PROFILING OF ABC (CDR1) TRANSPORTER GENES OF AZOLE RESISTANT STRAINS OF CANDIDA SPECIES ISOLATED FROM WOMEN OF REPRODUCTIVE AGE

ABSTRACT

Although adjuvants are a common component of many vaccines, there are few adjuvants licensed for use in humans due to concerns about their toxic effects. The currently available HBV vaccines contain hepatitis B virus surface antigen (HBsAg) as the immunodominant antigen and alum as an adjuvant. Alum, the most commonly used adjuvant in vaccines licensed for human use, predominantly activates Th2-type humoral immune responses, but is poorly effective in inducing Th1-type cellular responses essential for the treatment of viral infections. Consequently, there is a considerable demand for an effective adjuvant for therapeutic vaccines to treat HBV infections. Several studies have reported various health benefits of chitin including its use for treatment of burns and various infections. The present study was conducted to determine whether the chitin extract would enhance the immunogenicity of a recombinant Hepatitis-B vaccine when combined with it and also the toxicological effect this combination would have on the liver. Female mice at the age of 6 to 8  weeks  were used  for  the study.  Seven  (7) days  after acclimatization,  the mice  blood samples were collected on day 0, day 14, day 21 and day 28 for biochemical analysis such liver marker enzymes, liver weight, IgG, IgG1 and IgG2a assay. Also, white blood cell differentials such as neutrophils, lymphocytes, monocytes, eosinophils and basophils were also determined. From days 0 to 21, there was a significant (p < 0.05) increase in IgG titre level of mice administered with 3 doses of HBV vaccine alone. IgG titre level of mice administered  with  2  doses  of  HBV-chitin  combination  showed  a  significant  (P  <  0.05) increase from days 14 to 21. Mice administered with 3 doses of HBV vaccine alone showed a significant (p < 0.05) increase in IgG1 titre level from days 14 to 21. IgG1 titre of mice administered  with  2  doses  of  HBV-chitin  combination  showed  a  significant  (p  <  0.05) increase from day 0 to 21. Mice administered with 2 doses of HBV-chitin combination showed a non-significantly (p > 0.05) different IgG2a titre level from days 0 to 28. Mice administered with 2 doses of HBV-chitin combination elicited a significantly (p < 0.05) lower IgG2a when compared to mice administered with 3 doses of HBV vaccine on days 14 and 21 respectively. From days 0 to 28, there was a significant (p < 0.05) decrease in the percentage population of eosinophils in mice administered with 2 doses of HBV vaccine alone. Mice administered with 2 doses of HBV-chitin combination elicited a non-significant (p > 0.05) differences in the percentage population of basophils when compared to mice administered with 3 doses of HBV vaccine alone. Mice administered with HBV-chitin combination  showed  a  non-significant  (p  > 0.05) differences  in  the  ALT activity  when compared to mice administered with 3 doses of HBV vaccine alone. Mice administered with 2 doses of HBV-chitin combination exhibited a non-significant (p > 0.05) differences in the AST activity when compared to mice administered with 3 doses of HBV vaccine alone.  The findings of this research revealed chitin may possess adjuvant properties for recombinant hepatitis B vaccine and its combination with the vaccine exert no toxic effect.

CHAPTER ONE

INTRODUCTION

The term immunity comes from the Latin word immunitas, means protection from legitimate prosecution. Immunity refers to protection from disease and other pathogens. The cells and molecules responsible for immunity are collectively make up the immune system and their efforts in regards to any etiological agent are called immune responses. Normally the immune responses are elicited against the foreign substances but occasionally to the self-molecules which is referred to as autoimmune response (Wekerle et al., 2012). Immunology is a branch of life science which deals with the cellular and molecular events occurring in the body after encounters of micro-organisms and other foreign substances.

The history of immunology is quite old. In ancient China, people often used skin lesions of patients recovered from small pox to cure small pox in young children. The first successful record of vaccination came from the work of Edward Jenner’s efficient vaccination against smallpox. Jenner observed that milkmaid who had recovered from cowpox never showed any symptom of smallpox. Following this observation he inoculated the cowpox pustules into the arm of a young boy who later did not show full progressive smallpox symptoms. Small pox was the first disease that was eradicated worldwide by vaccination (Barquet and Domingo,

1997). Recently the science of immunology has greatly advanced by the advent of new molecular biology tools. Our current understanding of the human and animal immune system and its functions has remarkably improved. Advances such as recombinant DNA technology, immunohistochemistry, monoclonal antibody production and x-ray crystallography have changed  the immunology to a broader area. The development of techniques to produce transgenic and knockout mice has also played a great role to understand many complex immunological pathways (Poltorak et al., 1998).

Additionally, thorough investigations of similar molecular pathways in mice considerably increased our knowledge about the confounding immune reactions in humans. For example, elucidation of signaling via toll-like receptor 4 in mouse strains provided important useful information about basic mechanisms in human innate immunity (Beutler, 2004).

The vertebrate immune system consists of well differentiated molecules that recognize and respond to parasitic invasion in a very complex manner (Hughes and Yeager, 1997). The immune system is classified as innate – consisting of barriers to prevent penetration and spread of infectious agents, and adaptive system – consisting of lymphocytes and immunoglobulins. Lymphocytes consist of T cells and B cells that regulate immune response

and impart cellular and humoral immunity to the organism. The B cells develop into plasma cells that secrete antibodies (Spiegelberg, 1974).

1.1 Organization of the human immune system

The human microbial defense system can be simplistically viewed as consisting of 2 levels:

•  Innate immunity;

•  Adaptive immunity.

Failure in any of these systems will greatly increase susceptibility to infection. Anatomic and physiologic barriers provide the crucial first line of defense against pathogens. These barriers include intact skin, vigorous mucociliary clearance mechanisms, low stomach pH, and bacteriolytic lysozyme in tears, saliva, and other secretions. The extreme vulnerability to infections observed in subjects with severe cutaneous burns or primary ciliary dyskinesia demonstrates that intact innate and adaptive immune systems are not able to compensate for failure of essential anatomic and physiologic barriers. (Pancer and Cooper, 2006).

