QR185.8 C95 H66 blthr C.l G363351 The B a lm e L ib ra ry ■■IIIIIlSlllI 3 0692 1078 5905 8 University of Ghana http://ugspace.ug.edu.gh SEVERE MALARIA-THE ROLE OF REGULATORY CYTOKINES A Thesis presented to The Board of Graduate Studies University of Ghana, Legon Ghana In partial fulfilment of The Requirements for the Degree of Master of Philosophy(M. Phil.) (Biochemistry) By PRINCE KWEKU HODO BSc. (Hons) Department of Biochemistry, University of Ghana, Legon, Accra. Ghana. SEPTEMBER, 1999 University of Ghana http://ugspace.ug.edu.gh DECLARATION I do hereby declare that except for other people’s investigations cited which have been acknowledged, this exercise is a result of my own original research, and that this thesis, either in whole or in part, has not been presented elsewhere for another degree. PRINCE KWEKU HODO (Student) )R. B. D. AKANMORI (Supervisor) (Co-supervisor) ' (Head, Department of Biochemistry) DR. J. KURTZHALS (Co-supervisor) University of Ghana http://ugspace.ug.edu.gh DEDICATION To the memory of the fallen heroes/heroine of MALARIA across the world, who could not get the chance to make an impact. Ironically, man who was made in the image o f God, harbors in his v ita l o rgans various fo rm s o f loa thsom e creatures, which riot in his flu ids and consum e the very substance o f his tissues. PHILIP HENRY GOSSE University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENT DECLARATION............................................................................... " DEDICATION.................................................................................. iii TABLE OF CONTENT................................................................... iv LIST OF FIGURES..,....................................................................... vii LIST OF ABBREVIATIONS........................................................... viii LIST OF APPENDICES.................................................................. ix ACKNOWLEDGEMENT................................................................. x ABSTRACT....................................................................................... xi CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1 1.1. In troduction ........................................................... 1 1.2. Literature review .................................................. 5 1.2.1 Malaria as a disease................................... 5 1.2.2. Malaria parasite........................................... 6 1.2.2.1. Human malaria parasite................ 7 1.2.2.2. Rodent malaria parasite................ 7 1.2.3. Vector.............................................................. 10 1.2.4. Mice.................................................................. 11 1.2.5. Immunity against malaria............................. 12 1.2.5.1. Cellular immunity............................. 12 1.2.5.2. Humoral immunity........................... 13 1.2.6. Immunopathology of severe malarial anaemia 15 1.2.7. Cytokines as regulatory factors................... 17 1.2.7.1. Interleukin 10.................................... 18 1.2.7.2. Interleukin 6...................................... 19 ,1.2.7.3. Tumour necrosis factor.................. 21 1.2.7.4. Interferon gamma............................ 22 CHAPTER TWO: MATERIALS AND METHOD............................ 25 iv University of Ghana http://ugspace.ug.edu.gh 2.1. Materials.................................................................. 25 2.1.1 Chemicals........................................................... 25 2.1.2 Equipment.......................................................... 26 2.2 Mice................................................................................. 28 2.3 Malaria parasites.......................................................... 28 2.4 Propagation of P. berghei berghei in BALB/C mice.............................................................. 28 2.5 Treatment of BALB/C mice with rmil-10.................. 29 2.6 Haematology................................................................... 29 2.6.1 Estimation of haematocrit in P. berghei ANKA parasitised mice.............................................. 29 2.6.1 Estimation of haemoglobin level.................... 30 2.6.2 Estimation of reticulocytosis............................ 30 2.7 Determination of parasitaemia.................................... 31 2.8 Antibody measurement.............................................. 31 2.8.1 Preparation of antigen slides.......................... 31 2.8.2 Antigen well preparation.................................. 32 2.8.3 Immunoflourescent antibody test for titration of anti-plasmodial IgG-subtypes and IgM 32 / 2.9 Spleen cell cultures...................................................... 33 2.9.1 Preparation of mononuclear cells from the spleen 33 2.9.2 Cell cultures....................................................... 34 2.10 Cytokine measurements in the serum and spleen cell culture supernatants............................ 35 CHAPTER THREE: RESULTS........................................................... 37 3.1. Haematological measurements................................. 37 3.1.1. Parasitaemia (the course of infection) 37 3.1.2. Anaemia.............................................................. 40 3.1.2.1. Haemoglobin levels..................... 40 3.1.2.2. Reticulocytosis............................. 40 University of Ghana http://ugspace.ug.edu.gh 3.1.2.3. Packed cell volume..................... 43 3.2. Mortality..................................................................... 43 3.3. Titres of anti-plasmodial IgG subtypes and IgM................................................... 46 3.4 Spleen cell proliferation.......................................... 49 3.5. Cytokines measurement......................... 51 3.5.1. Interleukin 10................................................. 51 3.5.2. Tumour necrosis factor-a............................. 52 3.5.3. Interferon gamma.......................................... 55 CHAPTER FOUR: DISCUSSION AND CONCLUSION................ 61 4.1. Discussion................................................................... 61 4.2. Conclusion................................................................. 67 REFERENCES................................................................................... 69 APPENDICES.................................................................................... 80 University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 1: Graph to justify that the mice are comparable in a group 38 Figure 2: Graph of parasitaemia for rmlL-10 treated and control BALB/C mice.................................................................. 39 Figure 3: Graph of Haemoglobin level for rmll_-10 treated and control mice.......................................................................... 41 Figure 4: Graph of % Reticulocytes in peripheral blood of rmll_-10 treated and control untreated BALB/C mice 42 Figure 5: Graph of % PCV for rmlL-10 treated and control BALB/C mice.................................................................. 44 Figure 6: Kaplan-Meyer survival graph for rmlL-10 treated and control mice.............................................. 45 Figure 7: IFAT graphs for rmlL-10 treated (Ts) and control (Cs) mice showing the various IgG’S and IgM......................... 47 Figure 8: Graph of mean spleen lymphocytes proliferation of IL-10 treated (Ts) and control (Cs)......................................... 50 Figure 9: IL-10 levels in sera of rmlL-10 treated and controls BALB/C mice.................................................................... 53 Figure 10: Graph of IL-10 levels in spleen cell culture supernates from rmlL-10 treated (Ts) and controls (Cs).............................. 54 Figure 11: TNF-a levels in sera of rmlL-10 treated and controls BALB/C mice................................................................... 57 Figure 12: TNF-a levels in spleen cell culture supernates from rmlL-10 treated (Ts) and controls (Cs)...................................... 58 Figure 13: IFN-y levels in sera of rmlL-10 treated and controls BALB/C mice.................................................................... 59 Figure 14: IFN-y levels in spleen cell culture supernates from rmlL-10 treated (Ts) and controls (Cs)......................................... 60 vii University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS CD Cluster of differentiation MHC Major Histocompatibility IFN-y Interferon gamma TNF-a Tumour Necrosis Factor IL Interleukin IFAT Immunoflourescence Antibody Test Ig Immunoglobulin PHA Phytohaemagglutinin ELISA Enzyme Linked Immunosorbent Assay PCV Packed Cell Volume cpm counts per minute BFU-E Burst-forming units of erythroid cells CFU-E Colony- forming unit of erythroid cells EPO Erythropoietin PBS Phosphate buffered saline 3H Tritium NMIMR Noguchi Memorial Institute for Medical Research Th T-helper cells LPS Lipopolysaccharide cAMP Cyclic Adenosine monophosphate CTLS Cytolitic T- lymphocytes PHA Phytohaemoglobin University of Ghana http://ugspace.ug.edu.gh LIST OF APPENDICES Table 1: Average haematological data for the IL-10 injected mice and controls............................................... 