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i EVALUATION OF THE IMMUNOMODULATORY ACTIVITIES OF THE AQUEOUS EXTRACT AND THE BETA–D-GLUCAN-RICH POLYSACCHARIDE FRACTION OF Pleurotustuberregium(Pleurotaceae) BY IHIM, STELLA AMARACHI PG/MPHARM/13/64775 DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA APRIL, 2015. ii TITLE PAGE EVALUATION OF THE IMMUNOMODULk2ATORY ACTIVITIES OF THE AQUEOUS EXTRACT AND THE BETA–D-GLUCAN- RICH POLYSACCHARIDE FRACTION OFPleurotustuberregium (Pleurotaceae) iii CERTIFICATION IHIM, STELLA AMARACHI, a postgraduate student in the Department of Pharmacology and Toxicology with registration number PG/MPHARM/13/64775has satisfactorily completed the requirements for the award of the degree of Master of Pharmacy (M.PHARM) of the Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka. The work embodied in this dissertation is original and has not been submitted for the award of any other diploma or degree in this or any other University. ---------------------------------------Dr. C.SNworu Dr.T.C Okoye (Supervisor) ----------------------------------------(Head of Department) iv DEDICATION This work is dedicated to God Almighty for his mercies and love in my life and to my supervisor, Dr.ChukwuemekaS. Nworu,for his fatherly support in making this work a success. v ACKNOWLEDGEMENTS I wish to express my profound gratitude to my supervisor, DrChukwuemekaNworufor making this research work a reality. I also want to appreciate Prof. Charles Okoye, Prof. P. A.Akah, Prof. C.O.Esimone, Dr. T.COkoye, Dr. (Mrs) A. C. Ezike, Rev (Pharm) E.Ezea,Dr (Mrs)Odoh, Dr. NickObitte, Mr Austin Okorieand other academic, technical and administrative staff in the Department of Pharmacology, University of Nigeria, Nsukka for the part they played in making this work successful. I will want to appreciate my colleagues and friends - Ife, Nenye, Azom, Lydia, Lawrence, Victor, Ebuka, Lovelyn, EzeChinenye, Chigo, Ebere, Peter, Oge, Nnamdi, Uche, Chika, John and others too numerous to mention for their effort and encouragement in making this master’s programme a reality. I wish to acknowledge the help of the academic and technical staff of Pharmaceutical Microbiology and Biotechnology,Faculty of Pharmaceutical Sciences,NnamdiAzikiwe University (Agulu Campus) I will not fail to acknowledge and thank my family; Engr and Mrs George Ihim- my Parents, my siblings- Miki, Urchman, Victor, Chioma, Promise and my friend Kelvin for their support, understanding and prayers during this period. Finally, I thank the Almighty God for his favour, for the good health and for everything He has done for me. Ihim, StellaAmarachi April, 2015 vi ABSTRACT Mushroom cuisines are global delicacies and have been used for decades, not only as food, but mostly for the health benefits which are claimed or scientifically proven. The present study explored the immunomodulatoryactivites of the hot aqueous extract of a local oyster mushroom, Pleurotustuberregium (Fr.) Singer (Pleurotaceae) (PT) and its β–D-Glucan-Rich Polysaccharide fraction (BGP).The effects of the aqueous extract (PT) and the β–D-Glucan-Rich Polysaccharide fraction (BGP) were tested on some specific and non-specific immune responses in immunecompetent mice and in culture of RAW 264.7 macrophage cells. The effect of the PT and BGP on specific cell mediated immune response was investigated by the delayed type hypersensitivity response (DTHR) whilethe effect of the extract on specific humoral immune responses of treated mice was determined by prime-boost immunization protocol using ovalbumin as antigen and followed by the determination of antibody titers in the sera using the enzyme-linked immunosorbent assay (ELISA).Their effect on non–specific immune responses was determined by the colloidal carbon-clearance assay in mice. The effect of BGP on functional maturation and activation of the monocytic cells was also determined by measuring the level of tumour necrosis factor (TNF)-α and inducible oxygen (iNO) expressed into culture supernatant using cytokine capture ELISA and Griess reagent respectively.Short-term oral administration of PT (100, 200 and 400mg/kg) or BGP (100 and 200mg/kg) elicited a dose-related increase in DTHR and increased the mean phagocytic clearance of colloidal carbon in mice as much as 7 – 10 fold when compared to the clearance in the untreated group of mice. In a homologous prime-boost immunization schedule, oral supplementation with PT (100, 200 and 400mg/kg) or BGP (100 and 200mg/kg) significantly (P<0.05) increased primary and secondary ovalbumin (OVA) – specific total IgG, IgG1 and IgG2a titres in treated mice as much as 2 – 4 folds compared to the untreated control. In cell culture, stimulation of RAW 264.7 macrophages with BGP (5-1000 µg/ml) induced a remarkable and significant (P<0.05) proliferation of total spleenocytes in a concentration-related manner. Stimulation of RAW 264.7 cells with BGP (5-100 µg/ml) significantly (P<0.05) increased the levels of expressed TNF-α and iNO in culture. Similarly, prestimulation of RAW 264.7 cells significantly (P<0.05) increased the phagocytic capacity of the macrophages in a neutral dye uptake assay. Acute toxicity tests revealed that even at doses of PT up to 5000mg/kg body weight (per os), there was no lethality or any sign of acute intoxication in the mice after 24 h. Put together, the result of these investigations suggest that hot aqueous extract of P tuberegium (PT) and its β–D-Glucan-rich polysaccharide fraction (BGP) possess some immunomodulatory activities in mice and could be further investigated and harnessed for therapeutic purposes in immunodeficiency conditions. vii TABLE OF CONTENTS TITLE PAGE - - - - - - - - - - i CERTIFICATION - - - - - - - - - iii DEDICATION - - - - - - - - - iv ACKNOWLEDGMENTS - - - - - - - - v ABSTRACT - - - - - - - - - vi TABLE OF CONTENTS - - - - - - - - vii LIST OF FIGURES - - - - - - - - - xi LIST OF TABLES - - - - - - - - xiii - - - - - - - - xiv - LIST OF APPENDICES CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1.1 Scientific Background - 1.2 - - - - - - 1 Overview of the Immune System - - - - - 3 1.2.1 Organs of the Immune System - - - - - 3 1.2.1.1 Primary Lymphoid Organs - - - - - 3 1.2.1.2 Secondary Lymphoid Organ - - - - - - 5 1.2.2 Cells of the Immune System - - - - - 6 1.2.2.1 Lymphoid Stem Cells - - - - - - - 9 1.2.2.2 Myeloid Stem Cells - - - - - - 11 Arms of the Immune System - - - - - - - - - - 14 - - - - 23 - - - - - 26 - - - - - 28 - - - - - 29 1.2.3 - 1.2.3.1 Innate Immunity - - 1.2.3.2 Adaptive Immunity - - - 1.2.4 Overview of Antibodies 1.2.4.1 Classes of Immunoglobulin 1.2.4.2 Roles of Antibodies 1.3 Mediators of the Immune System - - - - - 31 1.3.1 Cytokines - - - - - - 31 1.3.2 Complement System - - - - - 35 Disorders of the Immune System - - - - - 38 1.4.1 Hypersensitivity - - - - - - 38 1.4.2 Immune Deficiency Diseases - - - - - - 39 1.4 - - - - - 14 - viii 1.4.3 Autoimmune Diseases - - - - - - 41 1.4.4 Graft Versus Host Diseases - - - - - - 42 1.4.5 Immune Complex Diseases - - - - - 42 1.5 The Concept of Immunomodulation, Immunosuppression, Immunostimulation and - Immunotolerance - - - - - - - 43 1.5.1 Immunostimulation - - - - - - - 43 1.5.2 Immunosuppression - - - - - - - 44 1.5.3 Tolerance - - - - - - 45 1.6 Potentials of Mushroom as Immunomodulatory Substance of Natural Origin45 1.7 Beta (B) Glucans - - - - - 48 1.7.1 Beta Glucans and the Immune System - - - - 48 1.7.1.1 Beta GlucanImmunostimulating Activity - - - - 49 1.7.1.2 Beta Glucan Increases Resistance to Infectious Challenge - - 50 1.7.1.3 Beta GlucanAnticarcinogenic Activity - 51 1.7.1.4 Beta Glucan as Adjuvant to Cancer Chemotherapy and Radiotherapy 51 1.8 Botanical Profile and Review Of Pleurotustuberregium - - 52 1.8.1 Taxonomy of Pleurotustuberregium - - - - 53 1.8.2 Botanical Description of Pleurotustuberregium - - - 53 1.8.3 Geographical Distribution of Pleurotustuberregium - - - 55 1.8.4 Ethnomedicinal and Folkloric Uses of Pleurotustuberregium - 55 1.8.5 Pharmacological Studies, Phytochemical and Proximate Constituents - ofP. tuberregium - - - - - - - - - - - - - - 56 CHAPTER TWO: MATERIALS AND METHODS 2.1 Materials 2.1.1 - - - - - - - - 57 Chemicals and Reagents - - - - - - 57 2.1.2 Equipment - - - - - - - - 57 2.2 Method - - - - - - - - 57 2.2.1 Collection and Authentication of Plant Materials - - - 57 2.2.2 Preparation of Plant Material - - - - 58 2.2.3 Bioactivity Guided Fractionation of Crude Extract - - - 58 2.2.4 Extraction of the β glucan rich Polysaccharide Fraction of - - ix - - - - - 60 2.2.5 Phytochemical Analysis of Extracts - - - - - 60 2.2.6 Pharmacological Studies - - - - - - 61 2.2.6.1 Animals - - - - - - - - 61 2.2.6.2 Antigens - - - - - - - - 61 2.2.6.3 Acute Toxicity (LD50) Test of Extracts - - - - 61 2.2.7 Studies on the Hot Aqueous Extract of Pleurotustuberregium(PT) Pleurotustuberregium - - and the βGlucan Enriched Polysaccharide Fraction of Pleurotus tuberregium(BGP) - - - - - - - 62 2.2.7.1 Studies on Relative Spleen Weight - - - - - 62 2.2.7.2 Studies on Phagocytic Index - - - - - - 62 2.2.7.3 Studies on Delayed Type Hypersensitivity Response (DTHR) - 63 2.2.7.4 Studies on Humoral Antibody Response Induced by Ovalbumin - 63 2.2.8 In vitro Studies 2.2.8.1 Spleenocytes Proliferation Assay - - - - - - 65 - - - - - 66 2.2.8.2 Effect of BGP on Inducible Nitric Oxide (iNO) Production/Release - 66 2.2.8.3 Effect of BGP on Tumour Necrosis Factor (TNF- Α) Production /Release - - - - - - - - 67 2.2.8.4 Effect of BGP on Rate of Phagocytosis of Macrophages - - 67 2.9.0 Statistical Analysis - - - - - - - 68 - - - - - - - 69 CHAPTER THREE: RESULTS 3.1 Extraction 3.2 Phytochemical Analysis of Extract and Fraction - - - 69 3.3 Acute Toxicity Test - - - 69 3.4 Effect of Aqueous Extract of Pleurotustuberregium on Phagocytic Activity in Mice - 3.5 - - - - - - - - - - 69 - - - - - - - 73 The Effect of Pleurotustuberregium Extracts on Delayed Type Hypersensitivity response (DTHR) 3.7 - - The Effect of Hot Aqueous Extract of P. tuberregium on the Relative Spleen Weight ofMice 3.6 - - - - - - - The Effect of Hot aqueous extract (PT) and the β Glucan rich - 75 x Polysaccharide Fraction of Pleurotustuberregium(BGP)on Humoral Antibody Synthesis Induced by Ovalbumin 3.7.1 - - - 78 The Effect of Hot of Aqueous Extract of Pleurotustuberregium on Primary and Secondary Humoral Immune Responses to Ovalbumin in Mice 3.8 - - - - - - - - 78 Effect of Βeta (β)-Glucan rich Polysaccharide Fraction of Pleurotustuberregium (BGP) on Primary and Secondary Humoral immune Responses to Ovalbumin in Mice - - - - - 82 3.9 Invitro studies - - - - - - 89 3.9.1 Effect of Βeta (β)-Glucan rich Polysaccharide Fraction of P. - - tuberregium (BGP) on Spleenocytes Proliferation - - - 89 3.9.2 Efect of BGP ON TNF-α Production by RAW264.7 - - - 89 3.9.3 Effect of BGP on Nitric Oxide Production by Raw264.7 - - 90 3.9.4 Effect of BGP on the rate of Phagocytosis (Neutral Red Uptake) RAW264.7 - - - - - - - - 90 CHAPTER FOUR: DISCUSSION AND CONCLUSION 4.1 Discussion - - - - - - - - 95 4.2 Conclusion - - - - - - - - 103 References Appendices xi LIST OF FIGURES Figure 1: The immune system cells - - - Figure 2: The Fruiting Bodies of Pleurotustuberregium - - - - 8 - - - - 54 Figure 3: Schematic Representation of the Extraction of Pleurotustuberregium (PT) and its Beta Glucan Polysaccharide Enriched Fraction (BGP) - - 59 Figure 4: The Effect of Hot Aqueous Extract of Pleurotustuberregium(PT) on Phagocytic Index in Swiss Albino Mice - - - - - - 72 Figure 5: The Effect of Hot Aqueous Extract of P. tuberregium on the Relative Spleen Weight of Mice - - - - - - - - 74 Figure 6: The Effect of Hot Aqueous Extract of Pleurotustuberregium (BGP) on Delayed Type Hypersensitivity Response in Mice - - - - - 76 Figure 7: The Effect of Beta (β) GlucanrichPolysacharide Fraction of Pleurotustuberregium (BGP) on Delayed Type Hypersensitivity in Mice - - - 77 Figure 8: The Effect of Hot Aqueous Extract of P. tuberregium (PT) on Ovalbumin Specific Total IgG Response in Mice - - - - - - 79 Figure 9: The Effect of Hot Aqueous Extract of P. tuberregium (PT) on ovalbumin-specific total IgG1 response in mice - - - - - - 80 Figure 10: The Effect of Hot Aqueous Extract of P. tuberregium (PT) on ovalbumin-specific total IgG2a response in mice - - - - - - 81 Figure 11a: The Effect of Β-Glucan Rich Polysaccharide Fraction of P. tuberregium on Ovalbumin – Specific Total IgG Primary Response in Mice - - 83 Figure 11b: The Effect of Βeta (β) Glucan Rich Polysaccharide Fraction ofP. tuberregium on Ovalbumin – Specific Total IgG Secondary Response in Mice - - 84 Figure 12a: The Effect of Βeta (β) GlucanRich Polysaccharide Fraction ofP. tuberregium on Ovalbumin – Specific IgG1 Primary Response in Mice - - - 85 Figure 12b: The Effect of Βeta (β) Glucan Rich Polysaccharide Fraction of P. tuberregiumon Ovalbumin – Specific IgG1 Secondary Response in Mice - - 86 Figure 13a: The Effect of Beta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium on Ovalbumin – Specific IgG2a Primary Response in Mice - - - 87 xii Figure 13b: The Effect of Beta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium on Ovalbumin – Specific IgG2a Secondary Response in Mice - - 88 Figure 14: The Effect of Beta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium(BGP) on Spleenocytes Proliferation - - - - - - 91 Figure 15: The Effect of Beta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium(BGP) onTNF-α Production by RAW264.7 - - - - - 92 Figure 16: The Effect of Beta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium(BGP) on Nitric Oxide Production by RAW264.7 - - - - 93 Figure 17: The Effect of Beta Glucan Rich Polysaccharide Fraction of P. tuberregium on Rate of Phagocytosis by Raw 264.7 - - - - - - 94 xiii LIST OF TABLES Table 1: Sources and Activity of Cytokines - - - - - - 34 Table 2: The Biological Functions of Complement and its Fragments - - 37 Table 3: Some Mushrooms with Identified Immunomodulatory Activities - - 47 Table 4: The Yield and Phytochemical Constituents of Immune Active Extracts and Fractions of Pleurotustuberregium - - - - - - 71 xiv LIST OF APPENDICES Appendix 1: The Effect of Hot Aqueous Extract of Pleurotustuberregium (PT) on Phagocytic Activity in Swiss Albino Mice - - - - 127 Appendix 2: The effect of Hot Aqueous Extract of P. tuberregium on the Relative Spleen Weight of Mice - - - - - - - 128 Appendix 3: The Effect of Hot Aqueous Extract of P. tuberregium on Delayed Type Hypersensitivity Response in Mice - - - - 129 Appendix 4: The Effect of BGP on Delayed Type Hypersensitivity Response in Mice 130 Appendix 5: The Effect of Hot Aqueous Extract of Pleurotustuberregium (PT) on Primaryand Secondary IgGHumoral Antibody Response to Ovalbumin Mice 131 Appendix 6: The Effects of Hot Aqueous Extract of Pleurotustuberregium (PT) on Primaryand Secondary IgG1 Humoral Antibody Response to Ovalbumin in Mice 132 Appendix 7: The Effects of Hot Aqueous Extract of Pleurotustuberregium (PT) on Primary and Secondary IgG2a Humoral Antibody Response to Ovalbumin in Mice 133 Appendix 8: The Effects of ΒGP on Primary and Secondary Total IgGHumoral Antibody Response to Ovalbumin in Mice - - - - 134 - 135 Appendix 9: The Effects of ΒGP on Primary and Secondary Total IgG1 Humoral Antibody Response to Ovalbumin in Mice - - - Appendix 10: The Effects of BGP on Primary and Secondary IgG2a Humoral Antibody Response to Ovalbumin in Mice - - - - 136 - - - 137 - - 138 Appendix 13: The Effect of BGP on Nitric Oxide Production by RAW264.7 - 139 Appendix 14: The Effect of BGP on Rate of Phagocytosis RAW264.7 - 140 Appendix 11: The Effect of BGP on Spleenocytes Proliferation Appendix 12: The Effect of BGP on TNF-α Production by RAW264.7 - xv CHAPTER ONE INTRODUCTION AND REVIEW OF LITERATURE 1.1 Scientific Background The immune system is a network of cells,tissues and organs that work together to defend the body against attacks by “foreign invaders”. The human body provides an ideal environment for many microbes. The immune system consists of two categories of defense mechanisms- the innate (non- specific) and the adaptive (specific) systems (Janeway et al., 2005). Scientists continue to study how the body launches attacks that destroy invading microbes. The emergence of acquired immunodeficiency syndrome (AIDS) and the concern about bioterrorism have also increased the emphasis on the role of the immune system in defending individuals against infections. The two mechanisms of the immune system can be modified by substances to either enhance or suppress their ability to resist invasion by pathogens (William, 2001). There are however, limited strategies available to efficiently modulate the immune response. Nutritional interventions that involve optimizing the intake of essential nutrients and utilizing promising functional foods have become an increasingly favoured approach to the modulation of immune cell function. Mushrooms have long been suggested to possess immunomodulatory properties. More recent studies, however, report that oral supplementation with a variety of different mushroom species are effective in modulating certain immune functions.It has also been observed that immunomodulatory substances of natural origin could play a significant role in human disease prevention and treatment (Dixon, 2001) Of significant relevance and importance is the ability of particular mushroom-derived compounds to modulate the human immune response and to inhibit certain tumour growths (Wasser and Weis, 1999a, 1999b). Medicinal mushroom research has focused on discovering compounds that can modulate xvi positively or negatively the biologic response of immune cells. Thosecompounds which appear to stimulate the human immune response are being sought for the treatment of cancer, immunodeficiency diseases, or for generalized immunosuppression following drug treatment; for combinational therapy with antibiotics; and as adjuncts for vaccines (Jong and Birmingham, 1992). Those compounds that suppress immune reactions are potentially useful in the remedy of autoimmune (an abnormal immune response against self-antigens) or certain gastro-intestinal tract diseases (e.g. Crohns) (Badger, 1983).Compounds that are capable of interacting with the immune system to upregulate or downregulate specific aspects of the host response can be classified as immunomodulators. One of the most significant factors of many of the derived bioactive polymers from medicinal mushrooms is their role as immunomodulators (Yamaguchi, 1992). P. tuberregium is a popular edible mushroom and has been considered a profound health promoting mushroom in traditional Chinese medicine (Isikhuemhen et al., 2000a, 2000b; Huang, 2002). It is used in the preparation of cures for headache, stomach ailments, chest pain, colds and fever, asthma, small pox, and high blood pressure (Okhuoya and Okogbo, 1991; Fasidi and Olorunmaiye, 1994; Alobo, 2003).Pharmacologically, P. tuberregium polysaccharide attenuates hyperglycemia and oxidative stress in experimental diabetic rats (Hui-Yuet al., 2012). For most other species of Pleurotus, several medicinal properties have been reported. They include properties attributable to their polysaccharides; antigenotoxic, biomutagenic activities (Fillipie and Umek, 2002) anti-inflammatory activity, antilipidemic, antihypertensive and antihyperglycemic activities (Huet al., 2006), antibacterial and antifungal activities (Hu et al., 2006; Lee et al., 2010;Dahech et al., 2011; Wong et al., 2011)as well as hepato- and nephro-protective effects (Nworu et al., 2014). However there is dearth of scientific information on the effect of P. tuber-regium on the immune system. 1.2 Overview of the Immune System xvii The architecture of the immune system is multilayered with defenses on several levels. Basically, there are two divisions of immune protection - the natural (innate/nonspecific) and the adaptive (specific) mechanisms (Abbas and Lichtman, 2004). Both systems consist of a multitude of cells and molecules that interact in a complex manner to detect and eliminate pathogens, basically distinguishing between nonself and self(Matzinger, 1994; Matzinger, 1998). The immune system is closely tied to the lymphatic system. The term innate immunity is sometimes used to include physical, chemical and microbiological barriers, but more usually encompasses the elements of the immune system (Neutrophils, Monocytes, Macrophages, Complement, Cytokines and acute phase proteins) which provide immediate host defense. The highly conserved nature of the response which is seen in even the smallest animals, confirms its importance in survival. Adaptive immunity is the hall mark of the immune system of higher animals. Its response consists of antigen specific reactions through T lymphocytes and B lymphocytes (Delves and Roitt, 2000). 