαβ biological activity of hepatocyte growth factor (HGF) in patients with periodontitis [139]. We

38 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection speculate that the P. gingivalis-induced expression of several growth factors as a response to host-pathogen interaction does not necessarily correlate with an increased biological activity of these factors. However, the biological activity of P. gingivalis-induced GECs growth factors, need to be determined in future studies.

Effects of Anti-P. gingivalis Antibodies on GECs and During P. gingivalis Infection (Study IV) In study IV, we studied in vitro effects of anti-P. gingivalis antibodies in P. gingivalis infected GECs. We show that anti-P. gingivalis antibodies significantly decreased the cytotoxic effects of P. gingivalis on GECs and thereby increased cell viability. Although antibodies could not restore the P. gingivalis-mediated cleavage of IL-6 and CXCL-8, there was a significant increase in TGF- 1 levels. We suggest that anti-P. gingivalis antibodies opso- nize P. gingivalis leading to a more controlled handling of the bacterium, β and thereby decreased cytotoxic effects by the accumulation of TGF- 1.

P. gingivalis colonization increases the stress on GECs which actively secreteβ several cytokines, chemokines and growth factors that could activate other immune cells and their generation of inflammatory mediators [140]. Inter- estingly, we show that P. gingivalis induces several growth factors in GECs and pre-incubation with anti-P. gingivalis antibodies modulate these effects. Growth factors, such as IGFBP (2, 3 and 4), PDGF, VEGF-R, PLGF, IGF- 1R, stem cell factor (SCF) and granulocyte colony stimulating factor (G- CSF), were induced during P. gingivalis infection in GECs, however, these effects were antagonized by anti-P. gingivalis antibodies. In addition to cell proliferation, growth factors have previously been shown to induce chemotaxis of effector cells and expression of MMPs, contributing to exaggerated inflammatory conditions [137, 141]. Therefore, the suppression of P. gingivalis-induced growth factors in GECs by anti-P. gingivalis antibodies may help in controlling inflammatory responses and prevent tissue degradation.

Several studies have shown increased levels of P. gingivalis-specific antibodies in patients with periodontitis and reported that the antibodies are a potential risk for systemic diseases [142, 143]. However, our study demon- strates a protective role of anti-P. gingivalis antibodies during P. gingivalis infection of GECs.

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 39

Combined Effects of Anti-P. gingivalis Antibodies and PLNC8 αβ on P. gingivalis-infected GECs (Study IV) Our findings demonstrating antibacterial effects of anti-P. gingivalis antibodies and PLNC8 , respectively, in attenuating P. gingivalis led us to study the effects of these agents in combination and evaluate their possible synergistic action in αβmodulating cellular responses induced by P. gingivalis in GECs. However, we found that a combination of antibodies and PLNC8 is not more effective on P. gingivalis, compared to the individual effects of these agents. The combination of anti-P. gingivalis antibodies and PLNC8αβ decreased the cytotoxic effects of P. gingivalis in GECs, however, only a trend in increased cell viability was observed. Furthermore, a combinationαβ treatment with antibodies and PLNC8 did not alter the P. gingivalis-mediated effects on cytokine expression and was also less effective in modulating P. gingivalis-induced release of growthαβ factors. Interest- ingly, our data indicate that PLNC8 and anti-P. gingivalis antibodies compete with each other in binding to P. gingivalis. The ability of PLNC8 in lysing P. gingivalis by rupturing αβthe outer membrane was diminished when P. gingivalis was pre-incubated with antibodies. We speculate that αβantibodies and PLNC8 block their potential binding sites on the bacterium, resulting in decreased efficiency when used in combination. αβ

40 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

Conclusions The main findings and conclusions of the thesis are as follows:

• Anti-P. gingivalis antibodies are easily developed in vitro and may be used for efficient detection of P. gingivalis infection in patients with periodontitis, while the attenuating effects of these antibodies suggest a role in passive immunization to prevent periodontitis and associated diseases.

• L. plantarum NC8 and 44048 are effective in suppressing P. gingivalis. Lysine gingipain seems important for P. gingivalis survival. PLNC8 is effective in lysing P. gingivalis.