80 Table 2: Data for percentage survival for IL-10 treated and control mice................................................................ 80 Table 3: Mean splenic lymphocyte proliferation/ number of cells With (3-uptake counted per min....................................... 81 Table 4: Mean IFAT Values for IL-10 treated and control mice.. 82 Table 5: Cytokine levels in both sera and cell culture supernatant samples........................................................ 83 Table 6: Standard curve values...................................................... 84 Appendix 5: Life Cycle of Plasmodium species............................ 85 Appendix 6: Developmental Cycle of P. berghei in Anopheles stephensi............................................................................. 86 Appendix 7: Blood Smear of P. berghei....................................... 87 ix University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT To give full and fair recognition to all of the people and institutions who assisted in this thesis is a practical impossibility, but an attempt will be made to do so. My special thanks go to the Noguchi Memorial Institute for Medical Research (NMIMR) for permitting me to undertake the project in the Institute, more especially to the immediate past director of the institute Prof. F.K Nkrumah and the current director of the institute Prof. D. A Ofori-Adjei. I particularly wish to express my appreciation to the World Bank Research Fund for Universities for their financial support for the research. My sincere gratitude goes to my supervisors; Dr. Jorgen Kurtzhals of Copenhagen University, Denmark, Dr. B.D Akanmori, Head of immunology unit of NMIMR and Prof. F.N Gyang, Head of Biochemistry Department, University of Ghana, for their warm support, constant encouragement, textual criticism and intellectual guidance. / I would also like to acknowledge with gratitude the technical support offered by Dr. G. Armah, Head of electron microscopy, Abdulrahman Sarban Hammond of the Immunology unit and Mr. Quartey of Animal unit (NMIMR). This also goes to other members of the following units; Animal, Haematology and Immunology, all of NMIMR. Again, I am grateful to the entire Biochemistry department for their support. The debt I owe to my colleagues including those in the NMIMR; Michael Ofori, Ben Gyan, William Vanderpuije, John Arko-Mensah, Mrs. Judith Antwi, Miss Ruth Kpedor, Francis Owusu, Ben Quarshie and Mrs. Anita Ghansah is not just an intellectual debt. My profound appreciation to those I am indebted in a variety of ways including Lion Alhaji Fattau El-Aziz, Lion E. K. Adjei and Mr.Julius Krampah, Lambert Faabeloun, Mrs. Shirley Otenkorang, Moses Duphey and those who cannot be cited here. I am grateful to my family through whose support I have cleared yet another hurdle on the academic ladder. The most of all to J.C for he is and to him be the glory. University of Ghana http://ugspace.ug.edu.gh ABSTRACT An anti-inflammatory cytokine, interleukin 10 (IL-10) suppresses T-helper 1 cells (Th1) route and enhances the Th2 route in mice by boosting the antibody response. The present work studied the effect of exogenous IL-10 in the system of BALB/C mice during the development severe anaemia. BALB/C mice developed severe anaemia within 2-3 weeks after inoculation with 106 parasitised RBCs (pRBCs) intraperitoneally. BALB/C mice treated with recombinant IL-10 developed patent parasitaemia of 7% on day 7-post inoculation as compared to 10.5% in controls. Nevertheless, there was no significant difference between the two groups haematologically (parasitaemia P>0.01, haemoglobin levels P>0.01, reticulocytosis P>0.01 and in hematocrit values P> 0.01). There was generally a positive correlation between anaemia and the parasite count and reticulocytosis, but negative correlation with haemoglobin levels and the hematocrit levels measured. Cytokine levels in vivo and in vitro were determined using the ELISA technique. The endogenous IL-10 measured was higher in the IL-10 treated than the controls. However there was no difference statistically between the mortality of the two groups, though it appeared to be slower in the IL-10 treated BALB/C mice. Also, there was no significant difference between the TNF-a measured in both sera and the spleen cell culture supernatants of the two groups. IgG and IgM were measured by means of Immunoflourescence Antibody Test (IFAT) and using whole / blood stages of the parasites as antigens. The role of immunoglobulin in erythrophagocytosis was observed because there was enhanced University of Ghana http://ugspace.ug.edu.gh erythrophagocytosis between the 7th and 12th day, that was when lgG2a recorded the highest peak value in both test and control mice. On the other hand, lgG3 recorded low levels with a significant increase in TNF-a during the same period. The use of mitogen in vitro showed that activation of T cells results in the production of cytokines. University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 1.0 INTRODUCTION AND LITERATURE REVIEW 1.1 INTRODUCTION Malaria continues to pose the greatest single infectious disease threat to the health of the world. The tropical areas, mostly developing countries, are the most affected. Within the range of this tropical zone, malaria prevalence varies with different natural and socio-economic conditions. Malaria is characterized by fever and it is caused by sporozoa of the genus Plasmodium (Marsh et al., 1991). Infection is initiated by the bite of an infected female mosquito that injects sporozoites into the blood stream of the vertebrate host. Eventually, there is synchrony of schizont formation and red blood cell (RBC) rupture, and this is responsible for the regular repeated bouts of fever, which are almost diagnostic of the infection (Wakelin, 1984). In Africa, the most common complications of severe malaria are anaemia and cerebral malaria (Marsh et al, 1991). Malarial anaemia remains one of the major causes of morbidity and mortality in patients with acute Plasmodium falciparum infection, especially in children and pregnant women residing in malaria endemic areas (Bruce-Chwatt, 1980; Weatherall, 1988; Mota et al., 1998). Anaemia in Plasmodium falciparum malaria occurs when RBCs are destroyed more rapidly than they are replaced by the erythropoietic process (Miller et al., 1989a). The complete cyclical development of malaria parasites I University of Ghana http://ugspace.ug.edu.gh can be sustained indefinitely by uninterrupted blood or sporozoite (mosquito) induced transmission in laboratory rodents. Rodent malarias are important tools continually used for all kinds of research with appreciable success. Plasmodium berghei, induces an anaemia in its rodent hosts just as P. falciparum does, which is in excess of that which can be accounted for solely by direct destruction of erythrocytes by the mature schizonts at the time of merozoite release (Roth & Herman, 1979). In earlier studies, a number of mechanisms have been postulated as possibly contributing to such excessive anaemia in a variety of malarial infections. These include a possible concomitant infection (Me Ghee and Loftis, 1968; Ludford et a!., 1969), alteration of the erythropoietin mechanisms (McGhee, 1970; Frankenburg and Greenblatt, 1977), erythrocyte membrane alterations and/or hemolysis by the action of parasite products (Cox, 1966; Fife et al., 1972; Holz et al., 1977; Klein et al., 1977), autoimmunity (Zukermann, 1964, 1966) and reticuloendothelial hyperphagocytosis directed against host erythrocytes (Todorovic et al., 1967; Sheagren et al., 1970; Zukermann et al., 1973). Nevertheless, recent studies have focused on ineffective erythropoiesis and immune mediated haemolysis as major underlying factor for these anaemias (Woodruff et al., 1979; Clark and Chaudhri, 1988; Villeval et al., 1990). Research has so far not identified the detailed pathogenesis of severe malarial anaemia. Whilst it is possible to conduct most malaria research using human 2 University of Ghana http://ugspace.ug.edu.gh subjects, certain studies especially immunological interventions cannot be carried out using human subjects for ethical and other considerations. Experimentation with other alternative models of malaria involving non-human hosts has thus become essential for finding answers to the many questions in malaria research. Furthermore, correlation of housekeeping genes, similarities of biochemical (Homewood, 1978; Homewood & Neame, 1980) and genetic processes (Walliker, 1989) between rodent and human malaria parasites have justified the use of rodent parasites for malaria studies. The ease and safety of handling and manipulation of the different stages of the parasites in mice also lends itself to in vivo experimentation. A useful model for severe anaemia similar to that in humans is the infection of BALB/c mice with P. berghei. The hemopoietic and the immune systems are now known to be interrelated via a network of interleukin