1.2.1 Organs of the Immune System Several organs existing in the human body bear responsibility for the synthesis and maturation of immunocompetent cells. These include the bone marrow, thymus and lymph nodes. 1.2.1.1 Primary Lymphoid Organs The primary lymphoid organs (bone marrow and thymus)are concerned with production and maturation of lymphoid cells. a. Bone Marrow xviii All the cells of the immune system are initially derived from the bone marrow through a process known as hematopoiesis. During foetal development hematopoiesis occurs initially in yolk sac and para-aortic mesenchyma and later in the liver and spleen. This function is taken over gradually by the bone marrow. During hematopoiesis, bone marrow derived stem cells differentiate into either mature cells or into precursor of cells that migrate out of the bone marrow to continue their maturation in thymus. The bone marrow produces B cells, natural killer cells, granulocytes and immature thymocytes, in addition to red blood cells and platelets (Fu and Chaplin, 1999). Other white blood cells produced in the marrow include neutrophils, basophils, eosinophils, monocytes, and lymphocytes.The bone marrow also contains antibody secreting plasma cells which have migrated from the peripheral lymphoid tissue. b. Thymus The thymus is a gland located in the anterior mediastinum just above the heart which reaches its greatest size just prior to birth then atrophies with age. Immature lymphocytes begin to accumulate in the thymus of human embryos at about 90 -100 days after fertilization. Initially, most of these immature lymphocytes have come from the yolk sac and foetal liver rather than the bone marrow. Cells from the bone marrow later migrate to the thymus as precursors and develop into mature thymocytes through a remarkable maturation process sometimes referred to as “thymus Education”. Mature T cells migrate from the thymus into the blood stream (Fu and Chaplain, 1999; Abbas and Lichtman, 2004) and some to secondary lymphoid organs such as lymph node, peyer’s patches and spleen. About 98% of all T cells die in the thymus. The greatest rate of T cells production occurs before puberty. After puberty, the thymus shrinks and the production of new T cells in the adult thymus drops away. Children with no development of the thymus suffer from Digeorge’s syndrome that is characterized by deficiency in T cell development but normal number of B cells. xix 1.2.1.2 Secondary Lymphoid Organ The secondary lymphoid organ plays an important role in the immune system. The secondary or peripheral lymphoid organs are the lymph nodes and the spleen. Others are the Tonsils, Peyer’s patch. They are sites where lymphocytes localize, recognize foreign antigen and mount response against it. a. Spleen The spleen is situated in the upper left quadrant of the abdomen. It is the largest single lymphoid organ in the body. It is an immunologic filter of the blood. The spleen is ductless, vertebrate gland that is closely associated with the circulatory system where it functions in the destruction of old reds blood cells in holding a reservoir of blood. In addition to capturing foreign materials (antigens) migratory macrophages and dendritic cells, bring antigen to the spleen via the blood stream. An immune response is initiated when the macrophages or dendritic cells present the antigen to the appropriate B or T cells. In the spleen, B cells become activated and produce large amounts of antibodies (Abbas and Lichtman, 2004; USC, 2006). b. Lymph Nodes These are small oval shaped structures located along lymphatic vessels throughout the body. They are about 1-25mm in diameter. It functions as an immunologic filter for the body fluid known as lymph composed mainly of T cells, B cells, dendritic cells, and macrophages. The nodes drain fluid from most body tissues. Antigens are filtered out of the lymph in the lymph node before returning the lymph to the circulation. In a similar fashion as the spleen, the macrophages and dendritic cells that capture antigens present these foreign materials to T cells and B cells consequently an immune response is initiated (Abbas and Lichtmann, 2004; USC, 2006). c. Tonsils xx The tonsils are often the first organ to encounter pathogens and antigens that come into the body by mouth or nose. There are basically three pairs of tonsils in a ring above the pharynx. The fundamental immunological roles of tonsils have yet to be understood. Tonsil size may have a more significant impact on upper airway obstruction for obese children than those of average weight (Wang et al., 2010). d. Peyer’s Patches This is located in the wall of the intestine and the appendix, attached to the cecum of the large intestine. It intercepts pathogens that come into the body through the intestinal tract. In adults, lymphocytes predominate in peyer’s patch. T cell exposed to antigen in peyers patches also migrate into the lamina propria and the epithelium, where they mature to cytotoxic T cells, providing another mechanism for containing microbial assaults (Nagler, 2001). 1.2.2 Cells of the Immune System All cells of the immune system are derived from pluripotent stem cells in the bone marrow by a process known as haematopoiesis. Under the influence of cytokines, the pluripotent stem cells may become a lymphoid stem cell or a myeloid stem cell. The lymphoid stem cell develops into B cells and T cells. Natural killer (NK) cells are lymphocytes that act in a similar manner to cytotoxic T cells. These cells, however, are not T cells (USC, 2006). The lineage of the Nk cells is not well understood. The myeloid stem cell develops into platelets, red blood cells or the granulocyte-monocyte line. Monocytic cells include monocytes and macrophages. The granulocytes include neutrophils, eosinophils, mast cells and basophils. Monocytes circulate in the blood and lymphatics but become macrophages when they enter the tissues. The development of these cells from a stem cell involves cytokine growth factors and the development of a progenitor cell in each line. The progenitor cell is a committed cell, meaning that it is committed to that line of cell growth (A xxi neutrophil progenitor must become a neutrophil, it cannot become an eosinophil for instance) (USC, 2006)(Figure 1). xxii Figure 1: The immune system cells xxiii 1.2.2.1 Lymphoid Stem Cells a. B Cells/Lymphocytes The development and maturation of this cell group occurs in the bone marrow. In mammals, the early stage of Bcell maturation occurs in the fetal liver and the bone marrow. B cell development begins in the fetal liver and continues in the bone marrow throughout life. They account for 5 -15% of lymphocytes in circulation and 80 -90% in bone marrow, 20% -30% in lymph node and 50 -60% in the spleen. The most important surface marker on the surface of mature B cell is the surface immunoglobulin usually of IgM and IgD type(Roitt and Delves, 2001). The major function of B lymphocytes is the production of antibodies in response to foreign proteins of bacteria, viruses and tumor cells. Antibodies are specialized proteins that specifically recognize and bind to one particular protein. Antibody production and binding to a foreign substance or antigen, often is critical as a means of signaling other cells to engulf, kill or remove that substance from the body (Roitt and Delves, 2001). B cells may differentiate into plasma cells (which secrete large amounts of antibodies) or into memory B cells. Memory cells can survive 20years or more. Plasma cells are the effector cells of the B cell lineage and are specialized in secreting immunoglobulin. When activated, B cells divide; some of its progeny become memory cells and the remainder becomes immunoglobulin- secreting plasma cells (Allman et al., 2004). b. T Cells T cell is an abbreviation of “thymus dependent lymphocytes”. T lymphocytes arise in the bone marrow as T cell precursors then migrate to and mature in the thymus (A lymphoid organ beneath the breast bone). After entry into the thymus, the T cell precursorare also referred to as the “thymocytes”. T cells express different receptors on the surface (T cell receptors) which is followed by expression of either CD8 or CD4 xxiv surface molecule. The T cells accounts for 70-80% lymphocytes in peripheral blood, 5-10% in bone marrow, 70-80% in lymph node and 30-40% in spleen. T lymphocytes have two major subsets that are functionally and phenotypically different; Helper (CD4) and cytotoxic/suppressor (CD8) T cells (Jacqueline and Bryon, 2001). Helper T cells (Th) also called CD4+ T cell is a pertinent coordinator of immune regulation. The main function of the T helper cell is to augument or potentiate immune responses by the secretion of specialized factors (cytokines) that activate and promote the proliferation and differentiation of other white blood cells; cytotoxic T cell, B cells and macrophages and activation of inflammatory leucocytes. They are identified by the presence of the CD4 marker. They recognize antigen when presented along with class 11 MHC molecules. Based on the kind of cytokines produced T lymphocytes (Th) are further divided into Th1 and Th2 subsets(Jacqueline and Bryony, 2001). Cytotoxic /T killer/ suppressor subset (TC) or CD8+ T cells are important in directly killing certain tumor cells, viral infected cells and sometimes parasites. They lyse cells with foreign antigens. They are identified by the presence of the CD8 Marker. They recognize antigen when presented along with class 1 MHC molecules(Jacqueline and Bryony, 2001). The suppressor T cells have a role in the downregulation of immune response. Both types of T cells can be found throughout the bodies. They often depend on the secondary lymphoid organs (the lymph nodes and spleen) as sites where activation occurs but they are also found in other tissues of the body, most conspicuously the liver, lung, intestinal and reproductive tracts (USC, 2006). c. Non Specific Killer Cells xxv Several different cells including NK and LAK cells, K cells, activated macrophages and eosinophils are capable of killing foreign and altered self target cells in a non-specific manner. These cells play an important role in the innate immune system (Abbas and Lichtman 2004; USC, 2006). 1.2.2.2 Myeloid Stem Cells The myeloid stem cells develop into platelets, red blood cells or granulocyte-monocyte line. Monocytic cells include monocytes and macrophages. The granulocytes include neutrophils, eosinophils, mast cells and basophils. a. Granulocyte or Polymorphonuclear (PMN) Leucocytes Another group of white blood cells is collectively referred to as granulocytes or polymorphonuclear (PMNs) leucocytes. These granulocytes are so called because they have irregularly shaped nuclei. Granulocytes are composed of three cell types identified as neutrophils, eosinophils and basophils based on their staining characteristics with certain dyes. These cells are predominantly important in the removal of bacteria and parasites from the body. They engulf foreign bodies and degrade them using powerful enzymes (Peakman and Vergani, 1997). i. Neutrophils These cells have short life span. They circulate in the blood for 6-7 hours then migrate through the endothelial cell functions and reside in tissue spaces where they live for only for a few days and do not multiply. Neutrophils are the most abundant of the leukocytes normally accounting for 54-75% of the white blood cell. They are called neutrophils because their granules stain poorly with the mixture of dyes used in staining leucocytes. They are effector cells of innate immunity that enter sites of infection and work in anaerobic conditions. The content of neutrophils granules bind to neitheracidic nor basic stains. Neutrophils phagocytose and kill microbes upon phagolysosomal fusion (Urban et al., 2006). xxvi ii. Eosinophils These granulocytes are the second most abundant, 1-6% of proportion of leucocytes. They defend against helminthes worms and other intestinal parasites. They also stimulate the release of cytokines such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-13 and TNF alpha (α)(Rothenberg and Hogan, 2006; Hogan et al., 2008). The eosinophilic granules contain basic substances that bind to the acidic stain eosin. Eosinophils bind to the antibody using low affinity receptors(FCER11). They are not phagocytic (Jacqueline and Bryony, 2001). iii. Basophils: It constitutes less than 1% of the proportion of leucocytes making them the least abundant. It is also implicated in regulating the immune response to parasites and allergy but is so rare that relatively little is known of its contribution to immune defense. The basophil cytoplasmic granule contains acidic substance that binds basic stains such as hematoxylin during histological stains. They are involved in some of the most severe immunologic reactions such as angioedema and anaphylaxis (Jacqueline and Bryony, 2001). b. Macrophages Macrophages originate from the monocytes. They function both in innate and adaptive immunity. Once monocyte leaves circulation and enter tissue, they are called macrophages. They are distinguished from the granulocytes by being bigger, having a distinctive indented nucleus. The monocytes are the mobile progenitors of sedentary tissue cells called macrophages. They travel in the blood to tissues, where they mature into macrophages and take up residence. Tissue macrophages are large, irregularly shaped cells characterized by an extensive cytoplasm with numerous vacuoles often containing engulfed materials. They survive for months and can multiply(Mosser and Edwards, 2008). xxvii There are two types of macrophages: one that wanders in the tissue spaces and the other that are fixed to vascular endothelium of liver, spleen, lymph node and other tissue. Macrophages display remarkable plasticity and can change their physiology in response to environmental cues. These changes can give rise to different populations of cells with distinct function (Mosser and Edwards, 2008). Macrophages present in different organs have been given different names. They are histiocytes (in tissues), kupffer cells (in liver), alveolar macrophages (in lungs), peritoneal macrophages (in peritoneum), microglial cells (in brain), mesangial cells (in kidneys) and osteoclastis (in bone). Macrophages kill microbes, infected cells, tumor cells; secrete immunolodulatory cytokines, antigen processing and presentation to T cells. Macrophages respond to infections as quickly as neutrophils but persist much longer. Hence they are dominant effector cells at the later part of infections. They are also often referred to as scavengers or antigen presenting cells (APC) because they pick up and ingest foreign materials and present these antigens to other cells of the immune system such as T cells and B cells. This is one of the important first steps in the initiation of an immune response. Stimulated macrophages exhibit increased levels of phagocytosis and are also secretory (Abbas and Lichtman, 2004; USC, 2006). c. Dendritic Cells They are also known as antigen presenting cells (APC) and are more efficient APC’s than the macrophages. These are efficient stimulators of B and T lymphocytes (Jacques and Ralph, 1998). B cells, the precursor of antibody secreting cells, can directly recognize native antigen through their B cells receptors. T lymphocytes however need the antigen to be processed and presented to them by an APC. Dendritic cells in the periphery capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. They not only activate lymphocytes, they also tolerize T cells to antigens that are innate to the body (self antigens), thereby minimizing autoimmune reactions (Jacque and Ralph, 1998). Not much is known about dendritic xxviii cells because they are extremely hard to isolate which is often a prequisite for the study of functional qualities of specific cell types. Of particular importance is the recent finding that dendritic cells bind high amount of HIV and may be a reservoir of virus that is transmitted to CD4+ T cells during an activation event (Janeway et al., 2005; USC, 2006). 1.2.3 Arms of the Immune System There are two major subdivisions of the immune system; the innate or non specific immune system and the adaptive or specific immune system. The innate immune system is the body’s first line of defense against invading organisms while the adaptive immune system act as second line of defense and also affords protection against re-exposure to the same pathogen. Each of the major sub divisions of the immune system acts as second line of defense and also affords protection against re-exposure to the same pathogen. Each of the major subdivisions of the immune system has both cellular and humoral components by which they carry out their protective function. There is usually interplay between the two arms of the immune system(Abbas and Lichtman, 2004). 1.2.3.1 Innate Immunity This is also known as non-specific immune system and is the first line of defense (Janeway et al., 2005). It does not confer a long lasting or protective immunity to the host (Alberts et al., 2002). The elements of non specific (innate) immune system include anatomical barriers, secretory molecules and cellular components. A. Anatomical Barriers to Infection xxix The skin, external epithelial layers, the movement of the intestines and the oscillation of bronchopulmonary cilia are the mechanical anatomical barriers (Roitt et al., 2001). Associated with these protective surfaces are chemical and biological agents. i. Mechanical Factors The epithelial surfaces form a physical barrier that is very impermeable to most infectious agents. Thus the skin acts as our first line of defense against invading organisms. The regular desquamation of skin epithelium also helps remove bacteria and other infectious agents that have adhered to the epithelial surfaces (Goldsby et al., 2000). Movement due to cilia or peristalsis helps to keep air passages and the gastrointestinal tract free from microorganisms. The flushing action of tears and saliva helps prevent infection of the eyes and mouth. The trapping action of mucus that lines the respiratory and gastrointestinal tracts helps to protect the lungs and digestive systems from infection. ii. Chemical Factors Fatty acids in sweat inhibit the growth of bacteria. Lysozyme and phospholipase found in tears, saliva and nasal secretions can breakdown the cell wall of bacteria and destabilise bacterial membranes. The low pH of sweat and gastric secretions prevents growth of bacteria. Defensins (low molecular weight proteins) found in the lung and gastrointestinal tract have antimicrobial activity. Surfactants in the lung act as opsonins (substances that promote phagocytosis of particles by phagocytic cell) (USC, 2006). xxx iii. Biological Factors The normal flora of the skin and in the gastrointestinal tract can prevent the colonization of pathogenic bacteria by secreting toxic substances or by competing with pathogenic bacteria for nutrients or attachment to cell surfaces. B. Humoral Barriers to Infection The anatomical barriers are very effective in preventing colonization of tissues by microorganisms. However when there is damage to tissues, the anatomical barriers are breached and infection may occur. Once infectious agents have penetrated tissues, another innate defense mechanism comes into play, namely acute inflammation. Humoral factors play an important role in inflammation, which is characterized by edema and the recruitment of phagocytic cells. These humoral factors are found in serum or they are formed at the site of infection. i. Complement System The complement system is the major humoral non specific defense mechanism. Once activated, complement can lead to increased vascular permeability, recruitment of phagocytic cells, lysis and opsonisation of bacteria (Goldsby et al., 2000; USC, 2006). ii. Coagulation System Depending on the severity of the tissue injury, the coagulation system may or may not be activated. Some products of the coagulation system contribute to the non specific defenses because of their ability to increase vascular permeability and act as chemotactic agents for phagocytic cells. In addition, some