• PLNC8 suppressesαβ P. gingivalis infection in GECs and promotes cell proliferation by inducing growth factors. The anti- bacterialαβ and proliferative effects of PLNC8 suggest a potential ability of these peptides in prevention and treatment of P. gingivalis infection. αβ

• Anti-P. gingivalis antibodies and PLNC8 inhibit P. gingivalis- induced cellular responses in GECs. However, PLNC8 and the antibodies antagonized the effects ofαβ each other when used in combination possibly due to competition for common bindingαβ sites. PLNC8 and antibodies have different mechanisms in the interaction with bacteria and the associated regulation of immune responsesαβ and may thus individually have potential roles in the treatment of P. gingivalis infection and periodontitis.

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 41

Future Perspectives The studies of this thesis demonstrate a possible role of anti-P. gingivalis antibodies and PLNC8 in detection and prevention of P. gingivalis infection. However, further studies are needed to clarify involved mechanisms and to demonstrate the therapeuticalαβ applications of these agents in clinical use.

A better understanding of the role of antibodies in patients with periodontitis is necessary due to their strong association with several associated systemic diseases. Periodontal lesions are predominated by B and plasma cells and it would thus be interesting to elucidate the effects of P. gingivalis on B cell activation, maturation and differentiation. IgG subclass responses to different major virulence factors of P. gingivalis could be targeted in order to develop a pool of antibodies and study their effects in attenuating P. gingivalis infection and activating Fc receptors on immune cells.

PLNC8 is a recently characterizedγ bacteriocin and both and peptides have been reported to be needed for full plantaricin activity. However, it would beαβ intriguing to elucidate the role, the binding receptorsα andβ the antibacterial activity of these peptides, independently. It is also interesting to investigate if PLNC8 has a broad or narrow spectrum of antibacterial effects and if the bacteriocin enhances the efficiency of traditional antibiotics. αβ

The ability and mechanism of PLNC8 in promoting cell proliferation via inducing growth factors have to be further elucidated. Furthermore, it is interesting to clarify the role of PLNC8αβ in TGF- and akt signalling pathways of cell proliferation. αβ β Gingipains are very potent in cleaving several host molecules, including cytokines, chemokines and several other inflammatory mediators. So it will be interesting to elucidate the role of gingipains in cleaving PLNC8 which would be beneficial for future drug development and therapeutical applications. αβ

42 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection

Acknowledgements It is an overwhelming joy to extend my gratitude towards my colleagues, friends and family who helped, supported and inspired me to pursue my dream. I cannot ask for anything more and thanks to each one of you to make my dream come true!

Torbjörn Bengtsson, my supervisor, I genuinely would like to thank you for giving me this opportunity and for believing in my abilities. I am always grateful for your patience and kindness. You have never failed to encourage my ideas and have always appreciated my efforts. Thanks for all our discussions and you are the best supervisor I would ever wish for!

Hazem Khalaf, my co-supervisor, words fall less to appreciate your tremendous support. Thank you so much for giving me an opportunity to collab- orate with you and to let me learn and grow through your research guid- ance. You always had the best solution every time I walk through your door. It is always a pleasure to discuss science with you and your command in research is really inspiring. You have always been kind in giving me chances and more importantly have never failed to motivate me. Thanks for all your generous car rides and for also teaching me football.

Fariba Nayeri, my co-supervisor, thanks for providing me an opportunity to work and learn at PEAS. Your hard work and care for patients have always been inspiring. I believe, working at PEAS made me stronger and has prepared me for much tougher challenges in life ahead. I really appreciate all your help and for believing in me.

Johanna, my co-supervisor, thanks for being there all round the clock. I would not be able to have come this far without your support. Thanks for being a wonderful friend. I have always been inspired by your adventurous and carefree attitude. I will miss you a lot but I am so sure that we will be in touch wherever I go!

A special thanks to our dentist Carin, collaborators from Linköping Uni- versity-Daniel and Camille, for your tremendous support and timely help. Thanks for believing in our studies and for all your nice words. I am thank- ful to Dr. Svante Hugosson and Amanj Saber for your support. Thanks to Prof. Katharina Person for your generous help.