growth factors and cytokines regulating the proliferation and differentiation of cells in the network. These cytokines are regulatory peptides produced by the cells of the immune system (and other tissues). They play a vital role in the interaction between parasites and the immune systems (Miller et al., 1989b; Oppenheim 1994; Paul and Seder, 1994). There are five components involved in cytokine activity: the producer cell, the cytokine itself, a receptor on the target cell, the target cell and the response of the target cell (Oppenheim, 1994). Tumor Necrosis factor (TNF) is a cytokine released as a result of systemic activation of macrophages by malarial parasites and together with other cytokines have been implicated in dyserythropoiesis and increased phagocytosis in experimental malaria (Clark et al 1987a; Clark and Chandri, 3 University of Ghana http://ugspace.ug.edu.gh 1988; Miller et al 1989a). It has recently been shown that certain cytokines when inoculated into mice, modulate the immune response by increasing mainly the antibody-mediated Th2 response and accelerating the elimination of malaria parasites from mice (Akanmori et al., 1996) Severe malarial anaemia is associated with high levels of TNF leading to dyserythropoiesis (Miller et al., 1989a). The effects of TNF are counteracted by the anti-inflammatory cytokine IL-10. Recent studies have shown that low plasma IL-10 is associated with severe malarial anaemia in children in Ghana and Kenya (Kurtzhals et al., 1998; Othoro et al., 1999). In order to test the hypothesis that IL-10 can reverse or prevent TNF-induced dyserythropoiesis and anaemia, we examined the effects of injections of recombinant mouse (rm) IL-10 into mice experimentally infected with P. berghei in comparison with untreated mice. The major objective of the study is to provide more information on the pathogenesis of severe malarial anaemia. The specific objectives of the study were: • To determine the haematological, biochemical and immunological changes associated with P. berghei infection in BALB/c mice. • To determine the effects of injections of rmlL-10 on survival, course of parasitaemia, haematological and immunological changes in BALB/c mice infected with P. berghei. 4 University of Ghana http://ugspace.ug.edu.gh 1.2 LITERATURE REVIEW 1.2.1 MALARIA AS A DISEASE Malaria is a disease caused by protozoan parasites of the genus Plasmodium. It affects vertebrates in which the asexual form of the parasite is transmitted by an invertebrate vector (the mosquito) in which the sexual reproductive phase of the cycle takes place (Perrin et al., 1982; Wakelin, 1984) More than 100 species of Plasmodia are known, most of which have extremely restricted host ranges. Natural infection is initiated by the bite of an infected female mosquito that injects sporozoites carried in the insect’s saliva. The sporozoite enters hepatocytes/ parenchymal cells of the liver shortly after injection. Within the liver cells the sporozoite then divides asexually to form a large pre-erythrocytic schizont. The rupture of the affected parenchymal cell releases thousands of exoerythrocytic merozoites into circulation. After a period of growth, during which the parasite passes through the ring and trophozoite stages there is a schizogony and production of the erythrocytic schizont, with division of cytoplasm and nucleus to form a relatively smaller number (32 or less) of merozoites. The infected cell then bursts, releasing merozoites, and repeated cycles of schizogony and RBC infection ensues, so that a high proportion of available RBC may become infected (illustration of the life cycle is in Appendices 5 & 6). It has been accepted that the rupture of the erythrocytic schizont is associated with the periodic fever of the disease (Perrin eta!., 1982; Wakelin, 1984; Bruce-Chwatt, 1980). 5 University of Ghana http://ugspace.ug.edu.gh The patterns of pathology differ with changes in the degree of endemicity (Miller et al., 1994). In regions of high endemicity, the greatest suffering is borne by children less than 5 years of age, whereas in areas of low endemicity, the disease affects all age groups (Miller et. al., 1994). The interaction of the malaria parasite and the host system has evolved into various forms of the disease, as stated by Khurtzhals and colleagues (Khurtzhals et al. 1998). In all cases, complicated and uncomplicated malaria are recognized. Complicated malaria is further categorized into cerebral and the severe malarial anaemia. Cerebral malaria usually affects non-immune patients and African children i (Miller et al., 1994). They further stated that human P falciparum infection is common in areas where severe anaemia is a major problem. 1.2.2. MALARIA PARASITES The microorganisms causing malaria are commonly referred to as malarial parasites. Malaria parasites belong to the family Plasmodiidae within the order Coccidiidae, sub-order Haemosporidiidae, which comprises various parasites found in the blood of reptiles, birds and mammals. The zoological family of Plasmodiidae includes the parasites, which undergo two types of multiplication by asexual division (schizogony) in the vertebrate host and a single sexual multiplication (sporogony) in the mosquito host (Bruce-Chwatt, 1980). 6 University of Ghana http://ugspace.ug.edu.gh 1.2.2.1 HUMAN MALARIA PARASITES There are four recognized species of malaria parasites of man in general: P. malariae, P vivax, P. falciparum and P. ovale. Of these human malaria parasites only P. falciparum causes significant mortality. This mortality affects children in Africa, especially those under the age of 5 and pregnant women in the high endemic areas (Weatherall, 1988). However in the low endemic areas it affects all ages (Miller et al., 1994). P. falciparum occurs in the tropics and sub-tropics. P vivax is the most widely distributed parasite occurring in most temperate areas as well as parts of the tropics. P vivax uniquely is associated with clinical relapses due to reactivation of hypnozoites in the liver. Differently, is P. malariae, one of the human forms, which affects non-human primates t especially chimpanzees (Killick-Kendrick, 1978; Bruce-Chwatt, 1980). P. ovale however occurs largely in the western parts of Africa and produces a milder form of the disease. 1.2.2.2 RODENT MALARIA PARASITES There are a number of malaria parasites, which infect wild rodents and are transmitted by mosquitoes. Among these is Plasmodium berghei. P berghei was first isolated In the wild tree rats by Vincke and Lips (Vincke and Lips, 1948; Yoeli,1965) in the forest galleries of Democratic Republic of Congo (former Zaire) and has since been used for various research works. This was possible because, various animal species (37 and more) have been found susceptible to P berghei (Yoeli, 1965). A number of clinical patterns were 7 University of Ghana http://ugspace.ug.edu.gh observed during the course by infection of P. berghei in different hosts. These are grouped as follows: 1. A fulminating and fatal course terminating in death within 7 to 14 days accompanied by high and rising parasitaemia. Example in white mouse and the baby albino rat 2. A more prolonged clinical course, terminating in the death of the host within 2-3 weeks and accompanied by a rising parasitaemia e.g. golden hamster. 3. Chronic and latent course with spontaneous recovery. Rarely, death from the infection and fluctuation in parasitaemia were noticed e.g. adult albino rat. 4. Mild and transient P. berghei infection with low parasitaemia and complete recovery. 5. Short-lived survival of inoculated parasites in an alien host, without power of multiplication. Detection of “static” infection was detected when blood was sub- / inoculated into susceptible hosts. It is also reported that P berghei. Anka specifically produces neurological syndrome 7-14 days post inoculation in CBA mice whilst it takes 2-3 weeks to produce severe anaemia in BALB/c mice (Schneider, 1968 and Lucas et al., 1997) The complete cyclical development of malaria parasites can be sustained indefinitely by uninterrupted blood or sporozoic (mosquito) induced transmission / in laboratory reared rodents. This advance was realized by Vincke and Lips in 1948 (Vincke and Lips, 1948; Schneider, 1968). 