of the products of the coagulation system are directly antimicrobial. For example, beta-lysin, a protein produced by platelets during coagulation can lyse many gram positive bacteria by acting as cationic detergent (Goldsby et al., 2000; USC, 2006). xxxi iii. Lactoferrin and Transferrin Lactoferrin is a 703- amino acid glycoprotein originally isolated frommilk. Plasma lactoferrin is predominantly neutrophil derived but indications are that it may also be produced by other cells (Levay and Viljoen, 1995). By binding iron, an essential nutrient for bacteria, these proteins limit bacteria growth. iv. Interferons Interferons are protein that can limit virus replication in cells. They are a major class of cytokine that have a role in immunity. They are so called because they inhibit viral replication but they have many other functions. For instance, they also activate NK cells to kill virus infected host cells. They are divided into two: type 1(α and β interferons) and type 2(ɣ or immune interferon). Type 1 interferons have potent antiviral activity and are produced mainly by fibroblast and monocytes as a reaction to infection. They also have antiproliferative function and useful in the treatment of chronic hepatitis B and C infections in combination with antiviral drugs (Hoofnagle and Di, 1997) as well as in some forms of leukeamia (Brustein and McGlave, 2001; Minet al., 2001). v. Lysozyme Lysozyme is an enzyme found in the protective fluid (tears, saliva and mucus) of most animals. Lysozyme breaks down the cell wall of bacteria. vi. Interleukin 1 More than any other cytokine family, the interleukin (IL) – 1 family is closely linked to the innate immune response (Dinarello, 2009). IL -1 beta is the most studied member of the IL-1 family because of its role in mediating autoinflammatory diseases. IL 1 induces fever and the production of acute phase proteins, some of which are antimicrobial because they can opsonise bacteria. xxxii C. Cellular Barriers to Infection Part of the inflammatory response is the recruitment of polymorphonuclear cells, eosinophils and macrophages to sites of infection. These cells are the main line of defense in the non-specific immune system. Polymorphonuclear neutrophils (PMNs) are recruited to the site of infection where they phagocytose invading organisms and kill them intracelllularly. In addition, PMNs contribute to collateral tissue damage that occurs during inflammation(USC, 2006). Tissue macrophages and newly recruited monocytes which differentiate into macrophages also function in phagocytosis and intracellular killing of microorganism. In addition, macrophages are capable of extracellular killing of infected or altered self-target cells. Furthermore, macrophages contribute to tissue repair and act as antigen presenting cells, which are required for the induction of specific immune responses (USC, 2006). Natural killer (NK) and lymphokine activated killer (LAK) cells can non-specifically kill virus infected and tumor cells. These cells are not part of the inflammatory response but they are important in nonspecific immunity to viral infections and tumor surveillance. Eosinophils have proteins in their granules that are effective in killing certain parasites. D. Phagocytosis and Intracellular Killing Polymorphonuclear leucocytes (PMNs) are motile phagocytic cells that have lobed nuclei are identified by their characteristic nucleus or by an antigen present on the cell surface called CD66 (Goldsby et al., 2000; USC, 2006). They contain two kinds of granules, the content of which are involved in antimicrobial properties of these cells. The primary or azurophilic granules which are abundant in young newly formed PMNs contain cationic proteins and defensins that can kill bacteria, proteolytic enzymes like elastase and cathepsin G to break down proteins, lysozyme to break down bacteria cell walls and characteristically, xxxiii myelopreoxidase, which is involved in the generation of bacteriocidal compounds (Goldsby et al., 2000; USC, 2006). The second type of granule found in more mature PMNs is the secondary or specific granule. These contain lysozyme, NADPH oxidase components which are involved in the generation of toxic oxygen products and characteristically lactoferrin an iron chelaring protein and B12- binding protein (USC, 2006). Monocytes and macrophages are phagocytic cells that have characteristic kidney shaped nucleus. Unlike PMNs they do not contain granules but they have numerous lysosomes which have contents similar to the PMNs granules. Phagocytosis occurs through different steps and processes as shown below i. Circulating PMNs and monocytes respond to danger signals generated at the site of an infection. The signals include N-formyl methionine containing peptides released by bacteria, clotting system peptides, complement products and cytokines released from tissue macrophages that have encountered bacteria in tissue (Bona and Bonilla 1995; Goldsby et al., 2000). Some of the signals stimulate endothelial cells near the site of the infection to express cell adhesion molecules such as ICAM 1 and selectins which bind components on the surface of phagocytic cells and cause the phagocytes to adhere to the endothelium (Goldsby et al., 2000; USC, 2006). Vasodilators produced at the site of the infection causes the junctions between endothelial cells to loosen and the phagocytes then cross the endothelial barrier by squeezing between the endothelial cells in a process known as diapedesis. ii Phagocytosis: phagocytic cells have a variety of receptors on their membrane through which infectious agents bind to cells. These include Fc receptors, complement receptors, scavenger receptors and toll like receptors. Bacteria with IgG antibody on their surface have the Fc region exposed and this part of the Ig molecule can bind to the receptor on phagocytes. Binding to the Fc receptor requires prior interaction of the antibody with the antigen. Binding of the IgG coated xxxiv bacteria to Fc receptors results in enhanced phagocytosis and activation of the metabolic activity of phagocytes (respiratory burst)(Goldsby et al., 2000; USC, 2006). Phagocytic cells have a receptor for the 3rd component of the complement C3b. Binding of C3b coated bacteria to this receptor also results in enhanced phagocytosis and stimulation of the respiratory burst. Scavenger receptors bind a variety of polyanions on bacterial surfaces resulting in phagocytosis of bacteria. Phagocytes have a variety of Toll like receptors (pattern recognition receptors or PRRs) which recognize broad molecular patterns called PAMPS (pathogen associated molecular patterns) on infectious agents. Binding of infectious agents via Toll like receptors results in phagocytosis and the release of inflammatory cytokines (IL-1, TNF-alpha, IL-6) by the phagocytes (Peakman and Vergani, 1997;Goldsby et al., 2000). After attachment of a bacterium, the phagocytes begin to extend pseudopods around the bacterium. The pseudopods eventually surround the bacterium and engulf it and the bacterium is enclosed in a phagosome. During phagocytosis the granules or the lysosomes of the phagocyte fuse with the phagosome and empty their contents. The result is a bacterium in a phagolysosome which contains the contents of the granules or lysosomes (Roitt and Delves, 2001; USC, 2006). iii. Respiratory burst: During phagocytosis, there is an increase in glucose and oxygen consumption. This is referred to as respiratory burst. The consequences of the respiratory burst is that a number of oxygen containing compounds are produced which kill the bacteria being phagocytosed. This is referred to as oxygen dependent intracellular killing. In addition, bacteria can be killed by preformed substances released from granules or lysosomes when they fuse with the phagosome. This is referred to as oxygen independent intracellular killing (USC, 2006). a. Oxygen Dependent Myeloperoxidase Independent Intracellular Killing xxxv During phagocytosis, glucose is metabolized via the pentose monophosphate shunt and NADPH is formed (Janeway et al., 2005). Cytochrome B which was part of the specific granule combines with the plasma membrane NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the NADPH. The result is the production of superoxide ions. Some of the superoxide ions is converted to H2O2and singlet oxygen by superoxide dismutate. In addition, superoxide ions react with the H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The result of these reactions is the production of the toxic oxygen compounds superoxide anion(O2-), H2O2, singlet oxygen(1O2) and the hydroxyl radical (OH+) (Roitt et al., 2001; USC, 2006). b. Oxygen Dependent Myeloperoxidase Dependent Intracellular Killing As the azurophilic granules fuses with the phagosome, myeloperoxidase is released into the phagolysosome. Myeloperoxidase utilizes H2O2 and the halide ions (usually Cl-) to produce hypochlorite a highly toxic substance. Some of the hypochlorite can spontaneously breakdown to yield toxic substance. Some of the hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions is the production of toxic hypochlorite (OCl-) and singlet oxygen (1O2) (Roitt and Delves, 2001; USC, 2006). c. Detoxification Reaction PMNs and macrophages have means to protect themselves from the toxic oxygen intermediates. These reactions involve the dismutation of superoxide anion to hydrogen peroxide by superoxide dismutase and the conversion of hydrogen peroxide to water by catalase (USC, 2006). d. OxygenIndependent Intracellular Killing xxxvi In addition to the oxygen dependent mechanisms of killing there are also oxygen independent killing mechanisms in phagocytes: cationic proteins (cathepsin) released into the phagolysosome can damage bacterial membranes: lysosome breaks down bacterial cell walls, lactoferrin chelates iron, which deprives bacteria of this required nutrient: hydrolytic enzymes breakdown bacterial proteins. Thus, even patients who have defects in the oxygen dependent killing pathways are able to kill bacteria. However since the oxygen dependent mechanisms are much more efficient in killing, patients with defects in these pathways are more susceptible and get more serious infections (USC, 2006). e. Nitric Oxide Dependent Killing Binding of bacteria to macrophages particularly binding via Toll like receptors results in the production of TNF –alpha, which acts in an autocrine manner to induce the expression of the inducible nitric oxide synthetase gene resulting in the production of nitric oxide (NO). If the cell is also exposed to interferon gamma (IFN-gamma) additional nitric oxide will be produced. Nitric oxide released by the cell is toxic and can kill microorganisms in the vicinity of the macrophage (Roitt and Delves, 2001; USC, 2006). 1.2.3.2 Adaptive Immunity Adaptive immunity is another major subdivision of the immune system. It is the hall mark of the immune system of higher animals. The adaptive immune system evolved in early vertebrates. It allows for a stronger immune response as well as immunological memory where each pathogen is remembered by a marker antigen (Pancer and Cooper, 2006). Adaptive immunity is so called because it adapts or learns to recognize specific kinds of pathogens and retains memory of them for speeding up future responses. It is xxxvii the second barrier against infections. It is acquired later in life and is precise. The responseconsists of antigen specific reactions through T lymphocytes and B lymphocytes. The adaptive response has memory so that subsequent exposure leads to a more vigorous and rapid response but this is not immediate (Delves and Roitt, 2001). The cells of the adaptive immune system are special types of leucocytes, lymphocytes, B cells, T cells that are derived from heamopoeitic stem cell in the bone marrow. There are two basic components of the adaptive immune system; the cell mediated immunity and the humoral component of the adaptive immunity. B cells are involved in the humoral immune responses whereas T cells are involved in the cell mediated immune response (Janeway et al., 2005). Both T and B cells carry receptor molecules that recognize specific targets. A. Cell Mediated Immunity Adaptive T cell mediated immunity is driven by activation of T cells. Cytotoxic T cell is activated byendogenous antigen to stimulate macrophage killing of endosomal pathogens. The pathogens targeted by cellular immunity are protected from antibody and complement binding by their intracellular locations. T cells recognize a non-self-target such as a pathogen only after antigen (small fragments of the pathogen) have being processed and presented in combination with a self-receptor called a major histocompactibility complex(MHC) molecule. There are two major subtypes of T cells; the cytotoxic T cells (killer T cells) and the helper T cell. Killer T cells only recognize antigens coupled to class 1 MHC molecules while helper T cells only recognize antigens coupled to class 11 MHC molecules. These two mechanisms of antigen presentation reflect the different roles of the two major types of T cells. Only professional antigen presenting cells (dendritic cells, macrophages and B cells) have both class 1 and class 11 MHC and can deliver co stimulatory signals. xxxviii Activated T cells perform their effector functions when they encounter MHC presented peptide on their target cells. A third major subtype is the ɣð T cells that recognize intact antigens that are not bound to MHC receptors (Girardi, 2006). i. Helper T Cells (Th) The helper T cells regulate both innate and adaptive immune responses and help determine which types of immune response the body will make to a particular pathogen (Abbas et al., 1996). These cells have no cytotoxic potentials and do not kill infected cells or clear pathogens directly. They control the immune response by directing other cells to perform these tasks. Helper T cells express T cell receptors (TCR) that recognize antigen bound to class 11 MHC molecules. Helper T cells require long duration of engagement with an antigen presenting cell (Kovacs et al.,2002). Th cells are subdivided functionally by the pattern of cytokines they produce. On stimulation, precursor Th O lymphocytes become either Th1 or Th2 cells. The difference between these cells is only in the cytokines secreted. They are morphologically indistinguishable. However the response they generate is very different. Th1 cells produce interleukin 2 which induces T cell proliferation. Interleukin 2 stimulates CD8 T cell division and cytotoxicity by decreasing activation threshold. The other major cytokine produced by Th1 cells, interferon ɣ actiavtes macrophages to kill intracellular pathogens such as mycobacteria, fungi and protozoan and induces natural killer cells to cytotoxicity. The Th1 cytokines therefore induces mainly a cell mediated inflammatory response.There is a positive feedback loop as interferon ɣ stimulates other Th 0 cells to become Th 1 and inhibits Th 2 differentiation (Swain et al., 1991). The activation of a resting helper T cell causes it to release cytokines that influence xxxix the activity of many cell types. Cytokine signal produced by helper T cells enhance the microbiocidal function of macrophages and the activity of killer T cells (Albertset al., 2002). ii. Cytotoxic T Cells (Tc) Cytotoxic T cells (killer T cells) mediate antigen specific, MHC restricted cytotoxicity and are important for killing intra-cytoplasmic parasites that are not accessible to secreted antibody or to phagocytes. Examples include all viruses, rickettsias, some obligate intracellular bacteria (Chlamydia) and some protozoanparasites which export their proteins from macrophage vesicles to the cytoplasm (Toxoplasma gondii). The only way to eliminate these pathogens is to kill their host cells (Harty et al., 2000). As with B cells each type of T cell recognizes a different antigen. Killer T cells are activated when their T cell receptor (TCR) binds to this specific in a complex with the MHC class 1 receptor of another cell. Recognition of this MHC antigen complex is aided by a co-receptor on the T cell called CD8. The T cell then travels throughout the body in search of cells where the MHC 1 receptor bears this antigen. When an activated T cell contacts such cells it releases cytotoxins that form pores in the target cells plasma membrane allowing ions, water and toxins to enter. This causes the target cell to undergo apoptosis (Radoja et al., 2006). T cell killing of host cell is particularly important in preventing the replication of viruses. T cell activation is tightly controlled and generally requires a very strong MHC antigen activation signal, or additional activation signals provided by helper T cells (Radoja et al., 2006). iii. γgamma T Cells γ gamma T Cells posses an alternative T cell receptor(TCR) as opposed to CD4+ and CD8+ T cells and share the characteristics of helper T cells, cytotoxic T cells and NK cells. The conditions that produce responses from γ gamma T cells are not fully understood. Like other unconventional T cells subsets bearing invariant TCRs such as CD1d- restricted natural killer cells, γ gamma T cells straddle the border between innate and xl adaptive immunity (Girardi, 2006). On the other hand, γ gamma T cells are a component of adaptive immunity as they rearrange TCR genes to produce receptor diversity and can also develop a memory phenotype. On the other hand, the various subsets are also part of the innate immune system, as restricted TCR or NK receptors may use as pattern recognition receptors(Girardi, 2006). B. Humoral Immunity This is also referred to as antibody mediated immunity. It is regulated by B cells and the antibodies they produce. Antibody mediated reactions defend against invading viruses and bacteria. Antibodies are produced and secreted by plasma cells which are found mainly within lymph node and which do not circulate. Plasma cells are derived from B lymphocytes. Adaptive humoral immunity also usually involves T cell activation to produce cytokines that stimulate B cell antibody synthesis (Janeway et al., 2005). 1.2.4 Overview of Antibodies Antibodies (immunoglobulins abbreviated Ig) are proteins of molecular weight 150,000-900,000kd (Janeway et al., 2005; Abbas and Lichtman, 2004). They are also referred to as gammaglobulins. Antibodies vary from molecule to molecule with respect to which organism they bind to or with their specialized function in the body. Antibodies have a ‘Y’ shaped structure with three different regions. The arms of ‘Y’ are termed the variable regions while the tail of ‘Y’ is the constant region. The variable regionsare randomly generated so that they bind to specific pathogen epitopes(Janeway et al., 2005). The constant region on the other hand is not randomly generated but comes in a few structural varieties called isotypes. The constant region is the part of the antibody to which other immune system cells such as macrophages bind. Depending on the isotype, of the constant region, different responses will be triggered upon binding, so that it is this part of the antibody that determines effect function. One end of the xli immunoglobulin binds to antigens (Fab portion so called because it is the fragment of the molecule which is antigen binding) and the other end which is crystallizable and therefore called Fc is responsible for effector function. The immunoglobulin molecule consists of two heavy chains and two light chains each of approximate molecular weight 50000kd (Abbas and Lichtman, 2004). The heavy and light chain consist of amino acids sequences. In the regions concerned with antigen binding these regions are extremely variable, whereas in other regions of the molecule, they are relatively constant. Thus every heavy and light chain possesses a variable and constant region. The isotype of an Ig is determined by the constant region. There are five classes (isotypes) of immunoglobulins; IgM, IgG, IgA, IgD, and IgE plus four subtypes of IgG (Ig G1-4) and two of IgA (IgA1, IgA2). Immunoglobulins are found in serum and in secretions from mucosal surfaces (Abbas and Lichtman, 2004). 