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 43

Eleonor, thanks for your tremendous support and for all your suggestions during my thesis preparation. Thanks for your encouraging words and for always believing in me. I would cherish all our work-outs, walks in crazy freezing nights, dinners, Fb chats, never-ending talks, biking together, fika breaks, planning for never-happening trips together and gossips. Thanks for being a very kind and nice friend Kaddy, thanks for teaching me to fix bugs and for sharing your clinical expertise. I will always remember all our trips together, fierce debates and never-ending discussions. Thanks a lot for all your generous gifts and for your amazing Indian food. Boxy, you are such a wonderful person and I have always admired your happy-go-lucky attitude. It was fun during all our long lab days and wish you all success in life. Kristin, I still remember coming to your home and trying to jump to hang my coat. It was so much fun for all that we did together and you taught me swimming and thanks for always translating Swedish for me. Your con- fidence and organizing skills are truly amazing.

Isak, Ahhhh bro!! Thanks for being a very good teacher. Your passion for research, lessons of life, dedication and hard work really inspire me. I am so glad to know you and was lucky to learn so much from you. You are the world’s best brother and a fantastic human I have ever metWish you a good health. Don’t be glad that I am done and that I will leave you in peace, I will be like a shadow to bug you, forever. Geena, Bumble bee, I remember laughing hysterically when you are around. I am always amazed at your drive for research and good luck for your defense.

A special thanks to Kjell, Michael, Oswald, Faisal, Ashraf and Wu for all your wise conversations and fun at PEAS. I would like to thank Anna M, Madaleine, Lisa, Julia, Savanneh, John peter, Berhane, Naveed, Sezin, Car- oline, Lisa, Alex and everyone at KFL for a very friendly and supportive work environment.

Pavithra, Thanks for nurturing the thought of higher studies in me. It is so exciting to grow up, understand things with a different perspective now and to just pick up from where we had left. Thanks for all your prayers, love and care. Thanks to aunty, uncle and pati for their support. Ambika, we have known each other for almost 25 years now and I am so happy we still are the same! Priya, if it weren’t you, I would have not known about masters in Sweden. Thanks for taking me to cosmos and I am so happy for all that

44 SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection you have achieved now! Koushik, sathyama namma pesardu elame teva ila- dadu dan, but still is so much fun. Good luck with your movie & khi kha lam kanavu kanardu vitutu padam edu modaleh. Aarthi J, Sugumar and Mohan, for always being there to wish the best for me.

Thanks to all my teachers and everyone for all that you have taught me and above all, for molding me into a better human.

Sathish, Thanks for being there and for always letting me win an argument. I would always love you and I hope our friendship continues. Kalai, it all feels like a dream now thinking about how it all fell in place for us to come here. Thanks for always being just a text away and for all your help in shift- ing, cooking and for being a very nice friend. I always adore your quench to learn new things. Shuba, Darls! You are always nice to me (probably only me, ha ha) It is definitely an amazing experience together and is always nice to catch up with things, once in a while. Thanks to Viji, Sharmi, Eddy and Saravana, for your care, warmth. I cannot forget our first-ever flight journey to Sweden, gossips, fights, struggle, cooking something resembling food and what not! Priya ji and Mohan ji, thanks a lot, for all the care, hospitality and amazing time.

Sally and Sven-olov Stehager, you both never gave us any reason to miss home. Thanks to stand by us and for celebrating our success and happiness. I am really grateful to learn and get inspired by your strength, values and compassion. You both lit up our lives and gave us every reason to long to come back to Linköping for all our holidays. My heartfelt thanks to Rama pedamma, Chandram mamaiah and Ammama, you three did so much for me to do well in life. I believe you would bless and guide me from the heaven! Nanna, thanks for giving me the best education. Thanks a lot for encouraging me to pursue a career in Science  Amma, thanks for all your prayers, sacrifices, love, patience and kindness. I am indebted and overwhelmingly happy to have you both.

I extend my sincere gratitude to Örebro University, Erik Norgen (repro) and all the administrators for your very kind help.

Our studies are supported by The Knowledge foundation, Sweden, the Swe- dish Heart and Lung Foundation, the Foundation of Olle Engkvist, Sweden.