8 University of Ghana http://ugspace.ug.edu.gh Later, the classical histochemical research of Yoeli (1965) and other investigators (Laudan and Killick-Kendrick, 1966) led to full visibility of the exoerythrocytic growth stages in the liver of rodents and the sporogonic growth stages in mosquito vectors (Schneider, 1968). Differences among inbred strains of mice in response to infection with various rodent malaria species have been observed. Greenberg and colleague showed that the morbidity and mortality vary significantly with various inbred strains of mice after infection with Plasmodium berghei (Greenberg and Coatney, 1954). Nevertheless, the ultimate of such infection with this species of rodent malarial parasite is lethal in all mice (Stevenson et al, 1982). Carter et al (1973) studied the enzyme variation in rodent malaria parasites and put P. berghei in two groups, among the strains of malaria parasites studied; one representing P. berghei berghei (P berghei. Anka is found in this group) and P. b. yoelii, P. b. killicki and the Nigeria P. berghei in the other group. Carter and colleagues used the term isolate to refer to parasites derived from a single wild host species on a unique occasion whilst strain meant parasites of a single species derived from an isolate. Rodent malaria are vital tools that are increasingly used for investigating immunity, physiology, metabolism chemotherapy, long acting antimalarial drugs, ultrastructural morphological characteristics and drug resistance strains (Zuckerman and Yoeli, 1954; Fulton and Spooner, 1956; Bowman et al., 1960; Hawking and Gammage, 1962; Jacobs et al., 1963; Thompson et al., 1963; 9 University of Ghana http://ugspace.ug.edu.gh Zuckerman, 1964; Nagarajan, 1964; Peters, 1965; Jacobs, 1965; Landau and Killick-Kendrick, 1966 and Powell, 1966). 1.2.3. VECTOR The Anopheles belong to the order of Diptera, sub-order Nematocera, family Culicidae, sub-family Culicinae and tribe Anophelini in the zoological classification. Within the tribe Anophelini the genus Anopheles has several sub­ genera. There are about 400 species of Anopheles mosquitoes throughout the world, but only some 60 species are important vectors of malaria under natural conditions. The Anopheles mosquito is known to transmit the malaria parasites in general. The Anopheles mosquitoes are most frequent in tropical or sub­ tropical regions but they are also found in temperate climates and even in the arctic during the summer. As a rule Anopheles are not found at altitudes above 2000-2500 meters. However, different strains of the Anopheles are known to transmit the parasites to specific hosts. The female Anopheles gambiae is the most important vector / of human malaria in the African tropical areas (Bruce-Chwatt, 1980), though there are many others based on the twelve epidemiological zones of malaria as classified by Macdonald (Bruce-Chwatt, 1980). Anopheles mosquito transmits rodent malaria parasites. The P berghei sporozoite was first isolated in wild sylvatic anopheles dureni (Yoeli, 1965). Also the first infection was observed in the tree rat (Thamnomys surdaster). Other vectors were tried and Anopheles stephensi was found most susceptible to P berghei. Cyclical transmission by 10 University of Ghana http://ugspace.ug.edu.gh bite was successful through Anopheles quadrimaculatus and Anopheles stephensi. Nevertheless, blood passage is used in most laboratories since some strains apparently have lost the capacity to produce gametocyte (Wellde, et al., 1966) or unless sporogony work is of importance in a particular research. 1.2.4 MICE The natural host of P. berghei is Thamnomys surdaster (tree rats). Apart from its mammalian rodent host (Thamnomys), more than 37 animal species have been found susceptible to P. berghei, majority of which belong to the group of Rodentia from different parts of the world (Yoeli, 1965). / The rodent parasites were first identified in wild rodents and later adapted to laboratory rodents and specific models of the disease produced. The rodent malaria parasites have become models for studying human malaria. For example P. berghei infection in BALB/c mice produces anaemia models and P. berghei infection in CBA mice produces neurological syndrome (cerebral malaria). Nevertheless, regardless of the mode of infection, there are two categories identified, based on the mouse strain as well as the parasite strain (host-parasite combination); lethal and non-lethal malaria models: P. chabaudi chabaudi in C57BL/6 mice is non-lethal (Akanmori et al., 1996). Also, non- lethal models can be observed in P chabaudi chabaudi in BALB/c x C57BL/6 and P yoelii in the same mouse strain, whilst in P. berghei NK65 in CBA mice is lethal (Akanmori et al., 1994) as are P yoelii and P. berghei in BALB/c x C57BL/6 (de Souza et al., 1997). Based on these models it is possible to 11 University of Ghana http://ugspace.ug.edu.gh design immunological interventions to examine protection from morbidity or early resolution of disease in mice. For this study, the specific pathogen-free female BALB/c mice were purchased from Bomholtegaard, Denmark and used for the work when they were between 6-8 weeks old. 1.2.5 IMMUNITY AGAINST MALARIA 1.2.5.1 CELLULAR IMMUNITY Many microorganisms live inside host cells where it is impossible for humoral antibody to reach them. In combating intracellular infections by acquired immune responses, T cells exploit two main strategies. The secretions of soluble factors called lymphokines which activate the cells they combine with to enhance their contribution to microbicidal defence mechanisms and the production of cytolitic T cells which kill the infected target cells (Roitt, 1988). In line with these, there has been considerable evidence in animal experiments to indicate that T-cell mediated immunity is involved in both protective immunity and pathogenesis against malaria (Waki et al., 1992). Evidence from a rodent malaria parasite is fatal to nude or T cell deprived mice irrespective of the virulence of the parasite (Jayawardena et al., 1977; Clark and Allison, 1974; and Waki et al, 1982). i Waki and his colleagues (Waki etal., 1992) re-emphasized the antagonistic role played by CD4+ and CD8+ T-cells to each other in immunity to murine malaria; 12 University of Ghana http://ugspace.ug.edu.gh they showed that CD8+ T cells play a vital role in pathogenesis in Plasmodium berghei infection whilst CD4+ T cells are important for the induction of protective immunity to P. berghei infection. Immunity to malaria can be transferred adoptively by lymphocytes (e.g. P. berghei in rats). Taylor- Robinson and co­ workers in 1993 also showed that, a vital role is played by CD4+ T cells in protective immunity against blood stages of malaria however the mechanism was unclear. By adoptive transfer of T-cells they made known that both subsets of T-helper cells (Th1 and Th2) are involved distinctively. However, these t subset responses vary according to the type of parasite species concerned. In self- resolving infections of Plasmodium chabaudi, a sequential Th1-Th2 response is required for elimination of parasitaemia whilst in mice infected with attenuated non-lethal P. berghei or with P vinckei, Th1 subset activity appears to be crucial for parasite elimination (de Souza e ta i, 1997). Thus, relative importance of individual components of the immune response in protection against malarial parasites is very difficult to assess. Nevertheless it / is clear that immunity is T-cell dependent. Also in 1987, vaccine failure taught Hoffman and his colleagues that fighting the parasite with antibodies alone is like trying to stop an elephant with a handgun (Hoffman et a l, 1987). The antibodies need help from the other parts of the immune system (the cellular immunity). 1.2.5.2 HUMORAL IMMUNITY In as much as immunity is T-cell dependent, the vital role of the antibodies cannot be overlooked (Wakelin, 1984) e.g. blocking penetration of cells and 13 University of Ghana http://ugspace.ug.edu.gh agglutination/opsonization of infected RBCs. The effectiveness of the humoral arm was demonstrated in the now classical experiment of Cohen, who showed that transfer of immunoglobulins from adults immuned to malaria into infected children was followed by a rapid fall in parasitaemia (Cohen & Waren, 1982). Also, in general, despite variations between systems, adoptive transfer of B lymphocytes or mixed population of B and T cells, have given the best results and this reinforces the importance of antibody in immunity against malaria (Wakelin, 1984). Studies have shown that there are malarial antigens present on the surface of infected RBCs and also present in the serum of infected hosts. Labeling experimehts indicate that surface antigens originate primarily from the developing parasites within the cell, although it is not fully understood how transfer to the surface takes place. Antibodies which react with these antigens also precipitate antigens present in the ring, trophozoite, schizont and merozoite stages (Wakelin, 1984). Specifically, in rodent malaria Th2 protection is by the accelerated production of specific immunoglobulin G1 antibody (Taylor-Robinson et al., 1993). Other immunoglobulins have been implicated in the protection of rodents from malaria. Akanmori et al., 1996 suggested the involvement of the subclasses of IgG (lgG2a, lgG2b and lgG3) in the humoral immune mechanism against Plasmodium chabaudi chabaudii in C57BL/6 mice. The predominant role of lgG2a in immunity against Plasmodium berghei Nk65 in CBA mice was also noted (Akanmori et al., 1994). lgG2a was reportedly detected on the surface 14 University of Ghana http://ugspace.ug.edu.gh membrane of P. berghei- infected erythrocytes after incubation with hyperimmune serum (Waki, 1994). lgG3 was significant in protection of BALB/c mice infected with 17x strain of P yoelii (Majarian et al., 1984). Murphy and Logie (1984) also supported the view that humoral immunity to P berghei may be mediated by more than one soluble factor and effected through at least two different pathways. 