1.1.1 Innate immunity

Innate immunity operates in conjunction with adaptive immunity and is categorized by rapid response to aggression, irrespective of previous stimulus, being the organism first line of defense. Its mechanisms include physical, chemical and biological barriers, cellular components, as well as soluble molecules (Cerwenka and Lanier, 2001). The innate system has three main characteristics:

➢  It responds very rapidly to infection. This is because it either uses mechanisms, such

as the barriers, that are present all the time, or uses cells and molecules that become active within minutes of exposure to disease-causing organisms (pathogens).

➢  It responds in exactly the same way each time it encounters an infection.

➢  It uses a handful of molecules that recognize that infection is present.

Indeed, innate immune response have been complicated in the development of asthma and atopy, as well as a variety of autoimmune disorders, including type 1 diabetes mellitus, multiple sclerosis and systemic lupus erythomatosus (Frank, 2010).

1.1.2 Elements of the innate immune system

The innate immune protection is a task performed by cells of both hematopoietic and nonhematopoietic   origin.   Hematopoietic   cells   involved   in   innate   responses   include

macrophages, dendritic cells, mast cells, neutrophils, eosinophils, and natural killer (NK) cells. Phagocytosis, release of inflammatory mediators, activation of complement system proteins, as well as synthesis of acute phase proteins, cytokines and chemokines are the main mechanisms  in  innate  immunity  (Medzhitov  et  al.,  1997).  The  cells  of  innate  immune response destroy the pathogens or microbes by the effector cells (Abbas and Lichtman, 2003).

1.1.3 Antigen presenting cells

Cells that are specialized to initiate or promote the development of lymphocyte activation are often termed ‘professional antigen-presenting cells’ (Banchereau and Steinman, 1998). The term ‘professional antigen-presenting cells’ is usually used to describe dendritic cells, macrophages and B lymphocytes; these cells are bone marrow-derived cells involved within the lymphoid tissues in stimulation of the effector lymphocytes of the adaptive immune response (Banchereau and Steinman, 1998). Dendritic cells are the most efficient “professional” APC since they also provide costimulatory signals for effective T cell activation. They express MHC (major histocompatibility complex) molecules and have various other specialized characteristics, such as mechanisms for effective antigen uptake and expression of ‘costimulatory’ molecules that promote cellular interaction (Geijtenbeek et al.,

2000). The human major histocompatibility complex (MHC) is composed of a set of highly polymorphic genes called human leukocyte antigen (HLA). It provides the link between innate response and adaptive response (Germain, 1994). In humans, these genes are located on chromosome 6 and are traditionally divided into classes I, II, and III, whereas in mice these genes are located on chromosome 17(Klein and Sato, 2000). Only the genes of classes I and II are involved in presenting antigen protein to T lymphocyte. Class I molecules are present on the surface of all nucleated cells (Klein and Sato, 2000), while class II are found primarily on antigen presenting cells (macrophages, dendritic cells, and B cells) (Germain,

1994).

1.1.4  Dendritic  cells  (DCs)  are  APCs  present  in  virtually  every  tissue,  and  are  potent inducers of innate and adaptive immunity (Efron and Moldawer, 2003). Importantly, various subsets  of dendritic cells  exist  and  drive specific immunological  responses  (Kaisho  and Akira, 2003). These microbial recognition molecules, which include peptidoglycan, dsRNA, lipopolysaccharide(LPS), flagellin, ssRNA/nucleoside analogs, and unmethylated CpG DNA are recognized predominantly by TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9, respectively (Akira and Takeda, 2004). According to Zanoni et al. (2005), recognition of TLR ligands by

DCs induces maturation and migration to secondary lymphoid tissue where they recruit, interact with, and activate various cell populations including T cells and natural killer cells (NK cells). Recently, it has been shown that individual TLR agonists may induce differential responses through activation of distinct signaling cascades, driving  type 1 T helper(Th1) or type 2 T helper cell(Th2) responses in CD4+  T cells (Agrawal et al., 2003). Also, TLR- activated dendritic cells have been shown to stimulate NK activity, which in turn can produce interferon gamma (IFN-γ) and provide an additional Th1 priming signal (Zanoni et al., 2005). Schlecht et al. (2004), demonstrated that mature dendritic cells produce the chemokine dendritic cell-specific CC-chemokine (DC-CK1) and other similar mediators, which selectively attract naïve T cells. In vivo, naive T cells entering the cortex of lymph nodes via high  endothelial  venules  (HEV)  interact  transiently  with  a  succession  of  dendritic  cells which, in this locality, are often called interdigitating cells. Interactions between adhesion molecules such as dendritic cell-specific intercellular adhesion molecules-3-grabbing non- integrin (DC-SIGN), intercellular adhesion molecule 1 (ICAM-1), intercellular adhesion molecule 2 (ICAM-2), lymphocyte function-associated antigen 1 (LFA-1) and lymphocyte function-associated  antigen  3(LFA-3)  on  the  dendritic  cell  and  intercellular  adhesion molecule 3(ICAM-3), LFA-1 and cluster of differentiation 2 (CD2) on the T cell are involved in these interactions (Schlecht et al., 2004). The dendritic cells, as described by Zammit et al. (2005), also receive signal from the T cells. Cluster of differentiation 40 (CD40) ligand, induced on activated T cells, engages CD40 on dendritic cells and stimulates further maturation and production of cytokines, particularly IL-12. In these cases, interactions between the ligand for CD40 on an activated CD4+ T cell and CD40 on the dendritic cells may be required before the dendritic cells can activate CD8+ cells (Zammit et al., 2005).

1.1.5 Monocytes give rise to macrophages (histiocytes), which become fixed  in various tissues and attached to the inner walls of blood and lymphatic vessels.  C57BL/6 mice contain about 2% monocytes (Doeing et al., 2003). These relatively nonmotile, phagocytic cells, can divide and produce new macrophages, which are found in organs such as the lymph nodes, spleen, liver, and lungs. This diffuse group of phagocytic cells constitutes the reticuloendothelial tissue (reticuloendothelial system or RES) (Mosser and Edwards, 2008). In addition to their role as immune effector cell, macrophages are important for tissue homeostasis, antigen presentation, and immune regulation (Mosser, D. M. and Edwards,

2008). Macrophages stimulated by lipopolysaccharide in the presence of high concentrations of C5a show enhanced production of tumour necrosis factor alpha (TNF-alpha), interleukin 8

(IL-8), and interleukin 6 (IL-6), which could be associated with cell damage (Ward, 2004). The IL-6 is considered to be a reliable predictor of sepsis because it continues at elevated levels longer than TNF-alpha and has been correlated with the severity of infections and mortality (Nasraway, 2003).