SRAVYA NAKKA Prevention and Diagnosis of Porphyromonas gingivalis Infection 45

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122. Ogawa T, Kusumoto Y, Hamada S, McGhee JR, Kiyono H: Bacteroides gingivalis-specific serum IgG and IgA subclass antibodies in periodontal diseases. Clin Exp Immunol 1990, 82(2):318-325. 123. O'Brien-Simpson NM, Black CL, Bhogal PS, Cleal SM, Slakeski N, Higgins TJ, Reynolds EC: Serum immunoglobulin G (IgG) and IgG subclass responses to the RgpA-Kgp proteinase-adhesin complex of Porphyromonas gingivalis in adult periodontitis. Infect Immun 2000, 68(5):2704-2712. 124. Mazumdar SD, Hartmann M, Kampfer P, Keusgen M: Rapid method for detection of Salmonella in milk by surface plasmon resonance (SPR). Biosens Bioelectron 2007, 22(9-10):2040-2046. 125. Spangler BD, Wilkinson EA, Murphy JT, Tyler BJ: Comparison of the Spreeta (R) surface plasmon resonance sensor and a quartz crystal microbalance for detection of Escherichia coli heat-labile enterotoxin. Anal Chim Acta 2001, 444(1):149-161. 126. Taylor AD, Yu QM, Chen SF, Homola J, Jiang SY: Comparison of E-coli O157 : H7 preparation methods used for detection with surface plasmon resonance sensor. Sensor Actuat B-Chem 2005, 107(1):202-208. 127. Oh BK, Lee W, Chun BS, Bae YM, Lee WH, Choi JW: Surface plasmon resonance immunosensor for the detection of Yersinia enterocolitica. Colloid Surface A 2005, 257-58:369-374. 128. Nanduri V, Bhunia AK, Tu SI, Paoli GC, Brewster JD: SPR biosensor for the detection of L. monocytogenes using phage- displayed antibody. Biosens Bioelectron 2007, 23(2):248-252. 129. Wu C, Nakka S, Mansouri S, Bengtsson T, Nayeri T, Nayeri F: In vitro model of production of antibodies; a new approach to reveal the presence of key bacteria in polymicrobial environments. BMC Microbiol 2016, 16:209. 130. Koll-Klais P, Mandar R, Leibur E, Marcotte H, Hammarstrom L, Mikelsaar M: Oral lactobacilli in chronic periodontitis and periodontal health: species composition and antimicrobial activity. Oral Microbiol Immunol 2005, 20(6):354-361. 131. Cotter PD, Ross RP, Hill C: Bacteriocins - a viable alternative to antibiotics? Nature Reviews Microbiology 2013, 11(2):95-105. 132. Brogden KA: Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 2005, 3(3):238-250. 133. Pieterse R, Todorov SD: Bacteriocins - exploring alternatives to antibiotics in mastitis treatment. Braz J Microbiol 2010, 41(3):542- 562.

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Publications in the series Örebro Studies in Medicine