1.2.4.1 Classes of Immunoglobulin When a B cell encounters the kind of antigen that triggers it to become active, it gives rise to many large cells known as plasma cells which produce antibodies (Pier et al., 2004). Each immunoglobulin class has a special characteristic that provides it with special advantage: i) Immunoglobulin G (IgG): is a kind of antibody that works efficiently to coat microbes speeding their uptake by other cells in the immune system. It is formed in large quantity and last for over a month and travel from the blood stream to tissue easily. It is the only immunoglobulin class that crosses the placenta and passes immunity from the mother to the new born (Pier et al., 2004). xlii ii) Immunoglobulin M (IgM): They are the first antibodies formed in response to infection. They are important in protection during the early days of an infection. IgM are the most effective against bacterial infection (Pier et al., 2004). iii) Immunoglobulin A (IgA): This immunoglobulin is produced near the mucus membrane and find its way to the saliva, mucus and digestive tract- guarding the entrance to the body and giving protection against infection in the respiratory tract and intestines. iv) Immunoglobulin E (IgE):The natural job of this immunoglobulin is to protect against parasitic infections and is responsible for allergic reactions (Pier et al., 2004). v) Immunoglobulin D (IgD): They are present in the surface of most but not all B cells. Early in their development but little IgD is ever released into the circulation. It is not clear what function Ig D performs though it may play a role in determining whether antigen activate the B cell. 1.2.4.2 Roles of Antibodies Antibodies exist free in body fluids eg serum, and membrane bound to B lymphocytes. Their function when membrane boundis to capture antigen for which they have specificity, after which the B lymphocytes will take the antigen into its cytoplasm for further processing and presentation. Free antibodies perform the functions listed below (Roitt et al., 2001; USC, 2006). i) Agglutination of particulate matter, including bacteria and virus. IgM is particularly suitable for this as it is able to change its shape from a star form to a form resembling a crab. ii) Opsonisation (i.e coating) of bacteria for which the antibody’s Fab region has specificity (especially IgG). This facilitates subsequent phagocytosis by cells possessing Fc receptor e.g. xliii neutrophil polymorphonuclear, leucocyte, (polymorphs). Thus it can be seen that in opsonisation and phagocytosis, both the Fab and the Fc portions of immunoglobulin molecules are involved. iii) Neutralization: neutralization of toxin released by immunoglobulin G (eg tetanus toxin) is neutralized when specific IgG antibody binds, thus preventing the toxin binding to motor end plate and causing persistent stimulation, manifest as sustained muscular contraction, which is the hall mark of titanic spasm). In the case of viruses antibody can hinder the ability to attach to receptors on host cells. Here, only Fab is involved. iv) Immobilization of bacteria: antibodies against bacteria ciliae or flagella will hinder their movement and ability to escape the attention of phagocytic cells. Again only Fab is involved. v) Activation of complement (classical pathway) especially by the Fc region of IgM and IgG, leads eventually to death of bacteria by the terminal complement component which punch holes in the cell wall leading to an osmotic death. Complement component also facilitate phagocytosis by cells possessing a receptor for C3b e.g. polymorphs. vi) Mucosal protection: This is provided mainly by IgA and to a lesser degree IgG. IgA acts mainly by inhibiting pathogens from attaching to mucosal surfaces. This function is attributed to Fab. vii) Expulsion as a consequence of mast cell degranulation: As a consequence of antigen e.g. parasitic worms, binding to specific immunoglobulin E, attached to mast cell by their receptor for IgE Fc, there is release of mediators from the mast cell. This leads to contraction of smooth muscles which can result in diarrhoea and expulsion of parasite. Here we see involvement of both Fab parasite antigen and Fc anchoring the reacting participant. viii) Precipitation of soluble antigen by immune complex formation: This consists of antigen linked to antibody. Depending on the ratio of antigen to antibody, these can be of varying size. When fixed at one site they can be removed by phagocytic cells. They may also circulate prior to localization and removal, and can fix complement. Here Fab and Fc involved. xliv ix) Antibody dependent cell mediated cytotoxicity (ADCC): Antibodies bind to organisms via their Fab region. Large granular lymphocytes (Natural killer cells - NK cells), attach via Fc receptors and kill these organism not by phagocytosis but by release of toxic substances called perforins. x) Conferring immunity to the foetus by the transplantal passage of IgG: IgG is the only class (isotope) of immunoglobulin that can cross the placenta and enter the foetal circulation where it confers passive immune protection. This is of great importance to the foetus in the first 3 months. 1.3 Mediators of the Immune System 1.3.1 Cytokines Cytokines are small molecular weight messengers secreted by cell to alter the behavior of itself (Jacquelin and Bryony, 2001). Cytokines are a group of proteins and peptides that are used as signaling compounds in the body(Lee et al., 2004). These chemical signals are similar to hormones and neurotransmitters and are used to allow one cell to communicate with another. The cytokine family consists of smaller water soluble proteins and glycoproteins with a mass of 8-30kDa (Roitt et al., 2001; USC, 2006). They are particularly important in both innate and adaptive immune responses. Due to their central role in the immune system, cytokines are involved in a variety of immunological, inflammatory and infectious diseases. Cytokines are produced by a wide variety of cell types (both haemopoeitic and non haemopoeitic) and can have effects on both nearby cells or throughout the organism. Sometimes these effects are strongly dependent on the presence of other chemicals and cytokines(Leeet al., 2004)(Table1). Each cytokine binds to a specific cell surface receptor. Subsequent cascades of intracellular signaling then alter functions. This may include the upregulation and / or downregulation of several genes and their transcription factors in turn resulting in the production of other cytokines, an increase in the number of xlv surface receptors for other molecules or the suppression of their own effect by feedback inhibition (Roitt and Delves, 2001; Janeway et al., 2005). Interestingly, cytokines are characterized by considerable “redundancy” in that many cytokines appear to share similar functions. Generalization of functions is not possible with cytokines; nonetheless, their actions may be comfortably grouped as; autocrine, if the cytokine acts on the cell that secretes it; paracrine, if the action is restricted to the immediate vicinity of a cytokine’s secretion; endocrine, if the cytokine diffuses to distant regions of the body(carried by blood or plasma) to affect different tissues. Cytokines are made by many cell populations, but the predominant producers are helper T cells (Th) and macrophages (Roitt and Delves, 2001). Cytokines have been variously named as lymphokines, interleukins and chemokines, based on their presumed function, cell of secretion or target of action. Because cytokines are characterized by considerable redundancy and pleiotropism, such distinction, allowing for exception, are obsolete. The term interleukin was initially used by researchers for those cytokines whose presumed target are principally leukocytes. It is now used largely for designation of newer cytokines molecules discovered every day and bears little relation to their presumed function(Roitt et al., 2001). The term chemokine refers to a specific class of cytokines that mediates chemoattraction (chemotaxis) between cells. A more clinically and experimentally useful classification divides immunological cytokines into those that promote the proliferation and functioning of helper T-cells, type 1 (Th1) (e.g. IFN-y) and type 2 (Th2) (e.g. IL-4, IL-10, IL-13, TGF-β), respectively (Goldsby et al., 2000). A classification of cytokines receptors based on their three-dimensional structure has therefore been attempted. This classification, though seemingly cumbersome, provides with several unique perspectives for attractive pharmacotherapeutic target (Janeway et al., 2005). xlvi Immunoglobulin (Ig) superfamily which are ubiquitously present throughout several cells and tissues of the vertebrate body, and share structure homology with immunoglobulin (antibodies), cell adhesion molecules, and even some cytokines. Example: IL-1 receptor types. Haemopoitic Growth Factor (type 1) family, whose members have certain conserved motifs in their extracellular amino-acid domain. The IL-2 receptor belongs to this chain, whose γ-chain (common to several other cytokines) deficiency is directly responsible for the x-linked form of severe combined immunodeficiency (X-SCID). Interferon (type 2) family: whose members are receptors for IFN β and γ. Tumour Necrosis Factor (TNF) (type 3) family: whose members share a cysteine-rich common extracellular binding domain, seven transmembrane helix familes, the ubiquitous receptor type of the animal kingdom. All G-protein coupled receptors (for hormones and neurotransmitters) belong to this family (Goldsby et al., 2000)). It is important to note that chemokine receptors, two of which act as binding protein for HIV (CXCR4 and CCR5), also belong to this family. xlvii Table 1: Sources and Activity of Cytokines Cytokine Primary sources Primary activities IL-1 Macrophage T,B cell activation; fever, inflammation IL-2 T cells T cell proliferation IL-3 T cells Growth of many cell types IL-4 T cells B cell growth and differentiation IL-5 T cells B cell eosinophil IL-6 Macrophages , T Cells B cell stimulation, inflammation IL-7 Stromal cells Early B and T cell differentiation IL-8 Macrophages Neutrophil (PMN) attraction IL-9 T cells Mitogen IL-10 T cells Inhibits Th 1 cytokine production IL-11 Bone marrow stroma Hematopoeisis IL-12 APC Stimulates T, NK cells IL-13 T cells Similar to IL-4 IL-14 Dendritics cells, T cells B cell memory IL-15 T cells Same as IL-2 IFNα Most cells Anti-viral IFNβ Most cells Anti-viral IFNγ T, NK cells Inflammation, activates macrophages TGFb Macrophages, lymphocytes Depends on target TNFa Macrophages Inflammation, tumor killing TNFb T cells Inflammation; tumor killing; enhance phagocytosis GM-CSF Th cell Act on progenitor cells to stimulate growth and differentiation of monocytes and DC MIP-1α Macrophages Chemotasis of monocytes and T cells MIP-1β Lymphocytes Chemotasis of monocytes and T cells (Adapted from immunology briefcase, an e-book of Dalhousie University, 2006). xlviii 1.3.2 Complement System The complement system is a biochemical cascade that attacks the surface of foreign cells. Complementis the major humoral component of the immune response (Rus et al., 2003; USC, 2006) and contains over 30 different proteins and is named for its ability to “complement” the killing of pathogens by antibodies. Complement was discovered by Jules Bordet in 1900. It is produced by a variety of cells including, hepatocytes, macrophages and gut epithelial cells (Anderson, 2003). In human, this response is activated by the binding of complement proteins to carbohydrates on the surface of microbes or by complement binding to antibodies that have attached to these microbes. This recognition signal triggers rapid killing response (Liszewski and Atkinson, 1993). The speed of the response is a result of signal amplification that occurs following sequential proteolytic activation of complement molecules, which are also proteases. After complement proteins initially bind to the microbes, they activate their protease activity which in turn activates other complement proteases, and so on. This produces a catalytic cascade that amplifies the initial signal by controlled positive feedback (Sim and Tsiftsoglou, 2004). The cascade results in the production of peptides that attract immune cells, increase vascular permeability and opsonize (coat) the surface of a pathogens, marking it for destruction. This deposition of complement can also kill cells directly by disrupting their plasma membrane (Ruset al., 2003). Some complement proteins bind to immunoglobulin or to membrane components of cells. Others are proenzymes that, when activated, cleave one or more other complement proteins. Upon cleavage some of the complement proteins yield fragments that activate cells, increase vascular permeability or opsonize bacteria. (Abbas et al., 1994)(Table 2). Complement activation can be divided into four pathways: the classical pathway, the alternative pathway, the lectin pathway, and the membrane attack xlix (or lytic) pathway. Both classical and alternative pathways lead to the activation of C5 convertase and result in the production of C5b which is essential for the activation of the membrane attack pathway. l Table 2: The Biological Functions of Complement and its Fragments Components and fragments C5a, C3a, C5a-desArg, C4a Activity Increase of vascular permeability; smooth muscle contraction, degranulation of mast cells and eosinophils C5a, C5a-desArg Neutrophil activation and chemotaxis; stimulation of prostaglandin and leukotriene production C3b, C4b Opsonization of bacteris and immune complexes leading to phagocytosis C3a, C5a-desArg, Clq Stimulation of the respiratoty burst of professional phagocytes C5bC6C7C8C9 (membrane attack complex) Lysis of bacteria and foreign cells C3b, CR l Solubilization of circulating immune complexes (Adapted with modifications from Roitt et al., 2001; Francis et al., 2003). 1.4 Disorders of the Immune System li The immune system is a remarkably effective structure that incorporates specificity, inducibility and adaptation. However, failure of host defenses can occur and fall into five broad categories: hypersensitivities, immunodeficiencies, autoimmunity, immune complex disorders and host-graft reaction. 1.4.1 Hypersensitivity Hypersensitivity is an immune response that damages the body’s own tissues. They are divided into four classes (Type I-IV) based on the mechanisms involved and the time course of the hypersensitive reaction. Type I hypersensitivity is an immediate or anaphylactic reaction, often associated with allergy. Type I hypersensitivity is mediated by IgE released from mast cells and basophils (USC, 2006). Usually harmless substances can provoke inappropriate immune responses. The most common types of allergic reactionshay fever, some kinds of asthma, and hives are produced when the immune system responds to a false alarm. The role of IgE in the natural order is not known, although some scientists suspects that is developed as a defense against infection by parasitic worms. The first time an allergy-prone person is exposed to an allergen, he or she makes large amounts of the corresponding IgE antibody. These IgE molecules attach to the surfaces of mast cells (in tissue) or basophils (in the circulation). When an IgE antibody sitting on a mast cell or basophil encounters its specific allergen, the IgE antibody signals the mast cell or basophil to release chemicals mediators stored within its granules(Roitt and Delves, 2001; USC, 2006). These mediators include histamine, heparin and substances that activate blood platelets and attract secondary cells such as eosinphilis and neutrophils. The activated mast cell or basophil also synthesizes new mediators, including prostaglandins and leukotrines, on the spot. It is such chemical mediators that cause the symptoms of allergy, including wheezing, itching, sneezing and runny eyes. They can also produce lii anaphylactic shock, a life threatening allergic reaction characterized by swelling of body tissues, including the throat, and a sudden fall in blood pressure. Type II hypersensitivity occurs when antibodies bind to antigens on the patient’s own cells, marking them for destruction. This is also called antibody-dependent (or cytotoxic) hypersensitivity, and is mediated by IgG and IgM antibodies (Roitt and Delves, 2001; USC, 2006). Immune complexes (aggregations of antigens, complement proteins, and IgG and IgM antibodies) deposited in various tissues trigger Type III hypersensitivity reactions (Roitt and Delves, 2001). Type IV hypersensitivity (also known as cell-mediated or delayed type hypersensitivity) usually takes between two and three days to develop. Type IV reactions are involved in many autoimmune and infectious diseases, but may also involve contact dermatitis (poison ivy). These reactions are mediated by T cells, monocytes, and macrophages (USC, 2006). 1.4.2 Immune Deficiency Diseases An immune deficiency disease occurs when one or more cells within the immune system do not operate properly, or the system is absent altogether. Some immune deficiency diseases are relatively common, while others are extremely rare(Janeway et al., 2005). A primary immune deficiency occurs when the abnormalities of the immune system develop from an inborn defect in the cells. Affected cells include Tcells, B-cells, B-cells, phagocytic cells or the complement system (Janeway et al., 2005). Most primary immune deficiencies are inherited diseases; examples include X-linked agammaglobuliemia (XLA) and severe combined immunodeficiency (SCID) which appear to run in families. Other primary immune deficiencies, such as common variable immunodeficiency (CVID), appear less obviously inherited, but the causes are unknown and genetic factors cannot be ruled out. Secondary, immune deficiencies occur when damage is caused by environmental factors. Radiation, chemotherapy, burns and infections contribute to the many causes of secondary immune deficiencies liii (Goldsby et al., 2000; Roitt et al., 2001). Acquired Immune Deficiency Syndrome (AIDS) is a secondary immune deficiency caused by the Human Immunodeficiency Virus (HIV) that destroys T4 cells. AIDS infections are known as “opportunistic” because they are produced by “commonplace” organisms that are harmless in people with healthy immune system, but which take advantage of the “opportunity” provided by immunocompromised condition. The most common infection is an unusual and lifethreatening form of pneumonia; Pneumocystis jerovecii (a protozoan infection). AIDS patients are also susceptible to unusual lymphomas and Kaposi’s sarcoma, a rare cancer that results from the abnormal proliferation of endothelial cells in the blood vessels. Although there are quite a number of antiretroviral drugs which is used successfully in reducing viral burden, there is still no definite cure for AIDS. The use of immunomodulators, especially those of natural origin in tackling the menace of HIV holds a lot of promise in preventing or treating opportunistic infections (Yamaguchi, 1992). In Leukemia and multiple myeloma, abnormal, cancerous immune cells crowd out the normal stem cells of the bone marrow. These abnormal cells reduce the number of B cells and lead to hypogammaglobulienemia or secondary immune deficiency (Roitt and Delves, 2001; Janeway et al., 2005). Diets lacking sufficient protein are associated with impaired cell-mediated immunity, complement activity, phagocytes function, IgA antibody concentrations, and cytokine production. Deficiency of single nutrients such zinc; selenium; iron; copper; vitamins A, C, E, and B6; and folic acid (vitamin B9) also reduces immune responses (Chandra, 1997). 1.4.3 Autoimmune Diseases Sometimes the immune system’s recognition apparatus breaks down, and the body begins to manufacture antibodies and T cells directed against the body’s own constituents-cells, cell components, or specific organs. Such antibodies are known as autoantibodies, and the diseases they produce are liv called autoimmune diseases. Autoimmune reactions contribute to many enigmatic diseases. For instance, autoantibodies to red blood cells can cause anemia, autoantibodies to pancreas cells contribute to Type I diabetes, and autoantibodies to nerve and muscle cells found in patients with myasthenia gravis. Autoantibody known as rheumatoid factor is common in persons with rheumatoid arthritis. Persons with systemic lupus erythematosus (SLE), whose symptoms encompass many systems, have autobodies that affect the immune system at several levels. In patients with SLE, for instance, B cells are hyperactive while suppressor cells are underactive. Moreover, production of IL-2 is low, while levels of gamma interferon are high (USC, 2006). The cause of autoimmune disease is not well understood, but several factors are likely to be involved. These may include viruses and environmental factors such as exposure to sunlight, certain chemicals, and some drugs, all of which may damage or alter body cells so that they areno longer recognizable as self. Sex hormones may be important, too, since most autoimmune diseases are far more common in women than in men (USC, 2006). Heredity also appears to play a role. Autoimmune reactions, like many other immune responses, are influenced by the genes of the MHC. A high proportion of human patients with autoimmune disease have particular histocompatibility types. For example, many persons with rheumatoid arthritis display the selfmarker known as HLA-DR4 (Goldsby et al., 2000). Many types of therapies are being used to combat autoimmune diseases. These include corticosteroids, immunosuppressive drugs developed as anticancer agents, radiation of the lymph nodes, and plasmapheresis, a sort of “blood washing” that removes diseased cells and harmful molecules from the circulation (Goldsbyet al., 2000). 