During inflammation, macrophages act as APCs, potentiating the activation of T lymphocytes and B lymphocytes by the expression of costimulatory molecules, and release proinflammatory cytokines, such as IL-1, IL-6, IL-12, TNF-α, and chemokines (Abbas and Lichtman, 2003). The cytokine interferon γ (IFNγ), released by specialized immune cells, is able to trigger innate immune response in humans and rodents. In mice, IFNγ induces the expression of 18 different genes of GTPases, the so-called p47 immunity-related GTPase (IRG) proteins (Nasraway, 2003). These proteins induce host response against bacterial and protozoan pathogens as shown by a dramatic increase in susceptibility to pathogens in IRG knockout mice (Coers et al., 2009). Further, IRG proteins in mice induce vesiculation, destruction  of  Toxoplasma  gondii  parasitophorous  vacuolar  membranes,  (Martens  et  al.,

2005) and modify lipid traffic in immune cells after escaping pathogens (Coers et al., 2009).

1.1.6 Neutrophils are also called polymorphonuclear leukocytes, which means their nuclei take many forms. They are the most abundant leukocytes in human peripheral blood, with an important role in the early stages of inflammatory reaction and sensitive to chemotactic agents, such as cleavage products of complement fractions (C3a and C5a) and substances released by mast cells and basophils (Arnhold and Flemmig, 2010). C57BL/6 mice contain

10–25% neutrophils and 75–90% lymphocytes respectively (Doeing et al., 2003). Reports have also showed that CD-1 mice contain 15–20% neutrophils (300–2000 cells/µL) and 50–

70% lymphocytes (1000–7000 cells/µL) respectively (Haley, 2003). Humans have 50–70% neutrophils (3500–7000 cells/µL) and 20–40% lymphocytes (1400–4000 cells/µL) respectively (Haley, 2003). Projecting components of azurophilic granules in human neutrophils are defensins, small cysteine-rich cationic proteins that bind to microbial membranes and induce pore-like membrane defects. Three of the four human defensins in neutrophils (human neutrophil peptide HNP-1, HNP-2, HNP-3) account for 5–7% of total protein content and about 30– 50% of protein content in azurophilic granules (Lehrer et al.,

1993). Neutrophils from mice do not express defensins (Eisenhauer, and Lehrer, 1992). In mice, Paneth cells, present in the crypts of the small intestine, express at least six different defensins (Shanahan et al., 2011), whereas in humans only two kinds of defensins (HNP-5, HNP-6) are found in these cells (Ghosh et al., 2002).Furthermore, one major component of

human neutrophils, are the heme-containing enzyme myeloperoxidase (MPO) that is stored in large amounts in azurophilic granules of resting cells, differs in its expression between man and  mouse.  Myeloperoxidase  is  an  enzyme  which  triggers  apoptosis  in  neutrophils  to terminate inflammatory response. On the other hand, myeloperoxidase released from necrotic cells promotes inflammation by recruiting neutrophils and chemical modification of proteins and other tissue constituents (Arnhold and Flemmig, 2010). The myeloperoxidase level in mice  neutrophils  is  about  10–20%  that  of  human  cells  as  determined  by  activity measurements (Rausch and Moore, 1975). The composition of neutrophil granules significantly differs between human and mouse neutrophils. Various antimicrobial molecules, which contribute decisively to host defense in human neutrophils, are less abundant in mouse neutrophils (Ghosh et al., 2002).

1.1.7 The eosinophil granulocytes constitute only 1–3% of the white blood cell count both in humans and mice (Shamri et al., 2011). Upon production in bone marrow, they circulate in blood, and though in the absence of infection, they reside in different organs (e.g., secondary lymphoid tissue, thymus, gastrointestinal tract) with so far unknown function. Eosinophils are specific in host defense against parasites, but devastating activities have also been described in allergic response and asthma pathogenesis (Shamri et al., 2011).

1.2 Natural killer cells (NK Cells)

According to Orange and Ballas (2006), natural killer cells are lymphocytes of the innate immune system, because they do not rearrange their germline DNA to gain specificity. They do not express T cell receptor (TCR) or immunoglobulin genes, but instead use a variety of germline-encoded  receptors  to  induce  their  functions  (Orange  and  Ballas,  2006).  These include the natural cytotoxicity receptor family (NKp30, NKp44, NKp46, NKp80), the C- type lectin-domain containing receptor NKG2D, the CD2 superfamily receptor 2b4 (CD244), and the IgG receptor CD16. The ligands for some of these receptors are known and include molecules upregulated by cellular stress or infection. Important examples are MIC for NKG2D, viral hemagglutinin for NKp46, CD48 for CD244, and IgG for CD16 (Umetsu and DeKruyff, 2006).The expansion and activation of natural killer cells are stimulated by IL-15, produced by macrophages, and IL-12, potent inducer of IFN-γ and cytolytic action. Once activated, the natural killer cells lyse infected and tumoural cells and secrete proinflammatory cytokines  (IL-1,  IL-2  and  especially  IFN-γ) (Cerwenka and  Lanier,  2001).  In  mice,  the activity of natural killer cells in spleen and blood is high at 4–10 weeks of age. In humans,

their activity is relatively stable throughout the whole lifetime. In lung, mice have high activity of natural killer cells, whereas their activity is low in man. Expression of Fc-receptors on natural killer cells is much higher in humans than in mice (Trinchieri and Perussia, 1984). In mice, killer cells use Ly49 proteins as inhibitory receptors for major histocompatibility complex (MHC) I molecules. This protein is absent in humans. Natural killer cells have activation and inhibition receptors and one class of these receptors belongs to the immunoglobulin superfamily killer-cell immunoglobulin-like receptor (KIR), while the other belongs to the family of C-type lectins. In humans there are 14 killer-cell immunoglobulin- like receptors (KIRs), eight activators and six inhibitors (Yokoyama et al., 2004). Natural killer cells trigger the destruction of cells coated with IgG, through Fc receptors (FcγRIII or CD16), by a mechanism of antibody-dependent cellular cytotoxicity (ADCC) (Cerwenka and Lanier, 2001).