1. Bergemalm, Per-Olof (2004). Audiologic and cognitive long-term sequelae from closed head injury. 2. Jansson, Kjell (2004). Intraperitoneal Microdialysis. Technique and Results. 3. Windahl, Torgny (2004). Clinical aspects of laser treatment of lichen sclerosus and squamous cell carcinoma of the penis. 4. Carlsson, Per-Inge (2004). Hearing impairment and deafness. Genetic and environmental factors – interactions – consequences. A clinical audiological approach. 5. Wågsäter, Dick (2005). CXCL16 and CD137 in Atherosclerosis. 6. Jatta, Ken (2006). Inflammation in Atherosclerosis. 7. Dreifaldt, Ann Charlotte (2006). Epidemiological Aspects on Malignant Diseases in Childhood. 8. Jurstrand, Margaretha (2006). Detection of Chlamydia trachomatis and Mycoplasma genitalium by genetic and serological methods. 9. Norén, Torbjörn (2006). Clostridium difficile, epidemiology and antibiotic resistance. 10. Anderzén Carlsson, Agneta (2007). Children with Cancer – Focusing on their Fear and on how their Fear is Handled. 11. Ocaya, Pauline (2007). Retinoid metabolism and signalling in vascular smooth muscle cells. 12. Nilsson, Andreas (2008). Physical activity assessed by accelerometry in children. 13. Eliasson, Henrik (2008). Tularemia – epidemiological, clinical and diagnostic aspects. 14. Walldén, Jakob (2008). The influence of opioids on gastric function: experimental and clinical studies. 15. Andrén, Ove (2008). Natural history and prognostic factors in localized prostate cancer. 16. Svantesson, Mia (2008). Postpone death? Nurse-physician perspectives and ethics rounds. 17. Björk, Tabita (2008). Measuring Eating Disorder Outcome – Definitions, dropouts and patients’ perspectives. 18. Ahlsson, Anders (2008). Atrial Fibrillation in Cardiac Surgery. 19. Parihar, Vishal Singh (2008). Human Listeriosis – Sources and Routes. 20. Berglund, Carolina (2008). Molecular Epidemiology of Methicillin- Resistant Staphylococcus aureus. Epidemiological aspects of MRSA and the dissemination in the community and in hospitals. 21. Nilsagård, Ylva (2008). Walking ability, balance and accidental falls in persons with Multiple Sclerosis. 22. Johansson, Ann-Christin (2008). Psychosocial factors in patients with lumbar disc herniation: Enhancing postoperative outcome by the identification of predictive factors and optimised physiotherapy. 23. Larsson, Matz (2008). Secondary exposure to inhaled tobacco products. 24. Hahn-Strömberg, Victoria (2008). Cell adhesion proteins in different invasive patterns of colon carcinoma: A morphometric and molecular genetic study. 25. Böttiger, Anna (2008). Genetic Variation in the Folate Receptor-α and Methylenetetrahydrofolate Reductase Genes as Determinants of Plasma Homocysteine Concentrations. 26. Andersson, Gunnel (2009). Urinary incontinence. Prevalence, treatment seeking behaviour, experiences and perceptions among persons with and without urinary leakage. 27. Elfström, Peter (2009). Associated disorders in celiac disease. 28. Skårberg, Kurt (2009). Anabolic-androgenic steroid users in treatment: Social background, drug use patterns and criminality. 29. de Man Lapidoth, Joakim (2009). Binge Eating and Obesity Treatment – Prevalence, Measurement and Long-term Outcome. 30. Vumma, Ravi (2009). Functional Characterization of Tyrosine and Tryptophan Transport in Fibroblasts from Healthy Controls, Patients with Schizophrenia and Bipolar Disorder. 31. Jacobsson, Susanne (2009). Characterisation of Neisseria meningitidis from a virulence and immunogenic perspective that includes variations in novel vaccine antigens. 32. Allvin, Renée (2009). Postoperative Recovery. Development of a Multi-Dimensional Questionnaire for Assessment of Recovery. 33. Hagnelius, Nils-Olof (2009). Vascular Mechanisms in Dementia with Special Reference to Folate and Fibrinolysis. 34. Duberg, Ann-Sofi (2009). Hepatitis C virus infection. A nationwide study of assiciated morbidity and mortality. 35. Söderqvist, Fredrik (2009). Health symptoms and potential effects on the blood-brain and blood-cerebrospinal fluid barriers associated with use of wireless telephones. 36. Neander, Kerstin (2009). Indispensable Interaction. Parents’ perspectives on parent–child interaction interventions and beneficial meetings. 37. Ekwall, Eva (2009). Women’s Experiences of Gynecological Cancer and Interaction with the Health Care System through Different Phases of the Disease. 38. Thulin Hedberg, Sara (2009). Antibiotic susceptibility and resistance in Neisseria meningitidis – phenotypic and genotypic characteristics. 