1.4.4 Graft Versus Host Diseases lv Transplantation of tissues and organs can cause life-threatening reactions in recipients (Janeway et al., 2005). Graft cell reaction is specific and immunologic and has memory. It has been shown to be mediated by lymphocytes. The antigens that serve as the principal targets of rejection are proteins encoded in the major histocompatibility complex (MHC). The mainstay of preventing and treating the rejection of organ transplant is immunosuppression, designed mainly to inhibit T cell activation and effector function (Abbas and Lichtman, 2004). 1.4.5 Immune Complex Diseases Immune complexes are clusters of interlocking antigens and antibodies. Under normal conditions immune complexes are rapidly removed from the bloodstream by macrophages in the spleen and Kupffer cells in the liver. In some circumstances, however, immune complexes continue to circulate. Eventually they become trapped in the tissues of the kidneys, lung, skin, joints, or blood vessels depending on the nature of the antigen, the class of antibody, and the size of the complex. There they set off reactions that lead to inflammation andtissue damage (Roitt et al., 2001). Sometimes, as is the case with malaria and viral hepatitis, they reflect persistent low-grade infections. Frequently, immune complexes develop in autoimmune disease, where the continuous production of autoantibodies overloads the immune complex removal system (Goldsby et al., 2000). 1.5 The Concept of Immunomodulation, Immunosuppression, Immunostimulation and Immunotolerance The innate and adaptive immune mechanisms could be modified by substances to either enhance or suppress their ability to resist invasion by pathogens (William, 2001). Both immunostimulation and immunosuppression are clinically relevant and search for substances and leads with these activities are becoming a field of major interest all over the world (Naved et al., 2005). lvi Those compounds which appear to stimulate the human immune response are being sought for the treatment of cancer, immunodeficiency diseases, or for generalized immunosuppression following drug treatment; for combination therapy with antibiotics; and as adjuncts for vaccines (Jong and Birmingham, 1992). Those compounds that suppress immune reactions are potentially useful in the remedy of autoimmune (an abnormal immune response against self-antigens) or certain gastro-intestinal tract diseases (e.g. Crohns) (Badger, 1983). Whether immunomodulators enhance or suppress immune responses can depend on a number of factors such as dosage, route of administration, and timing and frequency of administration. 1.5.1 Immunostimulation The immune response can be modified to enhance the humoral or cell-mediated immune response against antigens. Immunostimulation reinforces the immune system. The mechanisms of potentiation of immune responses may include one or all of the following situations: an increase in the rate; a heightened intensity or level; an extension of normal immune response; and the induction of immune response to an otherwise non-immunogenic substance (Jong and Birmingham, 1992). 1.5.2 Immunosuppression The term immunosuppression refers to impairment of any component of the immune system resulting in decreased immune function. Clinical indicators of immunosuppression include: myelosuppression, such as pancytopenia, leucopenia, lymphopenia, and other blood dyscrasias; hypocellularity of immune system tissues and decreased organ weight (such as the thymus, spleen, lymph nodes, or bone marrow); decreased serum globulin levels; increased incidence of infections; and increased incidence of tumors(Badger, 1983). It is important to differentiate between unintended (adverse) immunosuppressive effects and intended (pharmacodynamic) effects. For example, many antitumor drugs are toxic to rapidly dividing cells. lvii Immunosuppression due to bone marrow toxicity would be considered an adverse effect during the treatment of a solid tumor, but not necessarily during treatment of a haematologic malignancy. For drugs intended to be used for prevention of transplant rejection (e.g. cyclosporine), immunosuppression is the intended pharmacodynamic effect. Although this distinction appears to be relatively obvious, there are examples of drugs in which the relationship between immunosuppression and pharmacodynamic effects appears subtly, yet is important e.g., nonsteroidal anti-inflammatory drugs, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and testosterone (Fimmel and Zouboulis, 2005). Methods to enhance detection of immunosuppression in standard toxicology studies have been described, including exact tissues that should be examined and effects that should be noted. These include determination of serum biochemical markers such as globulin levels, hematology (including differential), gross pathology findings, immune system-related organ weights, and histologic examination of immune system-related tissues. 1.5.3 Tolerance Tolerance refers to the specific immunological non-reactivity to an antigen resulting from a previous exposure to the same antigen (Miller, 1993). While the most important form of tolerance is nonreactivity to self-antigens, it is possible to induce tolerance to non-self-antigens. When an antigen or substance induces tolerance, it is termed tolerogen. Tolerance can be induced to antigenic components on both soluble proteins and cells (tissues) by injecting these materials into animals. Tolerance to tissue and cell antigens can be induced by injection of hemopoietic (stem) cells in neonatal or severely immunocompromised (achieved by lethal irradiation or drug treatment) animals. A state of tolerance to a variety of T-dependent and T-independent antigens has been achieved using some substances in various experimental models (Roitt et al., 2001). 1.6 Potentials of Mushroom as Immunomodulatory Substance of Natural Origin lviii The value of medicinal mushrooms to human health is increasingly gaining acceptance as researchers provide data on the array of bioactive compounds found within these fascinating fungi. Mushrooms grow wild in many parts of the world and also are commercially cultivated. It is low in calories and carbohydrate; high in vegetable proteins and essential amino acids; a source of some fibre; and rich in some important vitamins and minerals, including B vitamins, iron, potassium, selenium and zinc (Nworu et al., 2011). Researchers have found that mushrooms can directly stimulate both the lymphocytes, neutrophils and secondary immune responses (immunoglobulin IgE, IgG) of the immune system. This stimulus can increase production of immune defenders such as cytokines and macrophages, which play important roles in recognizing and removing foreign antigens, as well as releasing chemical mediators including interleukin-I (Adachiet al., 1994). Some substances with immunomodulatory activities have been isolated from different species of mushroom and include beta-glucans, lentins, polysaccharides, polysaccharide-peptide complexes, triterpenoids, nucleosides and other secondary metabolites. Many of these bioactive substances, through their stimulatory effects on the immune system, are showing powerful antitumour, antimutagenic and anticancer activity (Borchers et al., 2008). Of the hundreds of known mushroom varieties, several have been studied for their ability to enhance the human immune system and fight infections. Some well-known medicinal mushrooms with benefits for the immunesystem are presented in table 3. The healing and immunostimulating properties of mushrooms have been known for thousands of years. The extracts of these mushrooms were widely used for treatment purpose indifferentcountries. The number of mushrooms on earth is estimated to be 140000: approximately10%(about 14,000 named lix species) is known. These mushrooms contain biologically active polysaccharides in fruit bodies and cultured mycelium. lx Table3: Some Mushrooms with Identified Immunomodulatory Activities Name of Mushroom Common Name References on Immuno-Activity Cordyceps sinensis Cordyceps (Caterpillar fungus) Bo And Bau, 1980; Chang, 1981; Lui et al., 1992; Ying et al., 1987 Ganoderma Iucidum Reishi Chang, 1981; Lin et al, 2006; Dharmananda, 1988; Lui 1993, Miyazaki and Nishijima, 1981; Willard, 1990 Trametes versicolor Kawaratake (Turkey Tail) Arora, 1986 Auricularia auricular Kikurage Arora, 1986; Bo and Bau, 1980; Ying et al., 1987. Agaricus blazeii Himematsutake Arora, 1986; Bo and Bau, 1980; Ying et al., 1987. Trametes versicolor Kawaratake (Turkey Tail) Arora, 1986 Auricularia aruricular Kikurage Arora, 1986; Bo and Bau, 1980. Agaricus blazeii Himematsutake Arora, 1986; Bo and Bau, 1980; Ying et al., 1987. Lentinula edodes Shitake (Snake butter) Liu et al., 1992 Grifola frondosus Maitake Arora, 1986; Adachi et al.,1994; Hericium erinaceus Lion’s mane Hericium Yang and Jong, 1989 Grifola umbellata Choreimaitake (zhu ling) Arora, 1986; Suzuki, 1990 Flamulina velutipes Enokitake (Velvet Foot) Arora, 1986; Zhou et al, 1989 Tremella fuciformis White fungus Arora, 1986; Ying et al., 1987 Poria cocos Bukuryo (Hoelen) Narui et al., 1980 lxi 1.7 Beta (B) Glucans Polysachandes is produced universally by living organisms. They exhibit a large variety of complex chemical structures, physiological functions and a wide range of potential application. They are polymers, of sugars (monosaccharides) joined to each other by glycosidic linkages. These are very complex molecules because sometimes covalent bonds occur between many pairs of carbon atoms(Bohn and Miller, 1995; Celine and Philippe, 2007). Consequently, one sugar unit can be joined to more than two other sugars, which results in the formation of highly branched enormous macromolecules(Bohn and Miller, 1995; Celine and Philippe, 2007). The polysaccharides of mushrooms occur mostly as glucans. Some of which are linked by ß-(1-3), (1-6) glycosidic bonds and α – (1-3) glycosidic bonds but many are true heteroglycans. Beta glucans are found in such foods as oats, barley, cereals, seaweeds, mush rooms and yeasts (Bohn and Miller, 1995; Celine and Philippe, 2007). ß – (1,3) D glucans are also found both in the prokaryotes and eucaryotes. Betaglucans from fungi and yeast are found as cell wall components. They have a common structure comprising a main chain of (1,3) - Linked ß-D- glucopyranosyl units attached by (1, 6) linkages. Glucans from barley oats or wheat are also found as cell wall components of grains but exhibit a different structure, with a mixed ß- (1,3) and ß – (1,4) glycosidic bonds in the backbone. 1.7.1 Beta Glucans and the Immune System Beta glucans do not in themselves cure disease. Rather, they help stimulate the immune system to work better so that diseases can be prevented from attacking the body. Recent studies help us understand how β gucans stimulate the immune system: β glucans from fungi bind to specific membrane receptors of phagocytic cells and natural killer (NK) cells, stimulating their germ-killing abilities. lxii It also appears that β glucan molecules resemble the molecules found on bacterial cell walls. In effect, β glucan molecules make the body believe it is being invaded by a bacterium. β glucan have been known to scientists as a plant constituent for decade. It has been common knowledge in the scientific community that βglucan is the most powerful immune stimulant and a very powerful antagonist to both benign and malignant tumors (Dalia et al.,2007). β glucan are naturally occurring polysacharides. These glucose polymers are the constituents of the cell wall of certain pathogenic bacteria (Pneumocystis jerovecii, Cryptococus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, Candida albicans) and fungi (Saccharomyces cerevisiae). The main components of the fungal cell wall are polysacharides and glycoproteins. Sacharomyces cerevisiae cell wall consists of three layers: an inner layer of soluble B-glucan (30-35%), middle layer of soluble β Glucan (20-22%), external layer of glycoprotein (30%) (Tokunaka et al., 2002). B glucan has been purified from brewers and baker’s yeast, oats and barley bran(Baur and Geisler, 1996;Tokunaka et al., 2000). 1.7.1.1 Beta Glucan Immunostimulating Activity. Persons who suffer from systemic fungal infections including those caused by Candida, Aspergillus and Cryptococcus species have being described to possess high levels of circulating b-glucans in their plasma. It is possible that they may have modulating effects on the immune system by activatingmacrophages, phagocytosis of the pathogens, release of proinflammatory cytokines(Sato et al., 2006).B glucans have been established as a key molecular pattern recognized by neutrophils(or polymorphonuclear leukocytesPMNs) in response to Candida albicans, because antibody specific for β glucan, a major component of yeast cell wall, blocks this response(Lavigne et al., 2006). lxiii This mechanism to recognize and respond to their conserved structural components, particularly B – glucans has evolved in mammals as defense against fungal pathogen. Macrophages play a critical role in all phase of host defense that are both innate and adaptive immune response in case of an infection. Lysosomal enzymes and phagocyticactivity determine the macrophage function. The secreting of cytokines (1L-1,1L-6,1L-81,1L-12, TNF-α) and inflammatory mediators (nitric oxide-NO and hydrogen perodixe H2o2) are other effects of these cells. Therefore activation of macrophage functions by β glucans increase host immune defense. However, polysaccharides stimulate a dose dependent increase in NO and TNF – α but not in reactive oxygen intermediate production in peritonuem macrophages (Kim et al., 2004). It is suggested that the ability of polysaccharides upon the up regulation of these surface molecules involved in antigen presenting processes may by inference activate T cell mediated immunity against malignant cells invivo. Taken together, these results suggest that β glucan acts as an effective immunodulator and enhances the antitumoral activity of peritoneum macrophages (Kim et al., 2004). 1.7.1.2 Beta Glucan Increases Resistance to Infectious Challenge B glucan itself can elicit broad anti-infective effects. Staphylococcus aureus, Echerichia coli, Candida albican, Pneumocystis jerovecii, Listeria monocytogenes, Leishmonvia donovani, influenza virus are the micro-organisms against which a protective effect of β glucan has been established(Dalia et al., 2007). The potential antiviral effect of Sacharromyces cerevisiae b glucan was investigated on the pneumonia induced by swine influenza virus (SIV). The microscopic lung lesions induced by SIV infection were significantly more severe than those induced by infect in animals preadministered β glucan(Dalia et al., 2007). Studies have also shownthat systemic β glucan treatment would result in enhanced migration of neutrophils into a site of inflammation and improve antimicrobial function(Leblanc et al., 2006)as well as lxiv a priming effect for chemotaxis and respiratory burst in circulating neutrophils isolated from β glucantreated animals(Leblanc et al., 2006). 1.7.1.3 Beta Glucan AnticarcinogenicActivity Carcinogenesis can be separated to different stages. The initiation phase involves exposure to a mutagen and often requires its subsequent metabolic transformation into a biologically active form. This exposure even if resulting in permanent damage to DNA is often insufficient by itself to cause cancer. Studies have shown that polysaccharide mushroom extracts can inhibit cell transformation induced by a defined oncogene through a novel non cytocidal route(Wendy et al., 2004; Dalia et al., 2007). Natural killer cells (NK) aredirectly cytotoxic for tumor cells and play a primary role in regulating immune response. Experimental studies have also shown that β glucan may mediate their anticarcinogenic activity through one pathway called “Angiogenesis” which is a crucial to tumor growth and metastasis and interruption of this process is a prime avenue for therapeutic intervention of tumor proliferation. Natural killer(NK) cells are also directly cytotoxic for tumor cells and play a primary role in regulating immune responses. As immunostimulating agents which acts through the activation of macrophages and NK cells cytotoxicity, β glucan can inhibit tumor growth in promotion stage(Kodama et al., 2002; Ho et al., 2004; Kodama et al., 2005). 1.7.1.4 Beta Glucan as Adjuvant to Cancer Chemotherapy and Radiotherapy The major side effect of most chemotherapeutic drugs is neutropenia. The administration of anticancer drugs often impairs blood forming functions. These functions are important to maintain thedefense system ofthe individual. As a result, chemotherapy may accelerate risk of infection that decreases the quality of life for cancer patients. β glucan extracted from certain mushroom have been shown to reduce cyclophosphamide induced leucopenia(Harada et al., 2002). lxv Radiotherapy often results in hematopoietic and immune depletion. Patients often experience anemia, lymphocytopenia and thrombocytopenia. This leads to high risk of development of serious and lethal infections andincreasing the mortality andmorbidity of these patients. Experiment has demonstrated that soluble yeast β glucan could enhance the proliferation ofhaematopoietic cells transplantation in a CR3 dependent manner. Taken together these observations suggest a novel role for complement CR3 and b glucan in the restoration of hematopoeisis following bone marrow injury(Jin et al., 2003; Cramer et al., 2006). 1.8 Botanical Profile and Review of Pleurotus tuberregium Pleurotus tuberregium(Fr) Sing, is an edible basidiomycetes which grows in both tropical and subtropical regions of the world(Okhuoya and Okogbo, 1991). It is found in many parts of tropical Africa and forms a part of the local people’s food system. The sclerotia is collected from the forests, dried and stored for future use as food(Isikhuemhen and Okhuoya, 1995). The tuberous sclerotium can be used as a partial replacement for melon seed (Citrulus ianatus) or groundnut (Arachis hypogea) cake in traditional preparation of sauces and soups. P. tuberregium is used for medicinal purposes by traditional medical practitioners(Isikhuemhen and Okhuoya, 1995). lxvi 1.8.1 Taxonomy of Pleurotus tuberregium Kingdom: Fungi Phylum: Basidiomycetes Class: Agaricomycetes Order: Agaricales Family : Pleurotaceae Genus: Pleurotus Species: P. tuberregium Synonyms: Pachyma tuberegium Fr.1822 Lentinus tuber-regium(Fr) 1836 Common vernacular names: 1.8.2 usu(Igbo), awu(Igala), katala(Hausa), umoho(Igede). Botanical Description of Pleurotus tuberregium P. tuberregium is found in many parts of the world especially the tropical and subtropical regions of the world (Okhuoya and Okogbo, 1991). It produces mushroom from a unique globose sclerotium that is more like a giant truffle. The sclerotia or underground tuber are usually spherical to ovoid, subterranean sclerotia which sometimes measure up to 3cm in diameter (Okhuoya and Okogbo, 1991; Fasidi and Olorunmaiye, 1994). The sclerotium is dark brown on the outside and whitish on the inside (Okhuoya and Okogbo, 1991;Oso, 1977). The fruiting bodies of P. tuberregium is shown in figure 2 lxvii Figure 2: The fruiting bodies of Pleurotus tuberregium lxviii 1.8.3 Geographical Distribution ofPleurotus tuberregium P. tuberregium (Fr) Sing, occurs in both tropical and subtropical regions of the world (Okhuoya and Okogbo, 1991). The geographical distribution of P. tuberregium includes most of equatorial Africa, India, Sri Lanka, South East Asia and North Australia(Oso, 1977). 