1.3 Mast cells

Mast cells originate from CD34+  hematopoietic progenitors in bone marrow and are not found in circulation (Haley, 2003). Mast cells express IgE-specific Fc receptors (FcεR) on the cell surface and are activated by multivalent antigen recognition by IgE stimuli such as products of complement activation, basic substances including some animals’ poisons, certain neuropeptides and several physical agents (mechanical trauma, heat and cold). The binding of bacterial components to TLR1, TLR2, TLR3, TLR4, TLR6, and other specific receptors such as CD48 also activates mast cells. Upon stimulation and receptor cross-linking by immunoglobulin  E  and  others,  human  mast  cells  release  histamine,  lipid  mediators, cytokines, proteoglycans, and proteases. Histamine is unrestricted upon sensitization from mast cells in response to immunoglobulin E in humans. Reports have shown that histamine are found in mice, but serotonin is responsible for the physiological effects in anaphylactic reactions in this species (Haley, 2003). Murine mast cells contain significant amounts of serotonin, which is found only in few quantities in human mast cells (Kushnir-Sukhov et al.,

2007). Murine mast cells and human basophils are also an important source of IL-4, whereas under normal conditions IL-4 is not produced by human mast cells. Furthermore, murine mast cells express CD14, like human monocytes, and various Toll-like receptors which are not, or only scarcely, found in humans (Bischoff, 2007).

1.4 Innate Immune Recognition Strategies

The  innate  immune  system  serves  as  the  initial  immune  defense  against  foreign  and dangerous material. In the most simplistic view, the innate immune system is hardwired with germline encoded receptors for immediate responsiveness. At least 3 broad approaches are used by the innate immune system to recognize attacking microorganisms. In the first innate recognition strategy, the system relies on a limited repertoire of germline-encoded receptors to recognize ‘‘microbial nonself,’’ conserved molecular structures that are expressed by a large variety  of microbes.  Charles  Janeway  (1989)  coined the terms  pattern recognition receptors to collectively describe these receptors and pathogen associated molecular patterns (PAMPs) to denote the microbial structures recognized by the pattern recognition receptors.

A second approach used by the innate immune system is to detect immunologic danger in the form of damage-associated molecular patterns (DAMPs). DAMPs represent common metabolic consequences of infection and inflammation (Bianchi, 2007).

In the third innate immune recognition strategy, innate immune receptors detect ‘‘missing self,’’ molecules expressed by normal healthy cells but not expressed by infected cells or microbes. Inhibitory receptors specific for self– MHC class I molecules play a central role in missing-self recognition by NK cells, ensuring NK cells preferentially attack infected cells that downregulate their MHC class I proteins (Joncker, and Raulet, 2008).

1.5 Toll-like receptors

Recognition of pathogen- and damage-associated molecular patterns is important to minimize any threat to the host organism. TLRs are a family of pattern recognition receptors (PRRs) that function as primary sensors of the innate immune system to identify microbial pathogens. They were originally revealed as factors involved in the embryonic development and resistance of the fly Drosophila to bacterial and fungal infection (Lemaitre et al., 1997). TLR recognize distinct structures in microbes, often referred to as “PAMPs” (pathogen-associated molecular patterns). PRR also include intracellular proteins, eg, Nod-like receptors (NLRs), retinoic acid-inducible gene-1-like helicases (RLHs), and extracellular receptors such as; scavenger receptors and C-type lectin receptors (Pichlmair, and Reis e Sousa, 2007). The human TLR family consists of 10 receptors, while that of mice consists of 12 receptors (Takeda, and Kaisho, 2003). These receptors comprise a family of conserved membrane spanning molecules containing an ectodomain of leucine-rich repeats, a transmembrane domain, and an intracellular Toll/interleukin (IL)-1R (TIR) domain (Gilliet, and Liu, 2008).

According to Kawai and Akira (2006), each toll-like receptor recognizes different antigenic molecules. For example, TLR4 recognizes bacterial cell wall component lipopolysaccharide (LPS) through its ectodomain (Medzhitov et al., 1997), in addition to monophosphoryl lipid A (MPL A). Lipoprotein and lipoteichoic acid are recognized by TLR2 in combination with TLR1 and TLR6, respectively (Takeuchi et al., 1999). TLR5 recognizes bacterial flagellin (Hayashi  et al., 2001).  Furthermore,  certain TLR (TLR3, TLR7, TLR8, and TLR9) are located within the endoplasmic reticulum (figure 1) and rapidly recruited to endosomal- lysosomal compartments, where they can detect microbial nucleic acids (dsRNA, ssRNA, and ssDNA containing unmethylated CpG motifs, respectively (Kawai and Akira, 2006). TLR7 is active in mice, whereas TLR8 is active in humans; TLR9 ligands can activate plasmacytoid dendritic cells and B cells (Liu, 2005), whereas dsRNA can be recognized by various cells including dendritic cells, fibroblast, macrophages, and endothelial cells. All of these TLR agonists induce type I IFN production (Akira and Hemmi, 2003), which can induce chemokine-mediated cellular recruitment and substitute for costimulatory signals for CD4+ T cell activation (Lande et al., 2003). In humans, TLR9 is expressed on plasmacytoid dendritic cells (PDCs) but not on conventional or myeloid dendritic cells (MDCs), whereas both types of dendritic cells express TLR9 in the mouse (Kadowaki et al., 2001).

1.6. TLR agonists

TLR agonists are generally microbial molecules that stimulate TLR receptors to initiate specific immunoactivity (Kepp et al., 2011). The most frequently studied of these agonists include lipopolysaccharides (LPS; TLR4 agonist), lipopeptides (TLR1, TLR2, and TLR6 agonists), flagellin (TLR5 agonist), single stranded (ssRNA) (TLR7 and TLR8 agonist) or double stranded (ds) RNA (TLR3 agonist), and DNA containing the CpG motif (TLR9 agonist). Recent studies indicate that endogenous molecules released from stressed or dead cells such as heat shock proteins (HSP; TLR2 and TLR4) and high mobility group box 1 (HMGB1; TLR2 and TLR4) are also important TLR agonists (Asea et al., 2002). Single- stranded RNA is an agonist for TLR7 in mice but works through TLR8 in humans (Diebold et al., 2004). TLR3 is expressed on tissue and blood dentritic cells, monocytes, mast cells, NK cells, and epithelial cells. Polyribosinic:polyribocytidic acid (poly I:C) is a synthetic dsRNA complex, which directly activates dendritic cell and also triggers NK cells to kill tumor cells (Sivori et al., 2004). It induces high levels of type I IFNs and activates several nuclear and cytoplasmic enzyme systems (oligoadenylate synthetase, the dsRNA-dependent