39. Hammer, Ann (2010). Forced use on arm function after stroke. Clinically rated and self-reported outcome and measurement during the sub-acute phase. 40. Westman, Anders (2010). Musculoskeletal pain in primary health care: A biopsychosocial perspective for assessment and treatment. 41. Gustafsson, Sanna Aila (2010). The importance of being thin – Perceived expectations from self and others and the effect on self-evaluation in girls with disordered eating. 42. Johansson, Bengt (2010). Long-term outcome research on PDR brachytherapy with focus on breast, base of tongue and lip cancer. 43. Tina, Elisabet (2010). Biological markers in breast cancer and acute leukaemia with focus on drug resistance. 44. Overmeer, Thomas (2010). Implementing psychosocial factors in physical therapy treatment for patients with musculoskeletal pain in primary care. 45. Prenkert, Malin (2010). On mechanisms of drug resistance in acute myloid leukemia. 46. de Leon, Alex (2010). Effects of Anesthesia on Esophageal Sphincters in Obese Patients. 47. Josefson, Anna (2010). Nickel allergy and hand eczema – epidemiological aspects. 48. Almon, Ricardo (2010). Lactase Persistence and Lactase Non- Persistence. Prevalence, influence on body fat, body height, and relation to the metabolic syndrome. 49. Ohlin, Andreas (2010). Aspects on early diagnosis of neonatal sepsis. 50. Oliynyk, Igor (2010). Advances in Pharmacological Treatment of Cystic Fibrosis. 51. Franzén, Karin (2011). Interventions for Urinary Incontinence in Women. Survey and effects on population and patient level. 52. Loiske, Karin (2011). Echocardiographic measurements of the heart. With focus on the right ventricle. 53. Hellmark, Bengt (2011). Genotypic and phenotypic characterisation of Staphylococcus epidermidis isolated from prosthetic joint infections. 54. Eriksson Crommert, Martin (2011). On the role of transversus abdominis in trunk motor control. 55. Ahlstrand, Rebecca (2011). Effects of Anesthesia on Esophageal Sphincters. 56. Holländare, Fredrik (2011). Managing Depression via the Internet – self-report measures, treatment & relapse prevention. 57. Johansson, Jessica (2011). Amino Acid Transport and Receptor Binding Properties in Neuropsychiatric Disorders using the Fibroblast Cell Model. 58. Vidlund, Mårten (2011). Glutamate for Metabolic Intervention in Coronary Surgery with special reference to the GLUTAMICS-trial. 59. Zakrisson, Ann-Britt (2011). Management of patients with Chronic Obstructive Pulmonary Disease in Primary Health Care. A study of a nurse-led multidisciplinary programme of pulmonary rehabilitation. 60. Lindgren, Rickard (2011). Aspects of anastomotic leakage, anorectal function and defunctioning stoma in Low Anterior Resection of the rectum for cancer. 61. Karlsson, Christina (2011). Biomarkers in non-small cell lung carcinoma. Methodological aspects and influence of gender, histology and smoking habits on estrogen receptor and epidermal growth factor family receptor signalling. 62. Varelogianni, Georgia (2011). Chloride Transport and Inflammation in Cystic Fibrosis Airways. 63. Makdoumi, Karim (2011). Ultraviolet Light A (UVA) Photoactivation of Riboflavin as a Potential Therapy for Infectious Keratitis. 64. Nordin Olsson, Inger (2012). Rational drug treatment in the elderly: ”To treat or not to treat”. 65. Fadl, Helena (2012). Gestational diabetes mellitus in Sweden: screening, outcomes, and consequences. 66. Essving, Per (2012). Local Infiltration Analgesia in Knee Arthroplasty. 67. Thuresson, Marie (2012). The Initial Phase of an Acute Coronary Syndrome. Symptoms, patients’ response to symptoms and opportunity to reduce time to seek care and to increase ambulance use. 68. Mårild, Karl (2012). Risk Factors and Associated Disorders of Celiac Disease. 69. Fant, Federica (2012). Optimization of the Perioperative Anaesthetic Care for Prostate Cancer Surgery. Clinical studies on Pain, Stress Response and Immunomodulation. 70. Almroth, Henrik (2012). Atrial Fibrillation: Inflammatory and pharmacological studies. 71. Elmabsout, Ali Ateia (2012). CYP26B1 as regulator of retinoic acid in vascular cells and atherosclerotic lesions. 72. Stenberg, Reidun (2012). Dietary antibodies and gluten related seromarkers in children and young adults with cerebral palsy. 73. Skeppner, Elisabeth (2012). Penile Carcinoma: From First Symptom to Sexual Function and Life Satisfaction. Following Organ-Sparing Laser Treatment. 74. Carlsson, Jessica (2012). Identification of miRNA expression profiles for diagnosis and prognosis of prostate cancer. 