1.8.4 Ethnomedicinal and Folkloric Uses of Pleurotus tuberregium P. tuberregium is used for medicinal purposes by traditional medical practitioners(Zoberi, 1973; Oso, 1977; Okhuoya and Okogbo, 1990; Okhuoya and Isikhuemhen, 1995). Most studies on P. tuberregium have concentrated on the food and nutritional contents of the sclerotium and sporophores. In Nigeria, both the sclerotium and the mushroom are eaten. The sclerotium of P. tuber-regium has long being used for food and medicine by various tribes in Nigeria(Isikhuemhen and Okhuoya, 1995;Akpaja et al., 2003). The Igbo use it to treat heart problems while it is used to treat asthma cough and obesity among people of Edo State(Isikhuemhen and Okhuoya, 1995; Isikhuemhen et al., 2000a, 2000b). The sclerotium which is hard is peeled and ground for use in a vegetable soup(Fasidi and Olorunmaiye, 1994). lxix It is used in the preparation of cures for headache, stomach ailments, chest pain, colds and fever, asthma, small pox, and high blood pressure (Okhuoya and Okogbo, 1991; Fasidi and Olorunmaiye, 1994; Alobo, 2003). It is sometimes used for weight gains in malnourished babies. It is also used in the Asaba area of Nigeria in herbal preparation for pregnant women to aid the development of foetus. In Ghana, the slerotium is used mainly for fattening of malnourished babies and as one of the ingredients in the embalming of dead bodies(Okhuoya et al., 1998). It has also being reported that the pure culture of this fungus is able to kill and feed on nematodes(Hibbett and Thom, 1994). 1.8.5 Pharmacological Studies, Phytochemical and Proximate Constituents of P. tuber regium P. tuberregium is considered a popular edible mushroom and has been considered a profound health promoting mushroom in traditional Chinese medicine(Isikhuemhen et al., 2000a, 2000b; Huang, 2002).P. tuberregium polysaccharideattenuates hyperglycemia and oxidative stress in experimental diabetic rats(Hui et al., 2012). The fruit bodies ofP. tuberregium are rich in protein while the sclerotium is rich in fiber especially non starch polysaccharides(Kadiri and Fasidi, 1990) mainly composed of bioactive β glucans responsible for pharmacological actions(Cheung and Lee, 2000; Tao et al., 2006). For most other species of Pleurotus, several medicinal properties have been reported. They include properties attributable to their polysaccharides;antigenotoxic, biomutagenic activities(Fillipie and Umek, 2002) antiinflammatory activity, antilipidemic, antihypertensive and antihyperglycemic activities(Huet al., 2006) antibacterial and antifungal activities(Hu et al., 2006;Ngai and Ng, 2006; Lee at al., 2010; Dahech et al., 2011; Wong et al., 2011). lxx CHAPTER TWO MATERIALS AND METHODS 2.1 Materials 2.1.1 Chemicals and Reagents Ovalbumin (Sigma, Germany), Tween 20 (Sigma, Germany), ABTS Peroxidase Substrate System, Peroxidase-labelled antibodies (HRP anti-Mouse IgG (H+L), anti-mouse IgG1 (H+L); anti-mouse IgG2a) (Kirkegaard & Perry Laboratories, KPL, Gaithersburg, MD, USA), Peroxidase Stop Solution Concentrate (KPL, USA), Coating Buffer Concentrate (KPL, USA), Blocking Solution Concentrate (KPL, USA), absolute ethanol (sigma), Indian ink, aluminium hydroxide (Al(OH)3), sodium bicarbonate (Na2CO3 ), pyrogen free sterile normal saline, distilled water (service training centre unn), Fehlings solution I and II, Dragendorff’s reagent, Wagner’s reagent, Mayer’s reagent, Hagner’s reagent, tetraoxosulphate (vi) acid, ammonium hydroxide, ferric chloride, ethanol, lead subacetate, 2% dinitrobenzoic acid in 90% ethanol, glacial aceytic acid, aluminium chloride, arachis oil. All reagents and solvents used were of analytical grade. 2.1.2 Equipment Thermomax Microplate ELISA Reader (Molecular Device, USA), 96 well ELISA plates (Titertek®),centrifuge (Gallenkamp, Germany), UV spectrophotometer(UnicoTM UV2102PC). 2.2 Methods 2.2.1 Collection and Authentication of Plant Materials The fruiting bodies of the mushroom, Pleurotus tuberregium (Figure 2) were purchased commercially from a local market – “Eke Atta” in Ikeduru Local Government Area of Imo State in the months of November to January. The plant material was authenticated by Mr Alfred O. Ozioko, a taxonomist of the lxxi international Center for Ethnomedicine and Drug Development (Inter CEDD), Nsukka, Enugu State, Nigeria. 2.2.2 Preparation of Plant Material Collected fruiting bodies of the mushroom plant, Pleurotus tuberregiumwere thoroughly cleaned and dried on filter paper sheets under shade at room temperature for about 2 weeks. After drying, the plant materials were properly pulverized using a hammer mill. Thoroughly shade dried finely powdered parts of the plant were extracted using aqueous hot water extraction method by heating the powder with distilled water at 98oC for 4 h(Jones, 1998). The extract was then allowed to cool and filtered using Whatman filterpaper no 4. The extract concentration was then determined and aliquoted in air tight containers and stored at -20oC for further experimental studies. The extract yield was determined as 18.45%, w/w. 2.2.3 Bioactivity Guided Fractionation of Crude Extract Based on the result of a prior preliminary immunomodulatory screening of the crude extract of Pleurotus tuberregium (PT) as well as the evidence of immunomodulatory activity of β-glucan polysacharide rich fractions of some mushrooms (Pugh et al., 2001; Wasser, 2002; Borchers et al., 1999), aβ−1,3−D-glucanrich polysaccharide fractionof P. tuberregium was extractedand studied for immunomodulatory properties. The scheme used for this separation and activity guide is shown in Figure 3. lxxii PLANT MATERIAL (P.tuber-regium) Mascerate with 96% ethanol + 80oC + 3 hrs + allow to cool + filter Residue Filtrate Residue air dried+ hot distilled water + water + 98oC + 4 Hrs + Cool +filter Filtrate Residue Centrifuge at 5000 rpm for 10 min Residue supernatant 1.5 × Equal volume of ethanol + 4oC+Centrifuge Supernatant Residue Freeze dry β-glucan rich polysaccharide for further immunomodulatory activity screening lxxiii Figure 3: Schematic Representation of the Extraction of Pleurotus tuber-regium(PT)and its Beta Glucan Rich PolysaccharideFraction (BGP) 2.2.4 Extraction of the Beta-glucan (β) Rich Polysaccharide Fraction of Pleurotus tuberregium The β-glucan rich polysaccharide fraction of Pleurotus tuberregium was extracted from about 500 g portion of the powdered Pleurotus tuberregium. The plant material (500 g) was extracted with 96% ethanol (11.25 L) at 80oC for 3 h to remove low molecular mass compounds (Robyt, 1998). The air dried residue was extracted with 2.5 L of hot distilled water (98oC) for 4 h. The filtrate was centrifuged and the supernatant (PT) recovered after centrifugation. Subsequently, the β-glucan rich polysaccharide extract (BGP) was obtained using the modification of a previously described protocol(Wasterlund et al., 1993). Briefly BGP was precipitated from PT with the addition of 1.5 volumes of absolute ethanol. The precipitate was stored overnight at 4oC the precipitate was centrifuged and freeze dried to obtain BGP powder (10g, 2.0%[w/w]). A water soluble beta glucan rich polysaccharide extract of P. tuberregium was extracted and studied for immunomodulatory properties. 2.2.5 Phytochemical Analysis of Extracts Phytochemical studies were carried out on the hot aqueous crude extract and on the β-glucan rich polysaccharide fraction of Pleurotus tuberregium. Phytochemical studies were carried out using lxxiv appropriate procedures (Harborne, 1998;Evans, 2009). The extracts were screened for carbohydrate using Molisch test; reducing sugar using Fehling’s test; alkaloids using Dragendorff’s, Meyers and Wagner’s reagent; glycosides using hydrolysis, Bontrager’s, Modified Bontrager, Keddel’s and KellerKiliani test; flavonoids using ammonia and ammonium chloride tests; saponins using frothing and emulsion tests, tannins using ferric chloride and lead subacetate tests; sterols and triterpenes using Moleschott’s and Salkowski’s tests. 2.2.6 Pharmacological Studies 2.2.6.1 Animals Adult swiss albino mice of either sexes of 7-8 weeks old weighing 20 g - 30 g obtained locally from the laboratory animal facility of the Department of Pharmacology and Toxicology, University of Nigeria, Nsukka were used in the study. The animals were acclimatized for at least 5 days in the institutional animal facility under standard conditions of temperature (23oC ± 2oC) and 12:12 h of light and dark cycle. The animals were fed with standard animal feed (Livestock Feed PLc, Lagos, Nigeria) and water ad libitum. 2.2.6.2 Antigen Ovalbumin antigen (Sigma, Germany) was used for immunization and challenge. 2.2.6.3 Acute Toxicity (LD50) Test of Extracts The acute toxicity of PT and BGPwas estimated in mice using the appropriate method (Lorke, 1983). The procedure was carried out using the oral route. The first phase was determination of the toxic range. The mice were placed in three groups of 3 mice each and were given 10, 100 or 1000 mg/kg of PT or BGP. The treated mice were observed for 24 h for number of deaths. lxxv The death pattern in the first phase determined the doses used for the second phase. Since there was no death recorded in the first phase, for the oral route of administration, four mice received 1000, 1600, 2900 or 5000 mg/kg of the extracts at one dose per mouse. The treated animals were observed for 24 h for lethality and other signs of acute intoxication. The LD50 was then calculated as the geometric mean of the highest non-lethal dose and the least toxic dose. 2.2.7 Studies on the Hot Aqueous Extract of Pleurotus tuberregium (PT) and the Beta (β)-Glucan RichPolysaccharide Fraction of Pleurotus tuberregium (BGP) The hot aqueous extract of Pleurotus tuberregium was screened for immunomodulatory activity in mice using different models which showed the effect of the extract on some specific and non-specific immune responses. The effect on delayed type hypersensitivity response, specific primary and secondary antibody synthesis, carbon clearance, relative organ weight as well as in vivo leukocytes mobilization was studied at this stage. Further immunomodulatory studies were carried out on BGP using delayed type hypersensitivity model and antibody ovalbumin – specific determination (total IgG, IgG1 and IgG2a) in mice. 2.2.7.1 Studies on Relative Spleen Weight PT (100, 200 or 400 mg/kg)or Morinda citrifolia extract (Noni®, 100 mg/kg, per os; Good ′N Natural, Ronkonkoma, NY)were administered orally to each mouse group for 7 days. On the Day 8, the animals were sacrificed 24 h after the last dose and the spleen were freshly excised and weighed. The results are expressed as the relative organ weight (organ weight/100 g of body weight) for each animal(Davis and Kuttan, 2000). 2.2.7.2 Studies on Phagocytic Index lxxvi Phagocytic activity of the reticuloendothelial system in vivo was determined by carbon clearance test. PT (100, 200 or 400 mg/kg) or Noni® (100 mg/kg)were administered orally to each group of animal for 8 days. On the Day 8, after the last dose, the animals received an intravenous injection of carbon suspension (1:50 dilution of Indian ink, Hi-Media Laboratories Pvt. Ltd, Mumbai, India, in a dose of 1 ml/200 g of body weight through the tail vein). Blood was withdrawn from the retro orbital plexus before injection at 0 min and 15 min after injection of carbon suspension. Each blood sample (50 µl) was lysed with 4ml of 1% Na2CO3 solution. The absorbance was measured spectrophotometrically at 680nm wavelength. The results are expressed as phagocytic index: K = (Ln A t15 min) – (Ln At0 min) / (t 15 min– t0 min) Where, A t15 min and A t 0 minare the Absorbances at 15 min and 0 min respectively.Phagocytic indices (K) were normalized to to the mean index for the untreated control group. 2.2.7.3 Studies on Delayed Type Hypersensitivity Response (DTHR) Delayed type hypersensitivity was induced in mice using ovalbumin as antigen. PT (100, 200 or400 mg/kg) or BGP were administered orally 3 days prior to sensitization and continued daily after the challenge. Animals were sensitized by subcutaneous injection of 100 µg ovalbumin (OVA; 1 µg/µl; Sigma-Aldrich, Munich, Germany) in normal saline (Day 3) in the plantar region of the right hind foot paw and challenged on Day 13. After 24 h the oedema produced by antigenic challenge in the left hind paw was measured with a micrometer screw gauge(AticoTM, Advanced Technocracy Inc., Haryana, India). The DTH response was determined from the increase (swelling) relative to initial footpad size and expressed as mean percent thickness/edema (Barcotti et al., 1984). 2.2.7.4 Studies on Humoral Antibody Response Induced by Ovalbumin lxxvii The effect of PT and BGP on humoral immune responses was determined in mice in two separate experiments using ovalbumin (OVA) as the antigen in a homologous prime-boost strategy. Mice were immunized by an intraperitoneal (i.p.) injection of 100 µg of ovalbumin into their hind footpads (50 µl/footpad)on day 3 and challenged by injecting the same amount (i.p.) on Day 14. Primary antibody titer was obtained on day 14 before the boost immunization, and secondary antibody titres were determined twice on days 21 and day 28(7 and 14 days post booster)by the Enzyme Linked Immunosorbent Assay (ELISA). PT (100, 200 or 400 mg/kg) or BGP (100 or 200mg/kg) were administered orally 3 days prior to sensitization and continued on alternate daysafter the challenge.Blood samples were collected from the retro-orbital plexus of individual sensitized mouse on days 14, 21 and 28and centrifuged to obtain the serum specific immunoglobulins (total IgG, IgG1, and IgG2a) titres against ovalbumin which was determined by Enzyme Linked Immunosorbent Assay. Microtiter plates with 96 wells were coated with 100 µg OVA in bicarbonate buffer pH 9.6 and incubated overnight at 4oC with a proper cover. After the incubation the unbound OVA was washed off thrice with 0.01 M PBS buffer containing 0.05% Tween-20 (PBS-T). The nonspecific binding sites were blocked with 1% solution bovine serum albumin (BSA) in PBS and incubated for 1 h at room temperature. The wells were washed again with PBS-T and incubated with 100 μl of 1 in 25 diluted sera samples in duplicates for 1 h at 37oC. The unbound serum proteins and other constituents were washed off. To assess the antibody levels, 100 μl of HRP-conjugated goat antimouse total IgG, IgG1, and IgG2a secondary antibodies were added at a dilution of 1:1000 was and incubated for another 1 h at room temperature. Finally the unbound conjugates were washed off with PBS-T and 100 μl/well of freshly prepared ABTS Peroxidase Substrate System and stopped with 100 μl of peroxidase stop solution. The colour developed was read at 405 nm using an automatic ELISA reader (Thermomax ® ELISA Plate Reader). The data represent the mean absorbance values of the sera samples lxxviii assayed in duplicates. Naïve mice sera or preimmune sera were included in the assay as control (Beck and Spiegelberg, 1989). 2.2.8 InvitroStudies The immunomodulatory activities of BGP were further evaluated by in vitro studies in the laboratory facility of Prof. Andreal Gamboto, Vector Core Facility, Rangos Research Center, University of Pittsburgh Medical Center, Pennyslyvania, USA. The assistance of my project supervisor; Dr C.S Nworu in getting these studies done is acknowledged with gratitude. Cell lines and Culture medium Spleen cell and RAW264.7 cells were cultured in R-10, consisting of RPMI 1640 medium (Corning cellgro® RPMI; Mediatech Inc., Manassas, VA, USA) supplemented with 10% heat-FBS, 50 µM 2-mercaptoethanol (Gibco, Invitrogen, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin Preparation of lymphocytes from mouse spleens BALB/c mice, 8-10 weeks old, obtained from Charles River Laboratories, Inc., Wilmington, MA, USA, fed and kept in institutional animal facility were used for the study. Mice were euthanized and the spleens were removed aseptically. The spleens were minced and single cell suspension was prepared by gentle dispersion of the cells and straining through 70-μm BD Falcon cell strainer. Residual red blood was haemolysed with acetic acid lysing buffer. The spleen cell suspension was washed twice with Hank’s Balanced salt Solution (Corning cellgro® HBSS; Mediatech Inc., Manassas, VA, USA), and centrifuged twice at 1200 rpm at 4°C for 5 min. The resulting cell pellet was resuspended in R-10 medium and re-suspended lxxix in R-10 medium for subsequent experiments. Cell viability was determined by trypan-blue dye exclusion technique on a Neubauer hemocytometer and the culture adjusted to 1 × 106 viable cells/ml (De Jesus et al., 2013). 2.2.8.1 Spleenocytes Proliferation Assay The lymphoproliferative (mitogenic) effect of the BGP on murine splenic cells was determined by the MTT(3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) reduction assay. MTT is reduced to blue formazan product by mitochondrial dehydrogenase in viable cells which reflects the normal functioning of mitochondria and hence the metabolic rate of cells. Spleenocytes (1×105cells in 100 µl) were seeded in a 96-well U-bottom plates. Graded concentrations of BGP (5, 25, 50, 100, 250 or 1000 μg/ml).or 1 μg lipopolysaccharide/ml (LPS; serotype 0128:B12, L 4255; Sigma) as a standard mitogen. The negative control consists of similarly un-stimulated wells in which R-10 medium was added. The cells were incubated at 37°C in 5% CO2 for 72 h. Cell proliferation was measured by the MTT cell viability assay. Briefly, 20 μl of MTT tetrazolium bromide) (5 mg/ml in PBS) was added to each well and incubated for another 4 h at 37oC. The culture plate was centrifuged and culture uumedium gently discarded. Formazan crystals formed were dissolved by the addition of 150 μl of DMSO to each well. The plate was agitated for 30 minutes on a shaker and the optical density (OD) of the developed colour was read at 570 nm in ELISA plate reader (Bio-Kinetic Reader-E312e®; Bio-Tech Instruments, Winooski, VT) (Mossman T,1983). The proliferation index (PI) was calculated according to this relationship: = [ . .] [ . .] lxxx 2.2.8.2 Effect of BGP on Inducible Nitric Oxide (iNO) Production/Release Nitric oxide production and release by macrophages was determined by nitrite accumulation in culture supernatant measured by Griess reagent (Green, 1981). RAW264.7 cells (1×105 cells/well) were seeded in 96-well flat plate for 24 h. And then cells were treated for 24 h with BGP (0, 5, 25, 50 or 100 μg/ml)or LPS (1 μg/ml). After incubation, 100 μl of cell-free culture supernatants were mixed with an equal volume of freshly prepared Griess reagent in 96-well plate and incubated at 25℃ for 10 min. Griess reagent contains equal volume of 0.2% naphthylethylenediamine dihydrochloride and 2% sulphanilamide in 5% phosphoric acid. The absorbance was measured on a microplate reader at 570 nm (Bio-Kinetic ReaderE312e®; Bio-Tech Instruments, Winooski, VT). Nitrite concentration was quantified by extrapolation on a standard NaNO2 curve generated by different concentrations of NaNO2 included in the Griess reagent assay. Nitrite concentrations in culture supernatants were measured to assess iNO production in RAW264.7 cells. 2.2.8.3 Effect of BGP on Tumour Necrosis Factor (TNF- Α) Production /Release The effect of BGP on tumour necrosis factor (TNF-α) by macrophages was determined by ELISA. RAW264.7 cells 1×105 cells/well) were seeded in 96-well flat bottom plate for 24 h. The cells were pretreated for 2 h with BGP (0, 5, 25, 50 or 100 μg/ml) or LPS (1 μg/ml) was added to the wells. After incubation, cell-free supernatant was harvested and stored at -20°C for future analysis (Khanittha et al., 2004). The concentrations of TNF-α in the conditioned culture supernatant was measured by cytokine capture ELISA kits according to the manufacturer’s instruction (R&D Systems, Inc., Minneapolis, MN, USA). The concentration of TNF-α was calculated from a standard curve of the standard mouse TNF-α included ELISA assay.The level of sensitivity of the kit was 31.2 pg TNFα/ml. 2.2.8.4 Effect of BGP on Rate of Phagocytosis of Macrophages lxxxi The phagocytic ability of macrophage was measured by neutral red uptake (Kim et al., 2004; Chen et al., 2010). RAW264.7 cells were cultured in 96-well flat-bottom plate at a density of 1×105 cells/well at 37oC in a 5% CO2 atmosphere for 24 h. Thereafter, the cells were incubated with various concentrations of BGP (5, 25, 50, and 100 μg/ml) and LPS (1 μg/ml) at 37℃ for 48 h. Neutral red solution (100 μl; 0.01%) was added and incubated further for 1 h. The supernatant was discarded and the cells were washed twice with PBS and lysed at room temperature with 100 μl of cell lysate solution (1:1 ratio of ethanol and 0.01% acetic acid) added into 96-well plate for 2 h. The optical density was measured with a microplate reader at 570 nm. ℎ ! "# " " $ # 2.9.0 = [%. &. ]'()''*(+',(-' [%. &. ].