1.2.6 IMMUNOPATHOLOGY OF SEVERE MALARIAL ANAEMIA If the host system is regarded as a complex system of cells trying to maintain a steady state, then an attack of malaria must represent a monumental perturbation. The host responses to this insult are primarily immunoregulatory and protective (Jayarwardena, etal., 1977). Nevertheless, immunopathology isi the immune phenomenon occurring in response to infection, which is not protective or is responsible for causing pathology in the immune system. Anaemia is a major concomitant effect in the pathology of malaria as observed in humans (Jilly and Nkrumah, 1965; Rencricca et al., 1974) and in rodents (Cox et al., 1966; Singer, 1954; Topley et al., 1970). The degree of anaemia observed in many animal models was not in proportion to the destruction of infected red blood cells. Since erythropoiesis could not keep pace with the rate / of erythrocyte destruction, severe uncompensated haemolytic anaemia occurred (Miller et. al., 1989a). 15 University of Ghana http://ugspace.ug.edu.gh Central to this interaction are cytokines that are released by immunocompetent cells in a highly regulated fashion. One of these cytokines is TNF-a , which has been implicated in the immunopathology of severe malarial anemia. Earlier, Clark and his group had postulated that TNF-a release as a result of the systematic activation of macrophages by malarial parasites, may mediate in part the anemia of malaria through its ability to inhibit erythropoiesis (Clark et al., 1987b). This was accepted by Miller and colleagues and they went on to suggest that, the release of the soluble mediators, (e.g. TNF-a) inhibit erythropoiesis and therefore increase the severity of the anemia (Miller et al., 1989a). It has been established that during the erythrocytic cycle, soluble products of Plasmodium species known as malarial toxins cause the release of pro- inflammatory cytokines such as TNF-a. Also, parasite antigens stimulate T cells to directly secrete or induce production of cytokines from other cells (Miller et al., 1994). TNF-a production is known to increase during infection with malaria, and its production is down-regulated by anti-inflammatory cytokines such as interleukin-10 (Hyde, 1992; Corradin e ta l, 1993; Ho and Moore, 1994). It has also been shown that low IL-10 level is associated with severe malarial anaemia (Kurtzhals et al., 1998) and its absence causes severe anaemia in mice (Kuhn et al., 1993). Kurtzhals and colleagues also buttressed the point of well-defined mechanisms that it is not clear if severe malarial anaemia / represents the extreme of a continuum of pathology or constitutes a separate 16 University of Ghana http://ugspace.ug.edu.gh disease entity. The fine balance between cytokines appears to be important in the pathogenesis of cerebral malaria and severe malaria. This however, requires confirmation, which even though it is not possible in human subjects, can be tested in murine model of malaria. A study has shown that recombinant IL-6 boosts specific serum anti-plasmodial IgG subtype titres and suppresses parasitaemia in Plasmodium chabaudi chabaudi infection (Akanmori et al., 1996). It is obvious therefore that anaemia and cytokines which provide Scientists with an important target for research into malaria pathogenesis and the mechanisms are however not fully understood. 1.2.7 CYTOKINES AS REGULATORY FACTORS Cytokines are regulatory peptides that can be produced by virtually every nucleated cell type in the body and they have pleiotropic regulatory effects in haematopoietic and many other cell types that participate in host defense and repair processes. Constitutive production of cytokines is usually low or absent. Production per se is regulated by various inducing stimuli at the level of transcription or translation (Oppenheim, 1994). In mice, it has been shown that T helper cells may have different physical manifestations as indicated by their profiles of cytokine production and eventual effector functions (Ho and Moore, 1994). As indicated in earlier sections, basically T helper cells are categorized into Th1 and Th2. Th1 cells produce IL- 2, IL-12 and IFN-y and preferentially mediate cellular immunity whilst Th2 cells 17 University of Ghana http://ugspace.ug.edu.gh produce IL-4, IL-5, IL-6 and IL-10 and provide superior help for antibody responses. However, the cytokines of interest are; IL-10, IL-6, TNF-a and IFN-y with respect to the severity of the disease (malaria). 1.2.7.1 INTERLEUKIN 10(IL-10) IL-10 an anti-inflammatory cytokine was originally described as a cytokine synthesis-inhibiting factor produced by T-cells. Subsequent studies showed that it had pleiotropic activities (Oppenheim, 1994). It has been shown that mouse IL-10 and human IL-10 proteins are 73% identical (Ho and Moore 1994). Also, mouse and human receptors for IL-10 have been identified and characterized (Tan'e ta l., 1993; Ho etal., 1993; Liu et. al., 1994). IL-10 secreted from Th2 cells suppresses the production of Th1 cells and it has recently been assigned a further cross-regulatory role of inhibiting cytokine synthesis by Th1 cells (Hyde, 1992). It has been known to inhibit several macrophage functions (Owens et al., 1996; Fiorentino.ef al., 1991; de Waal Malefyt etal., 1991; Ding and Shevach, 1992). Also, IL-10 is involved in the stimulation of the growth of mast cells (Thompson- Snipes et al., 1991; Ho and Moore, 1994) and thymocytes (Macneill et al., 1990). It is a potent growth and differentiation factor for B lymphocytes (Go et al., 1990). Cells other than T-cells including B cells, macrophages and keratinocytes have also been shown to produce IL-10 (Owens et al., 1996). In all cases, T cells only produce IL-10 after stimulation with antigen or polyclonal 18 University of Ghana http://ugspace.ug.edu.gh activators. Importantly, IL-10 secretion is relatively late compared with other cytokines which may explain why macrophages are able to secrete substantial amount of various cytokines before IL-10 inhibition occurs (Oppenheim, 1994). IL-10 suppresses cytokines synthesis by murine T helper cells (Th1) and macrophages (de Waal Malefyt et al., 1991; Fiorentino et al., 1991). IL-10 and IFN-y antagonize 'each other’s functions and production in several systems (Chomarat et al., 1993). IL-10 has been reported to inhibit nitric oxide synthesis and microbicidal activity of interferon-y -stimulated macrophages by preventing the secretion of tumor necrosis factor-a (Corradin e ta i, 1993). It has been suggested that IL-10 could be a switch factor for human lgG1 and lgG3 production (Rousset et al., 1992; Defrance et al., 1992; Briere et al., 1994). Nevertheless, IL-10 did enhance the viability of mouse splenic B cells in culture and increased their expression of class 2 Major Histocompatibility Complex-MHC- (Go et al., 1990). 1.2.7.2 INTERLEUKIN-6 (IL-6) lnterleukin-6 (IL-6), a multifunctional and a pro-inflammatory cytokine is produced by both lymphoid and non-lymphoid cells and regulates immune responses, acute phase reactions and haemopoises. IL-6 had been referred to differently by many writers in the 1980s, until the cloning of proteins with names like IFN-(32, 26-kDa protein and BSF-2, which revealed that all these molecules were identical. Therefore it came to be known as IL-6 by the end of 1988 (Oppenheim, 1994). 19 University of Ghana http://ugspace.ug.edu.gh IL-6 is a glycoprotein with a molecular mass ranging from 21to 28kDa. Human IL-6 shows a homology with that of mouse by 65% at the DNA level. IL-6 production is induced in T cells or T-cell clones by T-cell mitogens or by antigenic stimulation (Horii et al., 1988; Hodgkin et al., 1988). Also, lipopolysaccharide (LPS) enhances IL-6 production in monocytes and fibroblasts, whereas glucocorticoids inhibit it (Helfgott etal., 1987). A variety of peptide factors such as IL-1, TNF and various agents causing elevation of intracellular cyclic Adenosine monophosphate (cAMP) levels also induce IL-6 production. However, IL-4 and IL-13 inhibit IL-6 production in monocytes. Anti IL-6 antibody inhibits IL-4 driven IgE production, suggesting that endogenous IL-6 plays an obligatory role in the IL-4 dependent induction of IgE (Vercelli et al., 1989). It has been demonstrated that IL-4 induces IL-6 production in normal human B cells (Smeland et al., 1989). Antigen-specific antibody production by primary murine B cells is dependent on IL-6 but not the secondary murine B cells (Hilbert et al., 1989). IL-6 is also reported to augment the in vivo production of anti-s RBC antibodies in mice (Takatsuki et al., 1988). IL-6 is involved in T cell activation, growth and differentiation. It acts on murine thymocytes to induce proliferation and also induces the differentiation of cytotoxic T lymphocytes (CTLS) in the presence of IL-2 from murine as well as from human, thymocytes and splenic T cells (Oppenheim, 1994). IL-6 has been found to play a central role in defence mechanisms, the murine response, haematopoiesis and acute-phase reactions. Conversely, deregulated 20 University of Ghana http://ugspace.ug.edu.gh expression of the IL-6 gene has been implicated in the pathogenesis of a variety of diseases. IL-6 production is enhanced by total unsaturated fatty acid intake t (Grimble and Tapia, 1998). 