75. Gustavsson, Anders (2012): Therapy in Inflammatory Bowel Disease. 76. Paulson Karlsson, Gunilla (2012): Anorexia nervosa – treatment expectations, outcome and satisfaction. 77. Larzon, Thomas (2012): Aspects of endovascular treatment of abdominal aortic aneurysms. 78. Magnusson, Niklas (2012): Postoperative aspects of inguinal hernia surgery – pain and recurrences. 79. Khalili, Payam (2012): Risk factors for cardiovascular events and incident hospital-treated diabetes in the population. 80. Gabrielson, Marike (2013): The mitochondrial protein SLC25A43 and its possible role in HER2-positive breast cancer. 81. Falck, Eva (2013): Genomic and genetic alterations in endometrial adenocarcinoma. 82. Svensson, Maria A (2013): Assessing the ERG rearrangement for clinical use in patients with prostate cancer. 83. Lönn, Johanna (2013): The role of periodontitis and hepatocyte growth factor in systemic inflammation. 84. Kumawat, Ashok Kumar (2013): Adaptive Immune Responses in the Intestinal Mucosa of Microscopic Colitis Patients. 85. Nordenskjöld, Axel (2013): Electroconvulsive therapy for depression. 86. Davidsson, Sabina (2013): Infection induced chronic inflammation and its association with prostate cancer initiation and progression. 87. Johansson, Benny (2013): No touch vein harvesting technique in coronary by-pass surgery. Impact on patency rate, development of atherosclerosis, left ventricular function and clinical outcome during 16 years follow-up. 88. Sahdo, Berolla (2013): Inflammasomes: defense guardians in host-microbe interactions. 89. Hörer, Tal (2013): Early detection of major surgical postoperative complications evaluated by microdialysis. 90. Malakkaran Lindqvist, Breezy (2013): Biological signature of HER2- positive breast cancer. 91. Lidén, Mats (2013): The stack mode review of volumetric datasets – applications for urinary stone disease. 92. Emilsson, Louise (2013): Cardiac Complications in Celiac Disease. 93. Dreifaldt, Mats (2013): Conduits in coronary artery bypass grafting surgery: Saphenous vein, radial and internal thoracic arteries. 94. Perniola, Andrea (2013): A new technique for postoperative pain management with local anaesthetic after abdominal hysterectomy. 95. Ahlstrand, Erik (2013): Coagulase-negative Staphylococci in Hematological Malignancy. 96. Sundh, Josefin (2013): Quality of life, mortality and exacerbations in COPD. 97. Skoog, Per (2013): On the metabolic consequences of abdominal compartment syndrome. 98. Palmetun Ekbäck, Maria (2013): Hirsutism and Quality of Life with Aspects on Social Support, Anxiety and Depression. 99. Hussain, Rashida (2013): Cell Responses in Infected and Cystic Fibrosis Respiratory Epithelium. 100. Farkas, Sanja (2014): DNA methylation in the placenta and in cancer with special reference to folate transporting genes. 101. Jildenstål, Pether (2014): Influence of depth of anaesthesia on postoperative cognitive dysfunction (POCD) and inflammatory marker. 102. Söderström, Ulf (2014): Type 1 diabetes in children with non-Swedish background – epidemiology and clinical outcome 103. Wilhelmsson Göstas, Mona (2014): Psychotherapy patients in mental health care: Attachment styles, interpersonal problems and therapy experiences 104. Jarl, Gustav (2014): The Orthotics and Prosthetics Users´ Survey: Translation and validity evidence for the Swedish version 105. Demirel, Isak (2014): Uropathogenic Escherichia coli, multidrug- resistance and induction of host defense mechanisms 106. Mohseni, Shahin (2014): The role of ß-blockade and anticoagula- tion therapy in traumatic brain injury 107. Bašić, Vladimir T. (2014): Molecular mechanisms mediating development of pulmonary cachexia in COPD 108. Kirrander, Peter (2014): Penile Cancer: Studies on Prognostic Factors 109. Törös, Bianca (2014): Genome-based characterization of Neisseria meningitidis with focus on the emergent serogroup Y disease 110. von Beckerath, Mathias (2014): Photodynamic therapy in the Head and Neck 111. Waldenborg, Micael (2014): Echocardiographic measurements at Takotsubo cardiomyopathy - transient left ventricular dysfunction. 112. Lillsunde Larsson, Gabriella (2014): Characterization of HPV-induced vaginal and vulvar carcinoma. 113. Palm, Eleonor (2015): Inflammatory responses of gingival fibroblasts in the interaction with the periodontal pathogen Porphyromonas gingivlis. 114. Sundin, Johanna (2015): Microbe-Host Interactions in Post-infectious Irritable Bowel Syndrome. 115. Olsson, Lovisa (2015): Subjective well-being in old age and its association with biochemical and genetic biomarkers and with physical activity. 