-)'/,.0+'(123-'*30 Statistical Analysis To demonstrate statistical significance of data, One-way Analysis of Variance (ANOVA; Fisher LSD post hoc test) using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA). Generally, differences between test and control treatments are considered significant at P< 0.05. lxxxii CHAPTER THREE RESULTS 3.1 Extraction The extraction of the mushroom plant, Pleurotus tuberregium (PT) with hot water gave a yield of 90.2 g of aqueous extract which represents 18.45% w/w. Further extraction and purification of PT yielded 10 g (2%,w/w) of the β-D glucan rich polysaccharide fraction (BGP). 3.2 Phytochemical Analysis of Extract and Fraction Phytochemical tests on P. tuberregium gave positive reactions for alkaloids, acidic compounds, carbohydrates, flavonoids, proteins, reducing sugars, resins, saponins, steroids, tannins and terpinoids. The β-glucan rich polysaccharide fraction showed positive reactions for alkaloids, carbohydrates, glycosides, proteins, saponins and steroids. The phytochemical constituents of PT and BGP are shown in Table 4. 3.3 Acute Toxicity Test In the acute toxicity tests in mice, PT and BGPadministered orally at doses up to 5 g/kg body weight did not cause deaths in the mice after 24 h observation period in the two phases of the tests. There was generally the absence ofsymptoms of toxicity. The LD50 is therefore deemed to be greater than 5000 mg/kg. 3.4 Effect of Aqueous Extract of Pleurotus tuberregium on Phagocytic Activity in Mice The phagocytic activity of the reticuloendothelial system invivo which is generally determined by the carbon clearance test was increased in the treated group of mice in a dose related manner when compared to the untreated control group.The mean nomalised phagocytic index was increased to 7.47, lxxxiii 10.07and 7.70 in the groups that were treated with PT (100, 200 or 400 mg/kg). Daily oral administration of PT (100, 200 or 400 mg/kg), increased the mean phagocytic clearance of colloidal carbon in miceby as much as 7-fold, 10-fold and 7-folds respectivelywhencompared to the clearance in the untreated group of mice(Appendix 1, Figure 4). lxxxiv Table 4: The Yield and Phytochemical Constituents of Immune Active Extracts and Fractions of Pleurotus tuberregium. Extract/Fractions Yield Phytochemical Constituents PT 90.2g Alkaloids, acidic compounds, (18.45%w/w) Carbohydrate, flavonoids, proteins, Reducing sugar, resins, saponins, Steriods, tannins, terpinoids BGP 10 g Alkaloids, carbohydrates, (2.0%w/w) glycosides, Proteins, saponins, sterols. Percentage yield in parenthesis lxxxv Phagocytic activity index 20 15 10 5 ) g/ kg 00 (4 PT PT (2 00 m g/ kg m m 00 (1 PT ) ) g/ kg on N U nt re at ed co nt ro l i 0 Treatment Figure 4: The Effect of Hot Aqueous Extract of Pleurotus tuberregium (PT)on Phagocytic Index in Swiss Albino Mice lxxxvi 3.5 The Effect of Hot Aqueous Extract of P. tuberregium on the Relative Spleen Weight of Mice. Short-term daily oral supplementation with PT (100 and 200 mg/kg) and Noni® (100mg/kg, p.o) for 7 days caused a remarkable and dose related increase in the mean relative spleen weight of mice. The mean spleen weight was increased from 0.46±0.04 in the untreated mice group to 0.494±0.012, 0.611±0.026 and 0.567±0.036 in the group that were treated with PT (100 200 and 400 mg/kg) respectively representing a 6.87%, 32.20% and 22.76% increase (Appendix 2, Figure 5). Treatment e 10 0 (4 00 (2 00 (1 00 N on i( PT PT PT N eg at iv m m m m g/ kg ) g/ kg ) g/ kg ) g/ kg ) co nt ro l Specific spleen weight (g/100 g body weight) lxxxvii 0.70 0.65 0.60 0.55 0.50 0.45 0.40 lxxxviii Figure 5: The Effect of Hot Aqueous Extract of P. tuberregium on the Relative Spleen Weight of Mice. 3.6 The Effect of Pleurotus tuberregium Extracts on Delayed Type Hypersensitivity Response (DTHR) Treatment with PT (100, 200 and 400 mg/kg) produced a marked stimulation of DTHR induced by ovalbumin in a dose dependent mannerwith paw thickness increasing from 0.288±0.049 mm, 0.420±0.032 and 0.402±0.101 respectively representing a 1.4%, 47.8% and 40.8% increase when compared to the untreated control mice group (Appendix 3, Figure 6 ). In a similar manner, BGP (100 and 200 mg/kg) caused a marked stimulation of DTHR induced by ovalbumin in mice in a dose dependent manner with paw thickness increasing from 0.618±0.084 mm and 0.658±0.052representing a 36.7% and 45.5% increase (Appendix 4, Figure7). The cell mediated immunity caused by PT and BGP is comparable to that produced by an immunostimulant drug Morinda citrifolia (Noni) used as a standard control. 0.6 0.5 0.4 0.3 0.2 kg ) PT (4 0 (2 0 00 m m g/ gk g kg ) g/ PT PT (1 0 0 m m N on i (1 00 C g/ kg ) 0.1 on tr ol Paw thickness (mm) lxxxix Treatment Figure 6: The Effect of Hot Aqueous Extract of Pleurotus tuberregium (PT)on Delayed Type Hypersensitivity Response in Mice as Measured by Hind Paw Thickness B G P P (2 00 m m tr o l g/ kg g/ kg Treatment ) ) g/ kg ) on (1 00 m (1 00 i G on B N C Paw thickness (mm) xc 0.65 0.60 0.55 0.50 0.45 xci Figure 7: The Effect of Beta (β) Glucan rich Polysacharide Fraction of Pleurotus tuberregium (BGP) on Delayed Type Hypersensitivity in Mice as Measured by Hind Paw Thickness 3.7 The Effect of Hot of Aqueous Extract of Pleurotus tuberregium(PT) on Primary and Secondary Humoral Immune Responses to Ovalbumin in Mice Alternate day oral administration of PT (100, 200 and 400mg/kg body weight) significantly (P<0.05) caused an increase in the levels of ovalbumin (OVA) – specific total IgG, IgG1 and IgG2a in the sera of treated mice which were immunized with OVA. The boost in specific immunization was observed on the 14th day after prime immunization – primary response, and on the 21st and 28th day after boost immunization (ie 7th and 14th day post booster- Secondary response) (Appendix 5, 6, 7) 2-4 folds increases in anti-OVA total IgG, IgG1 and IgG2a were observed (Figure 8, 9, 10). Maximum boost in the antibody response immune was observed in the group of mice administered PT 200mg/kg. The increases in antiOVA antibody titers were similar to the increases caused by daily oral administration of Noni® as the standard immunostimulant agent. Response in Mice Primary response Boost response (7th day) PT (400 mg/kg) PT (200 mg/kg) PT (100 mg/kg) Noni® (100 mg/kg) Negative control PT (400 mg/kg) PT (200 mg/kg) PT (100 mg/kg) Noni® (100 mg/kg) Negative control PT (400 mg/kg) PT (200 mg/kg) PT (100 mg/kg) Noni® (100 mg/kg) Negative control Anti-OVA total IgG titre (Normalised to preimmune naïve sera) xcii 12 10 8 6 4 2 0 Boost response (14th day) Figure 8: The Effect of Hot Aqueous Extract of P. tuberregium (PT) on Ovalbumin Specific Total IgG response in mice Primary response Boost response (7th day) Boost response (14th day) Figure 9: The Effect of Hot Aqueous Extract of P. tuberregium (PT)on ovalbumin-specific total IgG1 PT (400 mg/kg) PT (200 mg/kg) PT (100 mg/kg) Noni® (100 mg/kg) Negative control PT (400 mg/kg) PT (200 mg/kg) PT (100 mg/kg) Noni® (100 mg/kg) Negative control PT (400 mg/kg) PT (200 mg/kg) PT (100 mg/kg) Noni® (100 mg/kg) Negative control Anti-OVA total IgG1 titre (Normalised to preimmune naïve sera) xciii 14 12 10 8 6 4 2 0 response in mice Primary response Boost response (7th day) Figure 10: The Effect of Hot Aqueous Extract of P. tuberregium (PT)on ovalbumin-specific total IgG2a PT (400 mg/kg) PT (200 mg/kg) PT (100 mg/kg) Noni® (100 mg/kg) Negative control PT (400 mg/kg) PT (200 mg/kg) PT (100 mg/kg) Noni® (100 mg/kg) Negative control PT (400 mg/kg) PT (200 mg/kg) PT (100 mg/kg) Noni® (100 mg/kg) Negative control Anti-OVA total IgG2a titre (Normalised to preimmune naïve sera) xciv 8 6 4 2 0 Boost response (14th day) xcv 3.8 Effect of Βeta (β)-GlucanRichPolysaccharide Fraction of Pleurotus tuberregium (BGP) on Primary and Secondary Humoral Antibody Responses to Ovalbumin in Mice In a homologous prime boost schedule, alternate day oral supplementation with BGP (100 and 200mg/kg body weight) promoted an increase in the anti-OVA antibody titers of specific total IgG, IgG1 and IgG2a in the sera of mice treated and immunized with OVA. Observation recorded on the 14th day before boost immunization-primary response, as well as the 7th and 14th day after boost immunization-secondary response indicated a surge in the anti-OVA specific total IgG, IgG1 and IgG2a antibody sera level (Appendix 8, 9, 10). Increases as high as 1-4 folds were recorded (Figure 11, 12, 13). Administration of BGP 200mg/kg body weight was observed to show the maximum boost in the antibody immune response. The increases in anti-OVA antibody titers were similar to the increases caused by oral administration with Noni® as standard control. The result was significant at P<0.05. B B G P P ni G No (2 (1 (1 00 00 00 m m g/ g/ kg kg Treatment ) ) ) ol kg tr g/ on m C Total IgG titre xcvi 0.40 0.35 0.30 0.25 0.20 xcvii Figure 11a: The Effect of β-Glucan Rich PolysaccharideFractionof P. tuberregium on Ovalbumin – Specific Total IgG Primary Response in Mice BG BG ni P P No (2 (1 00 00 00 (1 m m m g/ g/ kg kg l Treatment ) ) ) ro kg nt g/ Co Total IgG titre xcviii 1.2 1.0 0.8 0.6 0.4 xcix Figure 11b: The Effect of Βeta (β) Glucan Rich Polysaccharide Fraction ofP. tuberregium on Ovalbumin – Specific Total IgG Secondary Response in Mice N on i( 10 0m g/ kg ) B G P (1 00 m g/ kg ) B G P (2 00 m g/ kg ) C on tr ol IgG titre c 1.5 1.0 0.5 0.0 Treatment ci Figure 12a: The Effect of Βeta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium on Ovalbumin – Specific IgG1 Primary Response in Mice 3.5 IgG1 titre 3.0 2.5 2.0 1.5 Treatment m g/ kg ) (2 00 B G P B G P (1 00 m g/ kg ) m g/ kg ) 10 0 N on i( N eg at iv e co nt ro l 1.0 cii Figure 12b: The Effect of Βeta (β) Glucan RichPolysaccharide Fraction of P. tuberregium on Ovalbumin – Specific IgG1 Secondary Response in Mice N on i( 10 0m g/ kg ) B G P (1 00 m g/ kg B ) G P (2 00 m g/ kg ) C on tr ol IgG2a titre ciii 1.0 0.8 0.6 0.4 Treatment civ Figure 13a: The Effect of Beta (β) Glucan RichPolysaccharideFraction of P. tuberregium on Ovalbumin – Specific IgG2a Primary Response in Mice 2 1 ) ) kg kg m g/ g/ 00 (2 BG P (1 P BG No ni (1 00 00 m m g/ nt kg ro ) l 0 Co IgG2a titre 3 cv Figure 13b: The Effect of Beta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium on Ovalbumin – Specific IgG2a Secondary Response in Mice cvi 3.9 INVITRO STUDIES 3.9.1 Effect of Βeta (β)-GlucanRich Polysaccharide Fraction of P. tuberregium (BGP) on Spleenocytes Proliferation Treatment with BGP (5 - 1000 µg/ml) produced a remarkable proliferation of spleen cells. Treatment of spleenocytes with BGP produced a marked increase of spleenocytes. Stimulation with BGP (5, 25, 50, 100, 250 and 1000 µg/ml) showed spleenocyte proliferation indices of 1.44±0.02, 1.57±0.01, 1.90±0.05, 2.25±0.14, 1.53±0.07 and 1.28±0.11 respectively representing a 44.4, 57.3, 90.2, 125.1, 53.1, 28.4 and 86.1% increase when compared to the unstimulated control. The response at P< 0.001 was significant (Appendix 11, Figure 14). The highest spleenocyte proliferation was produced by treatment of cell line with 100 µg/ml BGP. The proliferation of spleenocytes produced by β-glucan rich polysaccharide extract of P. tuberregium (BGP) is comparable to that produced by a Lipopolysaccharide used as a standard mitogen control. 3.9.2 Effect of BGP ON TNF-α Production by RAW264.7 Stimulation of RAW264.7 cells with BGP significantly increased (P<0.001) production of TNF-α by RAW264.7.Treatment with BGP (5, 25, 50 or100 µg/ml) increased the mean TNF-α levels in culture supernatant in a concentration-dependent mannerfrom 0.18±0.01 ng/ml in the unstimulated control to 1.180±0.029 ng/ml, 1.580±0.017 ng/ml, 2.064±0.088 ng/ml and 1.987±0.022 ng/ml, respectively. These represents 5-10 fold increases in the concentration of TNF-α over the unstimulated control (Appendix 12, Figure 15). Stimulation of TNF-α expression by 50 and 100 µg BGP/ml are comparable to the production elicited by by LPS (1 µg/ml) used as standard mitogen. 3.9.3 Effect ofBGP on Nitric Oxide Production by Raw264.7 cvii Treatment with BGPproduced in a significant (P<0.001) increase in the production of nitric oxide by RAW264.7. Stimulation of RAW264.7 with BGP (5, 25, 50 or100 µg/ml) increased the expression of nitrite in the culture supernatant from 0.600±0.057 µM in the unstimulated control to 6.933±0.088 µM, 9.767±0.218 µM, 16.200±0.230 µM and 19.670±0.433 µM respectively. These represent 10 – 30 folds increases in the nitric oxide release compared to the unstimulated control treatment (Appendix 13, Figure 16). Nitric oxide production by 100 µg BGP/ml is comparable to that elicited by LPS (1µg/ml) used as a standard mitogen. 3.9.4 Effect ofBGP on the rate ofPhagocytosis (Neutral Red Uptake) RAW264.7 Raw264.7 preteated with BGP (5, 25, 50 or100µg/ml) induced remarkably higher rate of phagocytosis measured indirectly by neutral red uptake assay. Relative phagocytic indices were increased in RAW264.7 pretreated with BGP from1.000±0.013 in the unstimulated cells to 1.119±0.011, 1.165±0.016, 1.181± 0.001, and 1.083±0.010, respectively. These represent 11.9, 16.5, 18.1, and 8.3% increases compared mean phagocytic index of untreated control cells (Appendix 14, Figure 17). The highest ratesof phagocytic indiceswere shown byRAW264.7pretreated with 25 and 50µg BGP/ml and these were similar to the modest increase in phagocytic index of 14.9 % produce by cells stimulated with LPS (1µg/ml). cviii Proliferation index (PI) (Spleenocytes) 2.50 2.25 2.00 1.75 1.50 1.25 1.00 LP C on tr S ol (1 B µg G /m P (5 l) µg 25 /ml (µ ) g/ m 50 l (µ ) g/ 10 m l 0 (µ ) g/ 25 m l 0 (µ ) g/ 10 m 00 l (µ ) g/ m l) 0.75 Figure 14: The Effect of Beta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium (BGP) on Spleenocytes Proliferation cix α (ng/ml) TNFα 2.5 2.0 1.5 1.0 0.5 C on LP tr ol S (1 µ g/ B m G l) P (5 µ B g/ G m P l) (2 5 µ B g/ G m P l) (5 0 µ B g/ G P m l) (1 00 µ g/ m l) 0.0 Treatment Figure 15: The Effect of Beta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium (BGP) onTNF-α Production by RAW264.7 cx 20 15 10 5 C on LP tr ol S (1 µg B /m G l) P (5 µg B G /m P l) (2 5 µg B G /m P l) (5 0 B µg G /m P (1 l) 00 µg /m l) µ M) NO conc. (µ 25 Treatment Figure 16: The Effect of Beta (β) Glucan Rich Polysaccharide Fraction of P. tuberregium (BGP) onNitric Oxide Production by RAW264.7 cxi 1.1 1.0 0.9 0.8 U ns tim ul at LP ed S (1 µg B /m G P l) (5 µg B G /m P l) (2 5 µg B G /m P l) (5 0 B µg G P /m (1 l) 00 µg /m l) Phagocytic activity index 1.2 Treatment Figure 17: The Effect of Beta Glucan Rich Polysaccharide Fraction of P. tuberregium on Rate of Phagocytosis by Raw264.7 cxii cxiii CHAPTER FOUR DISCUSSION AND CONCLUSION 4.1 DISCUSSION In this study the effects of a hot aqueous extract of Pleurotus tuberregium (PT) on some specific and nonspecific immune responses were investigated.Noni®,a polysaccharide-rich precipitate derived from the fruit of Morinda citrifolia,and beneficial in the treatment of immuno-inflammatory disorders (Hirazumi et al., 1996; Zhang et al., 2009; Brooks et al., 2009) was used as the positive control in the in vivo experiments. This popular edible mushroom has been considered as a profound health promoting mushroom. It is reputed as effective in a number of therapeutic areas (Hui et al., 2012). From the preliminary phytochemical studies, the crude aqueous extract of P. tuber-regium contains saponins, alkaloids, flavonoids (aurones, chalcones, flavones, flavonols and leucoanthocyanins), oligigosaccharides as well as phytates and tannins (Basu et al., 2007; Ijeh et al., 2009). All these have potential health promoting effects at least under some circumstances (Basu et al., 2007). In the traditional setting, P. tuberregium has been known for its nutritive and medicinal properties in a wide range of diseases such as in treating colds, malnutrition, anaemia, obesity, cough, asthma etc. Modulation of the immune system response through stimulation or suppression may help in maintaining a disease free state (Ghule et al., 2006). The crude Pleurotus tuberregiumextract showed significant activity on both the humoral and cell mediated immune system. This result encouraged further bioactivity guided fractionation of the crude extract. Many bioactive substances with immunomodulatory properties have been extracted from different mushroom species which include; β-glucans, lentins, polysaccharides, polysaccharide-peptide complexes, triterpenoids, nucleosides, and other secondary metabolites (Luiet al., 1998; Eoet al., 1999; cxiv Zhu et al., 1999).Most of the bioactive polysaccharides isolated from mushroom are water-soluble (1→3)β-D-glucans with (1→ 6)-β-linked side branches (Schmid et al., 2001). Thus, for the present study, the β-Dglucan-rich polysaccharide fraction of P. tuberregium (BGP) was extracted and subjected to analyses of immunomodulatory effects in mice and in cultures of monocytic/macrophage cells (RAW267.4). In general, there were someremarkable effects of PT on theimmune system of treated mice.DTHR induced by ovalbumin was stimulated by P. tuberregium extract in a dose related manner. DTHR which was measured as an indicator of T cell mediated immune response assesses the effect of P. tuberregium (PT) on cell mediated immune response which is initiated by actively sensitized T lymphocytes and is expressed locally by cellular infiltration and inflammation (Sharma et al., 1996). DTHR is an autoimmune response mediated by T cells, macrophages and monocytes. It requires the specific recognition of a given antigen by activated T lymphocytes, which subsequently proliferate and release cytokines. The released cytokines and other molecules increase vascular permeability, induce vasodilatation, macrophage accumulation, and activation, promoting increased phagocytic activity and increased concentrations of lytic enzymes for more effective killing (Elgert, 1996; Descotes, 1999). DTH comprises of two phases, an initial sensitization phase after the primary contact with ovalbumin antigen. A subsequent exposure to the ovalbumin antigen induces the effectors phase of the DTH response, where TH1 cells secrete a variety of cytokines that recruits and activates macrophages and other non-specific inflammatory mediators. The delay in the onset of the response reflects the time required for the cytokines to induce the recruitment and activation of macrophages. Results of this study shows that the extract at all doses increased the immune reaction to ovalbumin indicating that the extract could affect the cellular component of the local immune reaction including lymphocytes and monocytes recruited at the site of interaction (Ghazanfaret al., 2002). cxv Since cell mediated immune response is known to be critical to defense against infectious organism, infection of foreign graft, tumor immunity and immunity to many intracellular infections of microorganisms, especially those carrying chronic disease e.g Tuberculosis and DTHR (Elgert, 1972;Mitra et al., 1999; Dashputre and Naikwade, 2010; Wahi, 2010), the increase in DTHR indicates that short term administration of PT could augment the cell mediated immune responses. This implies enhanced ability to protect host against conditons requiring robust cellular immune protection, such as viral infections, tumors, and intracellular pathogens. Similarly, oral supplementation withBGP increased stimulatory activity on the ovalbumin- induced delayed type hypersensitivity reaction.Oral administration was possible in the study sinceβ-D-glucan is resistant to gastric acid and pass through the stomach virtually unchanged (Rahar et al., 2011) This result demonstrates and suggests that the P. tuberregium extract could owe its stimulatory activity on delayed type hypersensitivity reaction to its abundant beta glucan rich proteo-polysaccharide constituent. P. tuberregium extract showed an overall stimulatory effect on the reticuloendothelial system as indicated by the phagocytic index. Phagocytosis is a non-specific immune defense and is very important