1.2.7.3 TUMOR NECROSIS FACTOR-a (TNF-a) Tumour necrosis factor alpha (TNF-a) is one of the many pro-inflammatory cytokines, which occurs rapidly following trauma, or invasion of the body by pathogenic organisms (Grimble and Tapia, 1998). TNF-a, a 17 kDa protein is produced by the monocyte, lymphocytes, mast cells, basophils, eosinophils, Nt K cells, B cells, T cells (Oppenheim, 1994). TNF -a has been implicated in the mediation of a number of other diseases, one of which is severe malaria and its production has been observed in both human and murine malaria (Clark et al, 1987a; Grimble and Tapia, 1998). The pleiotropic nature of TNF-a prevents generalization about whether it is beneficial or injurious. The protective effect of TNF-a has been shown by the inhibition of the multiplication of malaria parasites in vivo (Clark and Chaudhri, 1988; Taverne et al., 1987). Clark postulated that the activation of macrophages by malarial parasites leads to excessive production of TNF-a which mediates much of the pathology associated with malaria through its action on a variety of cell types (Clark, 1987). On the other hand, Grau and colleagues (1987) abrogated the cerebral malaria in CBA/ca mice infected with P. b. Anka when they treated the mice with anti-TNF serum. 21 University of Ghana http://ugspace.ug.edu.gh The biochemistry of TNF-a in vivo is definitely not in isolation of that of the entire cytokine network. One of the key biological effects of TN F-a is to trigger the release of a series of other cytokines that amplify and extend the biological activities of TNF-a alone (cytokine cascade). TNF-a triggers the release of IL-6, while IFN-y and IL-1 enhance TNF-a production (Oppenheim, 1994) with IL-10 acting as a potent inhibitor of macrophage functions including TN F-a (Corradin et al., 1993). Haemopoietically, TNF-a has been mentioned in the development of the anaemia of chronic diseases. Evidence has also shown that TNF-a is involved in dyserythropoiesis and erythrophagocytosis in malaria infection in mice (Clark et al., 1987). - 1.2.7.4 INTERFERON-GAMMA (IFN-y) IFN-y is an important immunomodulatory cytokine that regulates natural, cell- mediated and humoral immunity by eliciting a number of biological responses in many cell types. IFN-y is a lymphokine that displays no molecular homology with the other interferons but shares some important biological activities. I Two different subsets of mouse Th cells have been described based on the array of lymphokines secreted. IFN-y is a Th1 cytokine, which is secreted by T- lymphocytes and natural killer cells (NK) and is produced during infection (Rhodes-Feuillette et a l, 1985), since it is detected in the serum of patients 22 University of Ghana http://ugspace.ug.edu.gh shortly after acute P. falciparum infection (Wenisch et al., 1995). Both in humans and mice, IFN-y synthesis has been observed in T cells of the cytotoxic/suppressor phenotype bearing either the CD8 or the Ly-2 antigen respectively (Oppenheim, 1994). IFN-y preferentially inhibits the proliferation of Th2 but not Th1 cells. This implies that during an immune response, IFN-y would stimulate the proliferation of Th1 cells (Gajewski and Fitch, 1988). / IFN-y indirectly enhances MHC II expression on macrophages as antigen presenting cells for triggering on T-helper cells. For example, intravenous injection of recombinant murine IFN-y to mice results in an increased expression of these antigens on macrophages and on many other cells (Oppenheim, 1994). IFN-y activates macrophages and neutrophils in phagocytosis and secretion of toxic factors because parasite killing is significantly activated when infected macrophages are exposed to IFN-y (Oppenheim, 1994). IFN-y in vivo inhibits development of exoerythrocytic parasites in the liver stages of P. berghei (Mellouk et al., 1991) and also of P. falciparum schizonts in in vitro cultures of human hepatocytes (Mellouk et al., 1994). IFN-y induces the formation and release of TNF-a by macrophages (Philip and Epstein, 1986). Production of reactive oxygen intermediates and secretion of hydrogen peroxide are correlated with the capacity of macrophages to kill intracellular parasites (Murray, 1981, Clark and Hunt, 1983). Nevertheless, the stimulation of the secretion of reactive oxygen intermediates seems to be an 23 University of Ghana http://ugspace.ug.edu.gh exclusive function,of IFN-y. IFN-y can induce production of reactive nitrogen intermediates by macrophages, neutrophils, kupffer cells and hepatocytes (Adams et al., 1990; Green et al., 1990; Liew and Cox, 1991). It has been demonstrated that nitric oxide released by human monocytes can contribute to the ability of these cells to kill malarial parasites (Gyan et al., 1994), though a direct effect of IFN-y on parasite growth has been reported it had conflicting results (Jones etal., 1989; Orago and Facer, 1993). IFN-y is one of the'natural B-cell differentiation factors since the addition of anti IFN-y antibodies to activated T-cell supernatants abrogates the capacity of these supernatants to stimulate B cells into production of antibody-forming cells (Sidman et al., 1984). Studies in gene knockout mice unequivocally show that IFN-y is essential for the generation of normal lgG2a and to a lesser extent lgG3 response (Huang etal., 1993). 24 University of Ghana http://ugspace.ug.edu.gh CHAPTER 2 2.0 MATERIALS AND METHODS 2.1 MATERIALS 2.1.1 CHEMICALS Sodium chloride Diethyl ether Ethanol RPMI 1640 Tris hydroxymethylaminomethane Ammonium chloride (NH4CI) Heparin , Hydrogen chloride Giemsa stain Brilliant cresyl blue Disodiumhydrogenphosphate (monobasic) Potassiumdihydrogenphosphate (monobasic) Sorbitol Glycerol Potassium cyanide Potassium ferricyanide Methanol Nonidet Sterox BDH Laboratory Supplies, England BDH, Chemical Ltd Poole England BDH Laboratory Supplies, England Sigma Chemical Co. St. Louis USA Fluka AG Bachs SG in Switzerland AnalaR Analytical Reagent Wako Pure Chemical Industries Ltd , Japan Wako Pure Chemical Industries Ltd , Japan BDH laboratory Supplies , Eangland Wako Pure Chemical Industries Ltd , Japan Wako Pure Chemical Industries Ltd , Japan Sigma chemical Co. St. Louis , USA Sigma chemical Co. St. Louis , USA Sigma chemical Co. St. Louis , USA Wako Pure Chemical Industries Ltd .Japan Wako Pure Chemical Industries Ltd .Japan BDH Laboratory Supplies, England Wako Pure Chemical Industries Ltd Japan Wako Pure Chemical Industries Ltd .Japan 25 University of Ghana http://ugspace.ug.edu.gh Microil immersion oil p -thymidine Foetal calf serum BDH Laboratory Supplies , England NEN Boston, MA USA Sigma Chemical Co. St. Louis , USA Tripan blue Penicillin-streptomycin L-glutamine Sigma Chemical Co. St. Louis , USA GIBCO BRL Life Tech. in Paisley Scotland Sigma Chemical Co. Ltd, U K ANTIBODIES Goat anti-mouse Ig G (Fab specific) Goat anti-mouse Ig G1 (heavy chain specific) Goat anti-mouse Ig G2a (heavy chain specific)/ Goat anti-mouse Ig G2b (heavy chain specific) Goat anti-mouse Ig G3 (heavy chain specific) Goat anti-mouse Ig M (n - chain specific) 2.1.2 EQUIPMENT CENTRIFUGE 1. Kubota Centrifuge KS- 2. Compur Microspin 3. Sakuma RSL-05A 4. Kubota 1120 Lot 067H4845, Sigma Chemical Company, USA. Lot 077H9022, Sigma Chemical Company, USA. Pdt.No M8269, Sigma Chemical Company, USA. Lot 046H8968, Sigma Chemical Company, USA. Lot 086H8886, Sigma Chemical Company, USA. Lot 084H8862, Sigma BioSciences, USA. 4000 Kubota Corporation, Japan. Bayer Diagnostics Mfg. Ltd., Ireland. Sakuma Corporation, Japan. Kubota Corporation, Japan. MICROSCOPY 1. Olympus CH-2, model CHT-G Olympus Optical Company Ltd. Japan. 26 University of Ghana http://ugspace.ug.edu.gh 2. Olympus BX6DF-3 Olympus Optical Company Ltd. Japan. SPECTROSCOPY 1.Photic-100 Erma Inc.,Tokyo Japan. COULTER (3-Coulter counter PACKARD A Canberra Company Australia CELL HARVESTER Filtermate 196 PACKARD A Canberra Company Australia Micromate 196 PACKARD A Canberra Company Australia INCUBATOR 1.C02 incubator (10-0211 type) ikemotoRika kogyo Ikemoto Scientific Technology Co. Ltd Japan 2.Te-Her cubic incubator (HT-120) Hirasawa-Japan 27 University of Ghana http://ugspace.ug.edu.gh 2.2 MICE The specific pathogen-free female BALB/c mice purchased from Bomholtegaard, Denmark were kept in the animal house of the Noguchi Memorial Institute for Medical Research (NMIMR). They received food and water ad libitum. All mice were six to eight weeks old at the commencement of the experiments. 2.3 MALARIA PARASITES The rodent malaria parasite Plasmodium berghei berghei Anka strain from a t frozen stock kept in liquid nitrogen in the immunology unit of the NMIMR was used after at least 20 passages through BALB/c mice. The strain was obtained from the Department of Infectious Diseases, Copenhagen University Hospital, Denmark. It has since been stored frozen in liquid nitrogen with occasional passage through mice. The mice were inoculated intravenously with a suspension of 106 parasitized red blood cells. 