protein  kinase,   retinoic  acid-inducible  gene  Helicase,   and   melanoma  differentiation- associated gene-5) that are involved in antiviral and antitumor host defenses (Kawai and Akira, 2006). Polyribosinic:polyribocytidic acid (poly I:C) its therapeutic effects through eliciting antibody responses, (Sloat and Cui, 2006), enhancing crosspriming (Schulz and Diebold, 2005), stimulating antitumor CD8+ T cells, and antigen-specific CD4+ T cells (Longhi et al., 2009). TLR4 can signal to cells through the MyD88 and the TRIF pathways (Beutler, 2004). Its special use in activating human MDCs was reported by (Napolitani et al.,

2005). The classic agonist for TLR4 is bacterial LPS, which refers to a family of substances containing lipid A and its congeners. Gorden et al. (2005), demonstrated that TLR7 agonists activate human plasmacytoid dendritic cells (pDCs) to produce interferon gamma (IFN-γ), whereas TLR8 agonists activate human myeloid dendritic cells (mDCs), monocytes, and monocyte-derived DCs (MDDCs) to make proinflammatory cytokines and chemokines, such as TNF, IL-12, and macrophage inflammatory proteins-1(MIP-1).

1.7 Signaling activation pathway of Toll-like receptor

Signaling pathway of TLR activation begins with the ligand binding either on the cell surface or in the endosome. The binding of ligand to TLR leads to dimerization of the TLR which further recruits the adaptor proteins such as MyD88 (Akira and Takeda, 2004). The adaptor proteins then activate the transcription factor such as nuclear factor- κβ (NF-κβ), activation protein-1, interferon response factor-3 (IRF-3) and IRF-7 (Ho et al., 2004). NF-κβand activation protein-1 stimulates the production of inflammatory cytokines (TNF and IL-1) while interferon regulatory factor 3(IRF-3) and interferon regulatory factor 7(IRF-7) promote the production of type-I interferon. Inflammasome is triggered by a wide variety of stimuli, culminating in the activation of caspase 1, which will then cleave pro–IL1b and pro–IL-18 to drive an inflammatory response.  Human mutations and polymorphisms in many of the genes encoding elements of these pathways appear to alter susceptibility to infectious and inflammatory diseases.

1.8 Complement system

The complement system is an integral part of the innate immune response and acts as a bridge between innate and acquired immunity (Tegla, 2011). It mediates responses to inflammatory triggers through a co-ordinated sequential enzyme cascade leading to clearance of foreign cells through pathogen recognition, opsonisation and lysis (Ehrnthaller, 2011). Mevorach (2014), demonstrated that complement possesses anti-inflammatory functions: it binds to immune complexes and apoptotic cells, and assists in their removal from the circulation and

damaged tissues. There are three known pathways for complement activation: Classical, Alternative and Lectin pathway.

1.8.1 Classical pathway

According to Arumugam (2006), classical pathway is initiated by IgM or IgG antigen/ antibody complexes binding to C1q (first protein of the cascade) leading to activation of C1r, which in turn cleaves C1s. This in turn activates the serine proteases that lead to cleaving of C4 and C2, leading to formation of C4b2a (C3 convertase), which in turn cleaves C3 into C3a and C3b (Arumugam, 2006). While C3a acts as a recruiter of inflammatory cells (anaphylatoxin), C3b binds to the C4b2a complex to form C5 convertase (C4b2a3b). The C5 convertase initiates the formation of the Membrane Attack Complex (MAC), which inserts into membrane creating functional pores in bacterial membranes leading to its lysis (Morgan, (2010). MAC can cause lysis of some cells (e.g. erythrocytes) with a single hit, but some nucleated cells required multiple hits, or rather, multiple channel formation to cause cell lysis (Morgan, 2010). Padilla (2007), demonstrated that classical pathway can also be activated by other danger signals like Creactive protein, viral proteins, polyanions, apoptotic cells and amyloid, thus providing evidence that classical pathway could be activated independent of antibodies.

1.8.2 Alternative pathway

The alternative pathway, as described by Stahl, (2003), begins with the spontaneous rupture of the C3 component into C3a and C3b fragments. A thioester binding in fragment C3b is exposed with this cleavage, which allows their covalent binding to the surface of invading microorganisms. The binding of C3b, enables binding to Factor B, which is then cleaved into fragments Ba and Bb by Factor D (Diepenhorst, 2009). The C3bBb complex (alternative pathway  C3  convertase)  cleaves  more  C3  molecules  and  remains  on  the  surface.  This complex is stabilized by properdin (Factor P), amplifying the breakdown of C3. C3bBb component cleaves C3, generating C3bBbC3b, a protease able to cleave C5, the last step of the alternative pathway (Ehrnthaller, 2011).

1.8.3 Lectin pathway

According to Sarma and Ward (2011), Lectin pathway begins by recognizing mannose on microorganism surface by mannose-binding lectin (MBL) bound to Mannan-binding lectin serine proteases 1 (MASP1) and Mannan-binding lectin serine proteases 2 (MASP2) serine proteases. Activation of these proteases results in the breakdown of CS components C2 and

C4 into smaller fragments (C4a and C2b) and larger fragments (C4b and C2a) (Hajela, 2012). C4bC2a complex is the C3 convertase of  the classical pathway, which cleaves C3 into soluble C3a and C3b, which in turn binds to C4bC2a at the surface of microorganism. The C4bC2aC3b complex, called C5 convertase, cleaves the C5 component, following on this pathway and culminating in the formation of MAC (Matsushita, 2010).

1.9 Inflammation

Inflammation is the basic process whereby tissues of the body respond to injury (Henson,

2005). It is the first line of defense of the organism to tissue damage, having characteristic clinical signs, such as redness, warmth, swelling, pain, and functional impairment. The purpose of this process is to remove the stimulus inducing the response and start local tissue recovery (Abbas and Lichtman, 2003).