in the overall immune protection against a variety of infections (Janeway et al., 2005) as active phagocytosis is the major defense mechanism against infection. The clearance rate of granular foreign bodies from circulation reflects the phagocytic function of mononuclear macrophages. Increased carbon clearance is an indicator of enhanced competency of the reticuloendothelial system and mononuclear macrophages in removal of foreign particle, dead or injured cells, thereby an indicator of good immunological response against foreign particles or antigens (Ponkshe and Indap, 2002; Thakur et al., 2007; Roitt et al., 1993; Furthvan and Bergvanden 1991; Sangle et al., 2004).Pretreatment with P. tuberregium extract resulted in an increase in clearance rate of carbon from the system of the animals compared to the untreated mice group. P. tuberregium was found to stimulate the phagoytic activity of cxvi the macrophages as evidenced by an increase in the rate of carbon clearance. This activity implies that PT increased phagocytic responses of host against debris and foreign invaders. Therefore, this result indicates significant enhancement in the phagocytic function of macrophages hence response of host against debris and foreign invaders and thus that P. tuberregium may have properties of influencing the non specific immunity. The spleen is an important organ of the immune system, and so the level of immune competence of an organism is closely correlated with the developmental and functional integrity of this organ.Among the different organs of the immune system, the spleen represents a major secondary lymphoid organ involved in elicitation of immune response. The relative organ weight is fundamental to diagnose whether the organ was exposed to an injury or not (Vaghasiya et al., 2010). The relative weight of each organ of the immune system to the body weight of an individual is a commonly used index to reflect the developmental status of the organ (Dong et al., 2007). Also in the absence of any infection and obvious pathology, the relative weight of the spleen reflects overall immune function. Immunostimulatory agents are known to increase the relative weights of lymphoid organs such as the spleen (Zhang et al., 2011). In this study, administration of PT showed an increase in organ weight (spleen). Unlike the lymph nodes which are specialized to trap localized antigen from regional tissue spaces, the spleen serves as a reservoir for blood and filters, traps blood borne antigen and purifies the blood and lymphatic fluid that flows through it, and thus respond to systemic infections (Anamika et al., 2010). The increase in spleen weight may be due to the stimulatory effect of the plant extract on the lymphocytes and bone marrow haematopoeitic system. Oral supplemmentattion with aqueous extract of PT and BGP boosted the titres of IgG and its isoforms (IgG1 and IgG2a). This indicates the enhanced effect of PT and BGP on the antibody producing humoral immune system of Ova sensitized animals. The estimation of serum immunoglobulin level is a direct cxvii measure of humoral immunity and is an index of the functional status of developmental phases of the humoral immune response viz antigen recognition, activation, and expression (Benacerral, 1978). Basically humoral immunity is mediated by secreted antibodies and its physiological function is defense against foreign bodies (Surendra et al., 2010).The IgG antibody isotype provides the majority of antibodybased immunity against invading pathogens. It is remarkable that PTand BGP were able to boost the titers of IgG and its isoforms (IgG1 and IgG2a) thereby indicating that the antibody-producing humoral immune system is strengthened with their short-term supplementation. Spleenocyte proliferation is a crucial event in the activation cascade of both cellular and humoral immune responses (Borchers et al., 2008) MTT assay thus provides a means of accessing the effect of beta glucan rich proteo-polysaccharide on cellular immune response (Yang et al., 2011). Resultsfrom the present study indicates that the proliferation of mice spleenocytes in beta glucan rich proteo-polysaccharide treated cell lines is significantly increased in a dose dependent manner compared with the control and concavalin treated spleenocyte cell lines. Stimulation of spleen proliferation could be associated with elevated production of proinflammatory cytokines by lymphocytes (B cells) as well as increased production of T cells (Ho et al., 2004). This suggests that BGP may modulate Th1-mediated immune response (Andres et al., 2011). Also it suggests the possibility of the BGP extract being used as a possible mitogen in murine spleen lymphocytes since its effect on spleenocyte proliferation was comparably higher than the standard mitogen concavalin (Ho et al., 2004). Although the surface expression of molecular activation markers CD69 and CD25 on spleenocytes were not ascertained, the measurement of the expression of these molecules which are inducible cell surface glycoproteins acquired by immune cells during lymphoid activation and involved in lymphocyte proliferation in further research studies may shed more light on the ability of such treatment with beta glucan rich proteo-polysaccharide to produce activation of these important immune cells (Nworu et al., 2012). cxviii Result shows that the production of nitric oxide by monocytic/ macrophage cell line (RAW264.7) in beta glucan rich proteo-polysaccharide treated cell lines was significantly elevated in a dose dependent manner compared with the control mice group and lipopolysaccharide treated spleenocyte cell lines. Lipopolysaccharide (LPS) is a potent inducer of inflammation in cell lines and systemically in whole animal experiment (Tokunaka et al., 2000). Particularly, LPS can quantitatively promote the stimulation of macrophages to secrete pro-inflammatory cytokines and secondary mediatory, such as nitric oxide (NO) as well as synthesis of reactive oxygen intermediates (Kilbourn et al., 1984; Duval et al., 1996).NO is a gaseous molecule synthesized from L-arginine by nitric oxide systhase (NOS) (Xle et al., 1992; Korhonen et al., 2005). It is a highly reactive free radical and it can form a number of oxidation products such as NO2, NO2¯, N2O3 and S-nitrosothiols. NO participates in the physiology and pathophysiology of many systems (Diouf et al., 2009). It is an important mediator of the non-specific host defense against invading microbes and tumors. Thus NO can be used as a quantitative index of macrophage activation. It mediates diverse functions including vasodilation, neurotransmission and inflammation. The role of NO in host defense against microorganisms and tumor cells is well recognised. NO has been shown to be the principal effector molecule produced by macrophages for cytotoxic activity and thus a quantitative index of macrophage activation (Kilbourn et al., 1984; Ding et al., 1988).Although the mechanism was not evaluated, it is speculative that part of the immunomodulatory activity of BGP extract may be due to activation of macrophages through augmentation in NO production and other cytokines by induction of iNOS mRNA gene expression.This is in agreement withseveral investigations indicating the biological properties of the extracts/or compounds derived from other medicinal plants occurs through this mechanism (Stimpel et al., 1984; Kang et al., 2002; Park et al., 2004; Kim et al., 2007).In addition, the increased carbon clearance index invivo which reflects the enhancement of phagocytic function of mononuclear macrophages and non specific immunity probably act through the release of NO as previously described in the invivo carbon clearance model . cxix Inflammation is a complex biological response of the body to harmful stimuli including microbial infections and chemical toxins. The inflammatory response is characterized by several steps including coordinate activation of signaling pathways, expression of proinflammatory cytokines, chemokines and adhesion molecules in resident tissue cells, as well as infiltration of leukocytes mainly macrophages, neutrophils and dendritic cells and mediators of inflammation from the vascular system to remove the harmful stimuli and to initiate the healing process (Sacca et al., 1997; Dinarello, 2010).Leukocytes or endothelial cells produce proinflammatory or anti-inflammatory cytokines depending on their regulatory activities on regulating inflammation. Proinflammatory cytokines includetumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Tumor necrosis factor-α (TNF-α) have been shown to play central roles in the initiation and amplification of inflammatory response (Hehlgans and Pfeffer, 2005; Park and Bowers, 2010). The effect of treatment with BGP on the expression of key inflammatory cytokine tumor necrosis factor-α (TNF-α) was evaluated and found to increase its production.TNF- α has being implicated in inflammatory processes. Inflammatory response is characterized by increased blood supply carrying more leukocytes (mainly activated monocytes/macrophages) and plasma molecules, increased permeability of capillaries allowing exudation of plasma proteins and increased migration of leukocytes to the affected tissue (Male et al., 2007). These activated macrophages produce TNF- α, IL-1, IL-6 and IL-12 (Evans, 1996; Bone et al., 1997). In order to initialize the inflammatory response, TNF-α and IL-1 induce vasodilation, increase vascular permeability and increase blood flow to the affected site thereby resulting in edema, redness and heat (Thao et al., 2008, Nworu et al., 2010; Nworu et al., 2011) leading to subsequent elimination of the antigen. Helper T cells are designated as Th1 and Th2 subpopulations based on the patterns of their cytokine secretion. Th1 cells, characterized by IL-2, IFNγ, and TNFα production, are mainly involved in cell-mediated immunity to intracellular infection (Borchers et al., 2004). Enhanced Th1 response is beneficial for antimicrobial and antitumor defense, increased innate immunity to tumors and cxx viral infections and it is likely responsible for the immunomodulatory activity of the extract as mediated by TNFα though this was not ascertained specifically. BGP significantly increased the proliferation of monocytic/macrophage cell line (RAW264.7) in a dose dependent manner. The result is comparable with the lipopolisaccharide proliferation of monocytic/macrophage cell line (RAW264.7). Macrophages are the first line of defense in the interaction between innate and specific immunity against infection (Yoon et al.,2003;Allan and Anders, 2008; Zhao et al., 2010). They therefore play an important role in host defense against infections (Yu et al., 2008). The extent of phagocytosis is a major indicator of macrophage effector activity as well as an important indispensable step in the immunological defense system (Campelo et al., 2002). Increased proliferation of monocytic/macrophage cell line (RAW264.7) further indicates the activity of BGP on cell mediated immunity. β-D-Glucans are recognized by “pattern recognition receptors” (PRR) in innate immune cells, including dectin-1, complement receptor-3 (CR3 or CD11b/CD18) and TLR2/6, thereby activating and triggering responses in several immune cells including monocytes, macrophages, dendritic cells, neutrophils, and NK cells (Brown and Gordon, 2001). It is therefore plausible to suggest that upon stimulation by β-DGlucans content of the mushroom extracts through the PRR, macrophages exhibit increased expression of iNO, TNFα, and phagocytic capability which are signs of improved functional maturation (Brown et al., 2002). These in vitro results with BGP strongly agrees with the in vivo models using the crude extract of Pleurotus tuberregium (PT). This is also indicative of the better and enhanced immunomodulatory potentials of the BGP. These benefits might be replicated in humans taking sufficient P. tuberregium for an appropriate period of time. However, the clinical immunomodulatory potential of PT and BGPrequires further study. 4.2 CONCLUSION cxxi AlthoughP. tuberregium is a popular folk medicine and has attracted great attention due to its role in the immune system activity against infections, there is little information available about this action. This report provides both in vitro and in vivo evidences of the therapeutic potentials of this medicinal mushroom plant. This study has shown that the PTpossesses immunomodulatory properties. Pleurotus tuberregium extract showed specific and non-specific immunomodulatory activity on both innate and acquired immune mechanisms. The study also revealed that BGP, a glycopolysaccharide was responsible for the immunodulatory activities of the mushroom P. tuberregium. BGP was shown to possess stimulatory effects on the innate and adaptive components of the immune system depending on the type of immune cells involved. These properties of P. tuberregium are clinically important and could be employed to regulate normal immunologic functioning. Thus cellular and humoral immunomodulatory mechanisms of action for the mushroom, P. tuberregium, justifies its inclusion in dietary supplement and local use in food. 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Ganoderma cxlvi cxlvii APPENDICES (Tabular summary of some of the key results presented in charts in chapter 3) cxlviii Appendix 1: The Effect of Hot Aqueous Extract of Pleurotus tuber-regium (PT) on Phagocytic Activity in Swiss Albino Mice. Phagocytic Index (Mean titre±SEM) Phagocytic Index Phagocytic Index (% increase Normalized to Treatment Dose (mg/kg) in Parenthesis) Control PT 100 0.011±0.005(647.9) 7.479 200 0.016± 0.010(907.5) 400 0.012± 0.002(670.0) 7.701 Noni® 100 0.021±0.008(1296.0) 13.968 Negative Control ___ 0.002±0.001 ___ 10.075 Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance cxlix Appendix 2: The Effect of Hot Aqueous Extract of P. tuber-regium on the Relative Spleen Weight of Mice. Relative Spleen Weight (mean titre ± SEM) in grams Relative Spleen Weight (% Treatment Dose increase in parenthesis) PT 100 0.494±0.012(6.87) 200 0.611± 0.026*(32.2) 400 0.568± 0.036(22.76) Noni® 100 0.503±0.013(8.79) Negative Control ___ 0.463±0.047 Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group cl *= level of significance cli Appendix 3: The Effect of Hot Aqueous Extract of P. tuber-regium on Delayed Type Hypersensitivity Response in Mice Delayed Type Hypersensitivity (mean titre ± SEM) in mm Treatment Dose(mg/kg) DTH (mm) % stimulation of DTHR PT 100 0.288± 0.049 1.41 200 0.420±0.032 47.88 400 0.402±0.101 40.84 Noni® 100 0.474 ±0.022 65.49 Negative Control ___ 0.284 ± 0.102 ___ Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance clii Appendix 4: The Effect of BGPon Delayed Type Hypersensitivity Response in Mice Delayed Type Hypersensitivity (mean titre ± SEM) in mm Treatment Dose(mg/kg) DTH (mm) % stimulation of DTHR BGP 100 0.618±0.084 36.72 200 0.658±0.052* 45.57 Noni® 100 0.500±0.132 10.61 Negative Control ___ 0.452±0.154 Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance ___ cliii Appendix 5: The Effect of Hot AqueousExtract of Pleurotus tuber-regium(PT) on Primary and SecondaryIgG Humoral Antibody Response to Ovalbumin Mice Humoral antibody response (mean titre±SEM) Dose(mg Secondary response (7- Secondary Response Treatment /kg) Primary Response days Poost boost) (14-days Poost boost) PT 100 0.399±0.049(44.7) 1.105±0.139(41.9) 1.163±0.100(31.2) 200 1.052±0.196***(281.6) 1.437±0.106***(84.6) 1.301±0.106*(46.7) 400 0.760±0.037**(175.8) 1.282±0.115**(64.6) 1.200±0.139(35.4) 100 0.686±0.137*(148.7) 1.266±0.058**(62.6) 1.305±0.149*(8.6) Noni® cliv Negative Control ___ 0.275±0.030 0.778±0.073 0.886±0.090 Naïve ___ 0.154±0.003 (-43.9) 0.154±0.004***(-80.1) 0.154±0.003***(-82.5) Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance Appendix 6: The Effects of Hot Aqueous Extract of Pleurotus tuber-regium(PT) on Primary and Secondary IgG1 Humoral Antibody Response to Ovalbumin in Mice Humoral antibody response (mean titre±SEM) Dose(m Secondary response (7- Secondary Response Treatment g/kg) Primary Response days Poost boost) (14-days Poost boost) PT 100 1.446±0.176*(77.2) 2.756±0.146***(50.8) 2.557±0.162(16.3) 200 1.922±0.126***(135.5) 2.987±0.005***(63.4) 2.810±0.079**(27.7) clv 400 1.597±0.214*(95.7) 2.753±0.222***(50.7) 2.795±0.234**(27.2) 100 1.903±0.256***(133.2) 3.035±0.099***(66.1) 2.878±0.068**(30.8) Control ___ 0.816±0.091 1.827±0.202 2.199±0.022 Naïve ___ 0.239±0.005(-70.7) 0.239±0.006***(-86.8) 0.239±0.006***(89.) Noni® Negative Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance Appendix 7: The Effects of Hot Aqueous Extract of Pleurotus tuber-regium(PT) on Primary and Secondary IgG2a Humoral Antibody Response to Ovalbumin in Mice Humoral antibody response (mean titre±SEM) clvi Dose(m Secondary response (7- Secondary Response Treatment g/kg) Primary Response days Poost boost) (14-days Poost boost) PT 100 0.780±0.093(60.5) 1.130±0.198(82.1) 1.036±0.099(56.8) 200 1.483±0.153***(205.1) 1.753±0.159***(182.5) 1.611±0.115**(143.8) 400 0.937±0.039*(92.8) 1.174±0.198(89.2) 1.493±0.218**(126.0) 100 0.840±0.120(72.8) 1.358±0.159**(118.9) 1.172±0.131*(77.3) Control ___ 0.486±0.154 0.620±0.102 0.661±0.039 Naïve ___ 0.232±0.021(52.3) 0.232±0.021(62.6) 0.232±0.021(64.9) Noni® Negative Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance clvii Appendix 8: The Effects of ΒGP on Primary and Secondary Total IgG Humoral Antibody Response to ovalbumin in Mice Humoral antibody response (mean titre±SEM) Treatments Dose (mg/kg) Primary response Secondary Response BGP 100 0.268±0.019*(40.88) 0.623±0.046(9.12) 200 0.371±0.022***(93.98) 1.082±0.099***(89.49) Noni® 100 0.281±0.014**(47.55) 0.723±0.074(26.61) Negative Control ___ 0.190±0.013 0.571±0.041 Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance clviii Appendix 9: The Effects of BGP on Primary and Secondary IgG1 Humoral Antibody Response to Ovalbumin in Mice Humoral antibody response (mean titre±SEM) Treatments Dose (mg/kg) Primary response Secondary Response BGP 100 0.576±0.054(58.6) 2.350±0.195(27.6) 200 0.816±0.069**(124.6) 2.965±0.286*(61.0) Noni® 100 1.158±0.148***(218.6) 3.004±0.266**(63.2) Negative Control ___ 0.363±0.037 1.841±0.251 Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance clix Appendix 10: The Effects of BGP on Primary and Secondary IgG2a Humoral Antibody Response to Ovalbumin in Mice Humoral antibody response (mean titre±SEM) Treatments Dose (mg/kg) Primary response Secondary Response BGP 100 0.666±0.028***(59.0) 1.403±0.151*(75.6) 200 0.860±0.055***(105.5) 2.534±0.162***(217.1) Noni® 100 0.635±0.041**(51.6) 1.284±0.088**(60.7) Negative Control ___ 0.419±0.021 0.799±0.078 Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance clx Appendix 11: The Effect of BGP on Spleenocytes Proliferation Spleenocyte proliferation (titre±SEM) Spleenocyte Treatment Dose(µg/ml) Proliferation index % Proliferation BGP 5 1.444±0.020*** 44.4 25 1.573±0.012*** 57.3 50 1.902±0.051*** 90.2 100 2.251 ±0.146*** 25.1 250 1.531±0.070*** 53.1 1000 1.284±0.105* 28.4 2 1.861±0.033*** 86.1 Con A clxi Control ___ 1.000 ± 0.028 ___ Stimulation, n=8 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance Appendix 12: The Effect of BGP on TNF-α Production by RAW264.7 TNF-α production(titre±SEM) % increase in TNF-α Treatment Dose (µg/ml) TNF-α production Production BGP 5 1.180±0.029*** 544.8 25 1.580±0.017*** 763.3 50 2.064± 0.088*** 1027.8 clxii 100 1.987±0.022*** 985.7 LPS 1 1.822±0.030*** 895.6 Control __ 0.183±0.010 ___ Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance clxiii Appendix 13: The Effect of BGP on Nitric Oxide Production by RAW264.7 Nitric Oxide production(titre±SEM) % Increase in Nitric Treatment Dose (µg/ml) Nitric Oxide production Oxide Release BGP 5 6.933±0.088*** 1055.5 25 9.767±0.218*** 1527.8 50 16.200± 0.230*** 2600.0 100 19.670±0.433*** 3178.3 LPS 1 17.770±0.751*** 2861.6 Control __ 0.600±0.057 ___ Stimulation, n=6 ; *P<0.05, **P<0.001, ***P<0.0001 Where n= number of animals per group *= level of significance clxiv Appendix 14: The Effect ofBGP onRate of Phagocytosis RAW264.7 Rate of Phagocytosis (titre±SEM) Treatment Dose (µg/ml) Rate of Phagocytosis % Rate of Phagocytosis BGP 5 1.119±0.011*** 11.9 25 1.165±0.016*** 16.5 50 1.181± 0.001*** 18.1 100 1.083±0.010*** 8.3 LPS 1 1.149±0.011*** 14.9 Control __ 1.000±0.013 ___ Stimulation, n=6 ; *p<0.05, **p<0.001, ***p<0.0001 Where n= number of animals per group *= level of significance clxv