2.4 PROPAGATION OF P. BERGHEI BERGHEI ANKA IN BALB/C MICE For continuous in vivo maintenance of rodent malaria parasites in mice, blood passages were done every 6-8 days. An infected mouse with high peripheral parasitaemia (30-80% parasitaemia) was anaesthetised with diethyl ether. Blood was collected through cardiac puncture and the total number of red blood cells was counted in a Neubauer counting chamber microscopically. The inoculum was adjusted to 106 parasitized RBCs by diluting in Phosphate 28 University of Ghana http://ugspace.ug.edu.gh buffered saline (PBS) and injected intraperitoneally into 6-8 week old BALB/c naive mice. 2.5 TREATMENT OF BALB/C MICE WITH rm-IL-10 Recombinant mouse IL-10 (rmlL-10), lot number 066H6712 (Sigma Chemical Company St. Louis USA), with biological activity of 0.7ng/ml determined in a cell proliferation assay using mc/9 cells, of a mouse mast cell line was used. Each of the test groups of 15 BALB/c mice was injected intraperitoneally with 0.167(j.g of rm-IL-10 in 0.1ml saline, a day before and after parasite inoculation. Control mice of 15 in number were simultaneously injected with saline. i 2.6 HAEMATOLOGY 2.6.1 ESTIMATION OF HAEMATOCRIT IN P.BERGHEI ANKA PARASITIZED MICE Haematocrit level was monitored in two groups of 5 mice, IL-10 recipient mice and corresponding control non-recipient mice. The infected mice were bled from the caudal vein by making a tiny incision on the tail-tip and blood taken into heparinised micro capillary tubes on days 3,5,7,14 and 21. This was put in the Compur microspin'and spun at maximum speed of the centrifuge for 3 minutes (auto stop). The haematocrit level in each tube was read with an in-built haematocrit reader (Compur microspin, Bayer Diagnostics mfg. Ireland). 29 University of Ghana http://ugspace.ug.edu.gh 2.6.2 ESTIMATION OF HAEMOGLOBIN LEVEL Haemoglobin levels were determined for 5 rmlL-10 recipient mice and a corresponding number of control non-recipient BALB/c mice. Blood samples were collected with a micropipette from the caudal vein through a tiny incision made at the tip of the tail. These were diluted with Drabkin solution (.0.5g potassium cyanide, 0.2g potassium ferricyanide, 0.14g potassium dihydrogen i phosphate and 0.5ml Nonidet in 1L of distilled water). The diluted blood samples were allowed to stand at room temperature between 10 and 30 minutes, after which the absorbances were read at 540nm with a photic-100 spectroscope. The haemoglobin levels in grams per litre were determined from a standard curve of absorbance- concentration curve. 2.6.3 ESTIMATION OF RETICULOCYTES The percentage reticulocytes were determined for 5 of each of the rm-IL-10 recipient mice and non-recipient mice on the following designated days (3,5,7,14 and 21). Five microlitres (5|ul) of caudal vein blood were placed in a test tube containing a 0.04% brilliant cresyl blue in PBS at pH7.4. Later, an improved Neubauer chamber was filled with the diluted blood and observed under the microscope with x40 objective. Reticulocytes appeared bluish whilst normocytes appeared pinkish (Appendix 7). The percentage reticulocytes were determined with the formula below; % Reticulocytes = number of reticulocytes x 100 total RBCs count 30 University of Ghana http://ugspace.ug.edu.gh 2.7 DETERMINATION OF PARASITAEMIA The course of parasitaemia was monitored in 5 rm-IL-10 recipient mice and a corresponding number of control non- recipient mice. Blood smears were made on days 3,5,7,14 and 21, post-inoculation with parasites. These were deemed adequate based on previous trials where blood smears taken daily were compared to smears taken on the designated days. From a drop of caudal vein blood placed on a clean microscope slide, a thinly spread blood film was prepared, air-dried, fixed with methanol and air dried again before staining for 10 minutes with a 1:10 dilution of Giemsa stain (stock solution) to 0.1 M phosphate buffer, pH 7.2. After complete staining, the slide was washed with distilled water, dried and examined under the microscope with the oil immersion objective. 2.8 ANTIBODY MEASUREMENT The antibody levels against P berghei berghei ANKA blood stage antigens in the various blood samples were determined with the Immunoflourescent antibody test (IFAT) technique. The procedure involved initial preparation of antigen slides as described below and the actual IFAT. 2.8.1 PREPARATION OF ANTIGEN SLIDES Blood was taken from P berghei ANKA infected-BALB/c mice into heparinised tubes. The erythrocytes were washed five times with sterile saline. Centrifugation was done at 4000rpm for 10minutes. A suspension of University of Ghana http://ugspace.ug.edu.gh erythrocytes containing 106 parasites in 0.2ml saline was injected into non­ infected BALB/c mice and parasitaemia monitored as indicated in earlier sections. When average parasitaemia rose to about 20% on day 5, the mice were anaesthetized with diethyl ether and blood was sampled through cardiac puncture. The blood was pooled and three drops placed on a clean microscope slide, a thinly even spread blood film was prepared, air-dried, fixed with acetone and air-dried again. About 250 prepared slides were wrapped individually in a tissue paper and groups of five, sealed in plastic bags and repacked in the glass slide boxes. These were preserved in the -80°C freezer until required for I FAT. t 2.8.2 ANTIGEN-WELL PREPARATION The antigen coated-slides were removed from the -80°C freezer and allowed to thaw at room temperature. Islands of antigens (wells) were created by wiping strips of the blood film with an alcohol wetted tissue rod. Equal wells were created (a total of 16 wells) by carefully tracing the crevices with a non-soluble pen and then allowing them to dry for a few minutes. 2.8.3 IMMUNOFLOURESCENT ANTIBODY TEST (IFAT) FOR TITRATION OF ANTI-PLASMODIAL IgG SUBTYPES AND IgM The indirect IFAT was performed as described elsewhere (Akanmori et al., 1996). Briefly four-fold serial dilution of sera obtained from both IL-10 recipient and non-recipient mice in phosphate buffered saline (PBS; dilution range 1:4 to 32 University of Ghana http://ugspace.ug.edu.gh 1: 16384) were applied to wells of antigen coated slides in duplicate. After 30 minutes incubation period at 37°C, the slides were washed and goat anti-mouse IgG, lgG1, lgG2a, lgG2b, lgG3 or IgM monoclonal antibodies were added. The slides were incubated for another 30 minutes. After washing the slides, a fluorescent- conjugated rabbit anti-goat IgG was added and the slides were incubated for a further 30 minutes. After a final wash, mountant (50% glycerol) was applied and the slides were examined under a type BX6DF-3 incident light illuminating fluorescent microscope (Olympus, Japan). Each sample was tested in duplicate. 2.9 SPLEEN CELL CULTURES Spleen cell cultures were set up and the supernatants collected for cytokine assays. , 2.9.1 PREPARATION OF MONONUCLEAR CELLS FROM THE SPLEEN Two mice each were sacrificed from the two groups, IL-10 recipient and non­ recipient, on the designated days (3,5,7,14 and 21). The spleen was removed from anaesthetized mice by making an incision at the back and pulling gently to expose the spleen. The spleen was suspended in RPMI 1640 medium in petri dishes, crushed with the ends of two frosted slides and mixed well to separate cells and enable the removal of clumps. The cells were kept on ice until all the f spleens had been mixed. The suspension was centrifuged at 1500 rpm for 5min and the supernatant discarded. The pellets were resuspended in 5ml of 0.83% ammonium chloride (NH4CI) in Tris for 2min. RPMI 1640 was then added before centrifuging at 1500rpm for 5mins and the supernatant was 33 University of Ghana http://ugspace.ug.edu.gh discarded. The cells were washed three more times using RPMI 1640 before finally being resuspended in 3ml of complete medium (2ml penicillin in streptomycin, 4ml L-glutamine, 5% v/v foetal calf serum all in 500ml RPMI 1640). 2.9.2 CELL CULTURES For viable cell count, 0.44% tripan blue was added to an equal volume of cell suspension. This enabled the cell suspension to be adjusted to a population of 106 per volume. The cell suspension was aliquoted at 150^1 per well in a round bottom tissue culture plate. To each well 20p.l each of the mitogen (phytohaemaglobin (PHA)) or the antigen (P. berghei. ANKA) was added in triplicate. The plate was incubated for 24 hours in a C 02 incubator (10-0211 type Ikemoto Rika kogyo) at 37°C. After incubation, supernatants were collected from each well. The culture was then spiked with 3H-thymidine (1mCi per well) and the volume added up to 170|ul per well with complete medium. The plate was further incubated for 24 hours at 37°C in a C 02 incubator and the cells were harvested with S.F filtermate 96-cell harvester. The filter paper was dried in an oven at a temperature range of 50- 90°C. The nuclei adhered to the surface of the filter paper were counted using a p-counter to see how the labelled thymidine was utilised by DNA for the mitotic processes. The 3H utilised is proportional to the extent of proliferation by the cells which had imbibed the p-thyrr(idine. 