116. Klarström Engström, Kristin (2015): Platelets as immune cells in sensing bacterial infection. 117. Landström, Fredrik (2015): Curative Electrochemotherapy in the Head and Neck Area. 118. Jurcevic, Sanja (2015): MicroRNA expression profiling in endometrial adenocarcinoma. 119. Savilampi, Johanna (2015): Effects of Remifentanil on Esophageal Sphincters and Swallowing Function. 120. Pelto-Piri, Veikko (2015): Ethical considerations in psychiatric inpatient care. The ethical landscape in everyday practice as described by staff. 121. Athlin, Simon (2015): Detection of Polysaccharides and Polysaccharide Antibodies in Pneumococcal Pneumonia. 122. Evert, Jasmine (2015): Molecular Studies of Radiotheray and Chemotherapy in Colorectal Cancer. 123. Göthlin-Eremo, Anna (2015): Biological profiles of endocrine breast cancer. 124. Malm, Kerstin (2015): Diagnostic strategies for blood borne infections in Sweden. 125. Kumakech, Edward (2015): Human Immunodeficiency Virus (HIV), Human Papillomavirus (HPV) and Cervical Cancer Prevention in Uganda: Prevalence, Risk factors, Benefits and Challenges of Post- Exposure Prophylaxis, Screening Integration and Vaccination. 126. Thunborg, Charlotta (2015): Exploring dementia care dyads’ person transfer situations from a behavioral medicine perspective in physiotherapy. Development of an assessmement scale. 127. Zhang, Boxi (2015): Modulaton of gene expression in human aortic smooth muscle cells by Porphyromonas gingivalis - a possible association between periodontitis and atherosclerosis. 128. Nyberg, Jan (2015): On implant integration in irradiated bone: - clinical and experimental studies. 129. Brocki, Barbara C. (2015): Physiotherapy interventions and outcomes following lung cancer surgery. 130. Ulfenborg, Benjamin (2016): Bioinformatics tools for discovery and evaluation of biomarkers. Applications in clinical assessment of cancer. 131. Lindström, Caisa (2016): Burnout in parents of chronically ill children. 132. Günaltay, Sezin (2016): Dysregulated Mucosal Immune Responses in Microscopic Colitis Patients. 133. Koskela von Sydow, Anita (2016): Regulation of fibroblast activity by kera- tinocytes, TGF-β and IL-1α –studies in two- and three dimensional in vitro models. 134. Kozlowski, Piotr (2016): Prognostic factors, treatment and outcome in adult acute lymphoblastic leukemia. Population-based studies in Sweden. 135. Darvish, Bijan (2016): Post-Dural Puncture Headache in Obstetrics. Audiological, Clinical and Epidemiological studies. 136. Sahlberg Bang, Charlotte (2016): Carbon monoxide and nitric oxide as antimicrobial agents – focus on ESBL-producing uropathogenic E. coli. 137. Alshamari, Muhammed (2016): Low-dose computed tomography of the abdomen and lumbar spine. 138. Jayaprakash, Kartheyaene (2016): Monocyte and Neutrophil Inflammatory Responses to the Periodontopathogen Porphyromonas gingivalis. 139. Elwin Marie (2016): Description and measurement of sensory symptoms in autism spectrum. 140. Östlund Lagerström, Lina (2016): ”The gut matters” - an interdisciplinary approach to health and gut function in older adults. 141. Zhulina, Yaroslava (2016): Crohn’s disease; aspects of epidemiology, clinical course, and fecal calprotectin. 142. Nordenskjöld, Anna (2016): Unrecognized myocardial infarction and cardiac biochemical markers in patients with stable coronary artery disease. 143. Floodeen, Hannah (2016): Defunctioning stoma in low anterior resection of the rectum for cancer: Aspects of stoma reversal, anastomotic leakage, anorectal function, and cost-effectiveness. 144. Duberg, Anna (2016): Dance Intervention for Adolescent Girls with Inter- nalizing Problems. Effects and Experiences. 145. Samano, Ninos (2016): No-Touch Saphenous Veins in Coronary Artery Bypass Grafting. Long-term Angiographic, Surgical, and Clinical Aspects. 146. Rönnberg, Ann-Kristin (2016): Gestational Weight Gain. Implications of an Antenatal Lifestyle Intervention. 147. Erik Stenberg (2016): Preventing complications in bariatric surgery. 148. Humble, Mats B. (2016): Obsessive-compulsive disorder, serotonin and oxytocin: treatment response and side effects. 149. Asfaw Idosa, Berhane (2016): Inflammasome Polymorphisms and the Inflammatory Response to Bacterial Infections. 150. Sagerfors, Marcus (2016): Total wrist arthroplasty. A clinical, radiographic and biomechanical investigation. 151. Nakka, Sravya Sowdamini (2016): Development of novel tools for prevention and diagnosis of Porphyromonas gingivalis infection and periodontitis.