1.9.1 Adaptive immune response

Adaptive immune system consists of lymphocytes and immunoglobulins also called antibodies. The adaptive response is localized with precise homing function (Von Andrian and Mackay, 2000). T and B cells are the major components of the adaptive immune system, and both express highly specific antigen to different pathogens (figure 4). B cells produce specific antibodies which neutralize pathogens, facilitate phagocytosis by opsonization, and activate complement; the T cell response is mediated by the T cell receptor (TCR). The TCR responds to antigen peptides presented on macrophages following phagocytosis of pathogens (such as bacteria), and to peptides from cytosolic pathogens (such as viruses) presented on infected cells.

There are basically two types of adaptive immunity namely humoral and cell mediated. Humoral immunity is mediated by antibodies that are produced by B lymphocytes. It is the principal defense mechanism against extracellular microbes and their toxins, with secreted antibodies binding to microbes and toxins to assist in their elimination. Cell mediated immunity is mediated by T cells, with dendritic cells playing important roles in antigen presesntation. T cells can function by various methods: activating macrophages to kill phagocytosed microbes, directly destroy infected cells and by releasing cytokines and alter the milieu around them.

1.9.2 B lymphocytes are a population of cells that express clonally diverse cell surface immunoglobulin (Ig) receptors recognizing specific antigenic epitopes. According to Hardy et al. (2007), B-cell development in mice and humans has been extensively studied, and the

functional rearrangement of the Ig loci is a sine qua non. In mice and humans, this occurs in fetal liver and  adult marrow,  culminating in the development of a diverse repertoire of functional VDJH and  VJL rearrangements encoding the B-cell receptor (BCR) (LeBien,

2000). Early progenitors committed to the B-cell lineage (pro-B cells) begin recombination at the immunoglobulin heavy-chain loci (Blom and Spits, 2006). In mice, autoreactive immature B cells undergo receptor editing, deletion, and anergy induction to condition tolerance. B-cell activating factor (BAFF) and related costimulatory signals positively influence transitional B- cell survival and promote mature B-cell development (Miller et al., 2006). B cells provide humoral immunity against extracellular pathogens through antibody production. Antibodies neutralize pathogens and toxins, facilitate opsonization and activate complement. The B cell receptor (BCR) is capable of binding large conformational epitopes discontinuous in primary structure that include nonpeptide antigens such as polysaccharides and nucleic acids. Naïve follicular B cells reside in the follicles of secondary lymphoid tissues. Antigen arrives in lymphoid organs by circulation of soluble molecules or immune complexes or is transported by dendritic cells (Bergtold et al., 2005). B cell receptor cross-linking initiates receptor- mediated  endocytosis,  antigen  processing,  and  presentation  in  MHC  class  II.  Antigen- engaged follicular B cells migrate to the T-B interface, the border between the T-cell zone and B-cell follicle, increasing the probability of encounter with primed helper T cells of cognate specificity (McHeyzer-Williams and McHeyzer-Williams, 2005).   Signals from T cell-derived cytokines and CD40L:CD40 binding sustain B-cell activation and promote immunoglobulin class switch recombination (CSR). Activated B cells migrate into the follicle and, with continued T-cell help, inniate the germinal center (GC) reaction, or migrate to the marginal zone and differentiate into short-lived plasma cells. Short-lived plasma cells secrete antibody for 2 to 3 weeks, providing a rapid but transient source of effector molecules (Landers et al., 2005).

1.9.3 Immunoglobulins are a group of structurally and functionally similar glycoproteins that confer humoral immunity in humans and mice respectively (Spiegelberg, 1974). They are composed of 82 – 96% protein and 4 – 18% carbohydrate. The immunoglobulin protein “backbone” consists of two identical “heavy” and two identical “light” chains. Five classes of immunoglobulins (IgG, IgA, IgM, IgD, and IgE) have been distinguished on the basis of non- cross-reacting antigenic determinants in regions of highly conserved amino acid sequences in the constant regions of their heavy chains (Goodman, 1987).

1.9.3.1 Immunoglobulin G (IgG) is the predominant antibody class present in mouse and human serum. It is also the main class used in development of antibody therapies, especially those for cancer (Weiner, 2015). IgG is also used to treat immune deficiencies and autoimmune diseases in the form of intravenous immunoglobulin (IVIg), generated from a plasma pool of thousands of donors Barahona Afonso and Jo~ao, 2016).  Human IgG can be divided in 4 subclasses, IgG1, IgG2, IgG3 and IgG4, and in mouse IgG1, IgG2a, IgG2b and IgG3, with IgG2c being the equivalent of IgG2a in some mouse strains such as C47Bl/6 mice (Vidarsson et al., 2014). All subclasses mediate effector functions slightly different due to variable specificity and affinity for the different IgG binding partners mentioned above (Bruhns et al., 2009). Most of the currently approved therapeutic monoclonal antibodies are IgG1, but also IgG2 and IgG4 are used (Ecker et al., 2015). IgG2 and IgG4 are preferred when Fc-mediated effector functions are not needed or not preferred, as these subclasses bind with lower affinity to the FcgR and activate complement less actively (Bindon et al., 1988). The human FcgR locus contains 5 activating FcgR (Fc gRIa, FcgRIIa, FcgRIIc, FcgRIIIa and FcgRIIIb) associated with an immunoreceptor tyrosine-based activation motif (ITAM, except FcgRIIIb) in the intracellular domain (FcgRIIa and FcgRIIc) or the associated common FcR g-chain (FcgRIa and FcgRIIIa) (Bruhns et al., 2009). Mice have 3 activating receptors containing an ITAM motif (FcgRIa, FcgRIII and FcgRIV) and also one inhibitory FcgR containing an ITIM motif (FcgRIIb) (Nimmerjahn et al., 2005). The human response to antigens can be limited to one or more of the four classes of human IgG (IgG1, IgG2, IgG3, and  IgG4).  Each  subclass  shows  differences  in  the  ability  to  participate  in  biological functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement- dependent cytotoxicity (CDC). ImmunoglobulinG-antibody responses to bacterial capsular polysaccharide antigens can be almost completely restricted to IgG2 (Ferrante et al., 1990). Though IgG3 has been revealed to bind human Clq better than the other subclasses, IgG1 was much more effective than other IgG subclasses in complement-dependent cytotoxicity (CDC) (Bindon et al., 1988). IgG1 was also the most active in antibody-dependent cell-mediated cytotoxicity (ADCC). (Steplewski et al., 1988). IgG4 was shown to inhibit complement activation (Lu et al., 2007). These differences in activity were associated with differences in solution conformation of the IgG subclasses (Tao et al., 1993).

1.9.3.2 Immunoglobulin Ms (IgMs) constitute the natural antibodies to the oligosaccharide blood-group antigens and together with IgD serve as one of the major receptors for antigens on the surface of mature B lymphocytes (Pernis et al., 1974). They exists in two forms

namely: membrane bound IgM (L-H)2  and secretory IgM.  Membrane bound IgM (L-H)2  is made up of a pair of dimers, each of which comprises a single light (L) and a single heavy (H) chain. Secretory IgM found in serum is a pentamer of 5 identical (L-H)2 units covalently linked to each other and to a J-chain protein of molecular weight 15,600 (Koshland, 1975), giving a total molecular weight for the pentamer of about 950,000. There are 4 constant domains rather than the 3 found in α or y chains of IgA or IgG, respectively.  Specific IgM antibodies can have important protective functions against E. coli. The IgM secreted by B-1 cells without specific stimulation shows widely variable binding avidities and represents an initial line of defense against pathogens (Baumgarth et al., 2000). The IgM mediates direct neutralization of some bacteria and viruses in circulation (Barrington et al., 2001), enhances phagocytosis  of  pathogens  (Navin  et  al.,  1989),  and  activates  the  complement  system (Austem et al., 2003). In addition, IgM antibody–antigen complexes are efficiently filtered in the spleen and lymph nodes (Ochsenbein et al., 1999) to prime the immune response.

1.9.3.3 Immunoglobulin E (IgE) is the immunoglobulin isotype that has the lowest abundance in vivo and its levels are tightly regulated. Concentrations of free serum IgE are

~50–200 ng per ml of blood in healthy humans compared with ~1–10 mg per ml of blood for other immunoglobulin isotypes; a similar proportion of immunoglobulins are also present in mice  (Dullaers, 2012).  In  addition,  the  serum  half-life  of  IgE  is  the  shortest  of  all immunoglobulin  isotypes,  ranging  from  ~5–12 hours  in  mice  to  ~2 days  in  humans, compared with ~10 days in mice and ~20 days for IgG in humans (Dullaers, 2012). IgE binds to  IgE  receptors  on  immune  cells  in  the  tissues  and  in  the  circulation  (Gould  and Sutton, 2008). There are two receptors for IgE: the high-affinity Fc receptor for IgE (FcεRI) and the low-affinity Fc receptor for IgE (FcεRII; also known as CD23) (Acharya, 2010). FcεRI is expressed by mast cells and basophils, on which its activation mediates cellular degranulation, eicosanoid production and cytokine production (Kraft and Kinet, 2007). In humans, FcεRI is also expressed by dendritic cells and macrophages, on which its activation mediates the internalization of IgE-bound antigens for processing and presentation on the cell surface, as well as the production of cytokines that promote T helper 2 (TH2)-type immune responses (Kraft and Kinet, 2007). FcεRII is expressed by B cells, on which it regulates IgE production and facilitates antigen processing and presentation.

1.9.4. T lymphocytes

T, B, natural killer cells and their respective subsets originate from the bone marrow-derived progenitors. Progenitors that migrate to the thymus and receive signals through the Notch receptor commit to the T-cell lineage (La Motte-Mohs et al., 2005). In human beings, lineage development is critically dependent on IL-7 for T cells (Blom and Spits, 2006) and IL-5 for natural killer cells (Freud and Caligiuri, 2006). Mature T lymphocytes, known as naïve T cells, circulate through blood and the lymphatic system. Naïve T cells are those that have not yet encountered foreign antigen and have not yet been activated. APC generate antigenic peptides from a pathogenic agent or a self-Ag by antigen processing, and display them on the cell surface in the context of MHC molecules (Chen, 2004). Recognition of MHC-peptide complexes on the antigen presenting cells by Naïve T cells leads to their activation. The activated T cells rapidly proliferate (clonal expansion), migrate through the tissues to the sites of Ag presence, and perform effector functions such as cell-mediated cytotoxicity and production of various cytokines (soluble mediators of the immune response) (Nel, 2002).

1.9.4.1 T cell subsets

Thymic selection results in the appearance of T cells with two types of TCR. The majority express Ag-binding αβ chains in the TCR, which are disulfide-linked heterodimers of Ig superfamily proteins, forming unique structures on each T cell. T cell receptor complex consists of αβ heterodimers responsible for antigen recognition and CD3 molecules involved in   intracellular   signaling.   Immunoglobulin-like   αβ   chains   are   formed   upon   gene rearrangement and have high variability among individual T cells. Non-polymorphic CD3 chains (ζ, δ, ε, γ) contain intracellular immunoreceptor tyrosine-based activation motifs (ITAMs) initiating cascades of signal transduction. αβTCR T cells are subdivided into several groups on the basis of lineage markers and functional activities. Two major surface co- receptor molecules, CD4 and CD8, define two separate T cell lineages with different functions.

1.9.4.2 CD4+ helpers

CD4+ cells recognize antigens in the context of MHC class II molecules (only expressed on so-called professional APC such as B cells, macrophages and dendritic cells) and produce cytokines as effector T helper cells (Germain, 1994). Activated CD4+ T helper cells can be subdivided into Th1, Th2, Th17 and Treg subsets (Weaver et al., 2006). Th1 cells produce mainly IFN-γ, IL-2, TNF-α, (figure 5) and lymphotoxin. Experiments in mice revealed that loss of the Th1–IFN-γ pathway does not confer resistance to inflammatory autoimmunity and

suggests the presence of an additional pathogenic effector T-cell subset. Th1 cells enhance pro-inflammatory cell-mediated immunity and were shown to induce delayed-type hypersensitivity (DTH), B cell production of opsonizing isotypes of IgG, and mediate the response to some protozoa like Leishmania and Trypanosoma. Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13 and promote non-inflammatory immediate immune responses; they have been shown to be essential in B cell production of IgG, IgA, and IgE (Soumelis et al.,

2002).They derive B-cell proliferation by IL-4 and contact-dependent CD40 ligand: CD40 binding, augmenting humoral defenses against extracelluar pathogens. Furthermore, IL-4 and IL-5 enable IgE production and eosinophilic inflammation, important for the clearance of helminthic infestations, but highly relevant to the allergic response (Weaver et al., 2006). It was found that IL-23 induced production of CD4+  T cells that secrete proinflammatory cytokine IL-17A (Palmer and Weave, 2010). Contact with IL-17 leads to production by the latter cell types of IL-6 and chemokines like chemokine (CXC motif) Ligand 8, (CXCL8) and chemokine (CXC motif) Ligand 2 (CXCL2) and granulocyte macrophage colony stimulating factors (GM-GSF). This leads to recruitment of neutrophils and macrophages into the site of infection and enhances the bone marrow production of these cells. IL-22 produced by Th17 cells co-operates with IL-17 in the induction of antimicrobial peptides, such as β-defensins in epidermal keratinocytes, thereby enhancing the innate acute inflammatory response in infection (Sakaguchi, 2005). IL-4 in combination with STAT6 and GATA-3 generates Th2 cells (figure 5).  Th17 cells develop in the presence of TGF-β,  IL-6  and  IL-23  and are characterized by the transcription factors RAR-related orphan rececptor gamma (RORγt), RAR-related orphan rececptor alpha(RORα) and signal transducer and activator of transcription 3 (STAT3). Recently, Th9 cells also were proposed, a subset that develops under the influence of IL-4 and TGF-β and that produces IL-9 (Veldhoen et al., 2008). Colitis in mice is produced by Th1, Th2, Th17 and Th9 cells. T follicular helper cells (TFH) can mediate the pathogenic antibody response in experimental lupus models (Linterman et al.,

2009).

1.9.4.3 CD8+ cytotoxic T lymphocytes

CD8+  lymphocytes are activated by antigenic peptides presented by MHC class I molecules (expressed on all nucleated cells) and form effector cytotoxic T lymphocytes (CTL). CD8+ lymphocytes also can be assigned to Tc1 or Tc2 subsets according to their cytokine profile (Croft et al., 1994), although they do not produce the same quantities of cytokines as CD4+ helpers and are not efficient in B cell activation (Croft et al., 1994). On priming, CD8+  T

cells produce cytotoxic proteins including perforin and granzymes and secrete them at the point of contact with the target cell (immunologic synapse), resulting in specific killing without bystander cell damage. In addition to cytolysis, CD8+  effectors produce IFN-γ and TNF. In mice, the transcription factors T-box expressed in T cells (T-bet) and Eomesodermin (Eomes) function in a cooperative and partially redundant  way to  regulate CD8+  T-cell differentiation (Intlekofer et al., 2005). CTL cytotoxicity can be mediated by two distinct pathways namely: Ca2+-dependent perforin/granzyme-mediated apoptosis, and Ca2+- independent Fas ligand/Fas-mediated apoptosis. Both pathways are mediated through TCR signaling   (MacDermott   et   al.,   1985).   In   Ca2+-dependent   perforin/granzyme-mediated apoptosis pathway, granzymes enter into the target cell directly through plasma membrane pores formed by perforin (PFN) or through receptor-mediated endocytosis. Perforin mediates the translocation of granzymes from endocytic vesicles into the cytosol. Proteoglycan serglycin serves as a chaperone of perforin until the complex reaches the plasma membrane of the target cells (MacDermott et al., 1985). Fas-mediated apoptosis is initiated through the interaction  between  CD95  (Fas)  and  CD95L  (FasL).  TCR-mediated  activation  induces CD95L expression on the CTL. Binding of CD95 on the target cells will induce sequential caspase activation, leading to apoptosis (MacDermott et al., 1985).

1.9.4.4 Regulatory T cells

Regulatory T cells (Treg), include more than one cell type critical in the maintenance of peripheral tolerance, down-modulation of the amplitude of an immune response, and prevention of autoimmune diseases. The majority of Treg appears within the CD4+ T cell set, suppressor activity was also reported among CD8+ T cells. Natural regulatory T cells (nTreg) arise from  the thymus  and  represent  about  10% of the total  CD4 population.  Foxp3  is essential in the development and function of nTreg. The absence of functional Foxp3 results in severe systemic autoimmune diseases in mice and man. Foxp3 inhibits il-2 transcription and induces up-regulation of Treg-associated molecules, such as CD25, CTLA-4 and GITR (Shevach and Stephens, 2006), that can down-regulate the immune response of adjacent cells. Type 1 regulatory T cell (Battaglia et al., 2006) depend on il-10 for their induction and their suppressive action, whereas Th3 cells (Weiner, 2001) depend on TGF-β for their suppressive action. The inhibitory effect of all Treg primarily requires stimulation of the TCR. Upon activation,  cells  may  mediate  their  function  via  direct  cell  contact  through  inhibitory molecules such as CTLA4, but they may also function via secretion of IL-10 and TGF-β. IL-

10 can suppress differentiation of Th1 and Th2 cells directly by reducing IL-2, TNF-α and

IL-5 production, and also indirectly by down-regulating MHC and costimulatory molecules on APC, thereby reducing T cell activation. The mechanism of suppression will most likely depend on the type of regulatory T cell, the nature of the immune response, the antigen and the site of inflammation (Vignali et al., 2008).

1.9.5 Hepatitis b virus infection

Hepatitis B Virus (HBV) is a DNA virus and was first identified in the 1960s. According to the  International  Committee  on  Taxonomy  of  Viruses  (ICTV)  classification,  this  virus belongs to the genus Orthohepadnavirus of the Hepadnaviridae family and along with the Spumaretrovirinae subfamily of the Retroviridae family, represents the only other animal virus with a DNA genome known to replicate by the reverse replication of a viral RNA intermediate (Seeger et al., 2007). Bowyer et al. (2011), demonstrated that hepatitis b virus is a blood-borne virus and is roughly 75-200 times more infectious than Human Immunodeficiency Virus (HIV). The virion (figure 9) consists of a viral envelope, nucleocapsid and a single copy of the partially double-stranded DNA genome. The nucleocapsid  is  comprised  of  120  dimers  of  core  protein  and  is  covered  by  a  capsid membrane embedded with 3 viral envelope proteins, the large (L), middle (M) and small (S) surface proteins (Seeger et al., 2007).

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