34 University of Ghana http://ugspace.ug.edu.gh 2.10 CYTOKINE MEASUREMENTS IN THE SERUM AND SPLEEN CELL - CULTURE SUPERNATANTS Interleukin-10 (IL-10), Tumour necrosis factor-alpha (TNF-a) and Interferon- gamma (IFN-y) were measured by commercial enzyme linked immunosorbent assay (ELISA) kits from R& D systems (Minneapolis, MN, USA). The assays detect both recombinant and natural mouse cytokines. Briefly, the assay employs the quantitative sandwich enzyme immunoassay technique as described by the manufacturers of the kits used. An affinity / purified polyclqnal antibody specific for mouse IL-10, TNF-a or IFN-y had been pre-coated onto separate microplates. Standards, controls specific for the cytokines and samples were pipetted into the wells and incubated for 2 hours at room temperature to allow binding to the immobilised antibody. After washing four times with buffer (25x concentrated solution of a buffered surfactant, with preservatives) to remove any unbound substances, an enzyme- linked polyclonal antibody specific for mouse IL-10, TNF-a or IFN-y was added to the wells. The plates were further incubated for 2 hours at room temperature and washed to remove any unbound antibody- enzyme reagent. A substrate solution (equal volume of stabilised hydrogen peroxide and stabilised tetramethylbenzidine) was added to the wells and the plates were incubated for 30 minutes at room temperature in the dark. A stop solution (diluted hydrochloric acid solution) was added and the optical density determined. 35 University of Ghana http://ugspace.ug.edu.gh The optical density was measured at 450nm with a correction factor at 570nm on a microplate reader (Corona Electric, Japan). The intensity of the colour measured was in proportion to the amount of mouse IL-10, TNF-a and IFN-y bound in the initial'step. The concentration of cytokine in the samples was then calculated from the standard curve. STATISTICAL ANALYSIS Student's t test was performed to determine statistically significant differences between groups. P< 0.01 was considered significant. 36 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3 3.0 RESULTS 3.1 HAEMATOLOGICAL MEASUREMENTS To study the effect of exogenous IL-10 on haematological, parasitological and immunological parameters, it was justified through repeated passages that the individual mice in a group were comparable to each other by measuring parasitaemia, haemoglobin levels (Hb), reticulocytes and packed cell volume (PCV). Figure 1 illustrates the justification to use the mean of the data from the individual mice in the group. IL-10 recipient mice and controls were then infected with 106 P. berghei Anka. This was followed by an injection of an equal amount of rmlL-10 into the IL-10 recipient mice and the course of infection followed. Figures 2,3,4 and 5 illustrate the profiles of mean parasitemia, Hb, reticulocytes and PCV observed/ in rmlL-10 recipient and control mice (5 in each group), after inoculation with P berghei Anka. 3.1.1. PARASITAEMIA (THE COURSE OF INFECTION) Mean patent parasitaemia in IL-10 recipient mice on day 5 was 0.2%, rising to 7% on day 7 and followed by a further rise sharply to 29.7% on day 12 (see figure 2). This tapered to 32.5% on day 14. All mice died before day 21 (100% mortality on day 19). The control mice developed a similar pattern of parasitaemia. >The onset of patent parasitaemia of 0.2% occurred on day 5, 10.6% on day 7, 21.0% on day 12 and a maximum of 31.4% on day 14 (100% mortality by day (Table of results in Appendix 1) 37 University of Ghana http://ugspace.ug.edu.gh F IG U R E la :G raphs to ju s t ify that the mice are comparable in a group KEY: M1,M2,M3 & M4 - Individual mice In graph 1a: the ascending lines denote % Parasitaemia rise and the descending lines also denote fall in Haemoglobin levels over a period In graph 1b: the ascending lines denote % Reticulocytes and the descending lines also denote fall in % Packed Cell Volumes over a period S 60 2 w 50 < 55< < 30 GrapMa: Parasitaemia with corresponding Haemoglobin levels of individual mice in a group 18 3 5 7 9 14 P o s t-in o cu la tio n /d a ys Graph 1b: % Reticulocytes with corresponding PCV of individual mice in a group 3 5 7 9 14 P o s t-in o cu la tio n /d a ys 38 University of Ghana http://ugspace.ug.edu.gh % P ar as ita em ia Figure 2:Graph of Parasitaemia for rmlL-10 treated and control balb/c mice 39 University of Ghana http://ugspace.ug.edu.gh 19). A point worth mentioning is that on day 7, parasitaemia was 10% in controls as compared to 7% in rmlL-10 recipient. ' 3.1.2 ANAEMIA 3.1.2.1 HAEMOGLOBIN LEVELS Controls were not significantly more anaemic than the IL-10 recipient mice (P>0.01). Haemoglobin levels remained stable in both groups until day 7. It then decreased sharply to approximately 6.00g/dl on day 12 and 14. From figure 3 it was clear that significant difference occurred on day 5, however there was no significant difference by day 14. The normal haemoglobin level measured in the animal unit of the NMIMR for similar mice was 12.0 ±2 g/dl. 3.1.2.2 RETICULOCYTES The normal percentage reticulocyte count measured in the animal unit of the NMIMR was 0.5% ±0.5. The reticulocyte counts were performed as a confirmation to the anaemic situation in the internal environment of the BALB/c mice. The percentage of reticulocytes in the blood as against the normal RBCs was an indication of synthesis of mature RBCs lagging behind the destruction of infected RBCs in the spleen. There was no difference between IL-10 recipient and control mice (P>0.01 for the means of percentage reticulocyte count). In both groups, the reticulocyte count fluctuated between 1.5 and 3.5% on day 5. It started rising on day 7 and rose drastically to over 25% on days 12 and 14. (See Figure 4) 40 University of Ghana http://ugspace.ug.edu.gh H ae m og lo bi n g/ dl Figure 3: Graph of Haemoglobin level for rmlL-10 Treated and control balb/c mice - m - rmlL-10 TREATED(Tf) — CONTROL(Cf) 5 7 Post-inoculation/days 41 University of Ghana http://ugspace.ug.edu.gh % R et ic ul oc yt es Figure 4: Graph of % Reticulocytes in peripheral blood of rmlL-10 treated and control untreated balb/c mice 42 University of Ghana http://ugspace.ug.edu.gh 3.1.2.3 PACKED CELL VOLUME (PCV) For further confirmation on the anaemic status in the BALB/c mice the packed cell volume was determined. Here again, the patterns were almost identical in the two groups (Fig.5). 3.2 MORTALITY It is known that Plasmodium berghei Anka has a predilection for the immature rbcs (reticulocytes) put into circulation compensatedly as a result of early onset of anaemia. This anaemia progresses and eventually terminates the life of the mice. The mortality of the BALB/c mice was determined from a group of 5 mice each; the IL-10 recipient mice and the controls after the inoculation of P. / berghei Anka. Both IL-10 recipient mice and control started dying after day 7. On day 12, percentage survival dropped sharply from 100% to 60% for the IL- 10 recipient mice, a further drop to 40% on day 14 and by day 19, all the mice were dead. There was identical pattern for control mice however with slight differences in value. Percentage survival was 40% on day 12, which dropped to 20% for day14 and stayed constant till day 18. All the mice were also dead by day 19. (Fig.6). Any significant difference in mortality started beyond day 7 and before day 18. Speculatively, any critical observation must lie beyond day 7 / since meaningful differences occurred here (P< 0.01). 43 University of Ghana http://ugspace.ug.edu.gh % PC V Figure 5: Graph of % PCV for rmlL-10 treated and control balb/c mice - m - rmlL-10 TREATED(Tf) — CONTROL(Cf) 3 5 7 12 Post-inoculation/days 44 University of Ghana http://ugspace.ug.edu.gh Pe rc en t su rv iv al Figure 6: Kaplan-Meyer survival graph for rmlL-10 treated and control mice 120 100 80 60 40 20 0 0 2 4 6 8 10 12 14 16 18 20 Day post infection IL-10 treated mice - • • • control mice 45 University of Ghana http://ugspace.ug.edu.gh 3.3 TITRES OF ANTI-PLASMODIAL IgG SUBTYPES AND IgM IgG subtypes and IgM were measured on day 3,5,7 and 14 by means of IFAT and using whole blood stages of the parasites as antigen. During the course of infection, anti-Plasmodial IgM titre rose to 8704 on day 3 which decreased to 4096 on day 5 and dropped drastically on day 7 to 160. However there was a transient increase of 256 by day 14 for the IL-10 non­ recipient BALB/c mice (control). In contrast, rmlL-10 recipient mice recorded almost twice as much for the IgM titre on day 3, which also traced the same graph pattern in the controls. This is as shown in fig. 7f. i With the anti-Plasmodial whole IgG, it rose to an average value of 10240 maintained over a period of 3 days, and decreased to a value of 4096 by day 14 in the rmlL-10 recipient mice whereas in controls, the peak was 16384 on day 5, and took a plunge to 4096 by day 7 This fell further to 256 on day 14 as it was on day 3 (Fig. 7a) and Appendix 3. In addition, there was a progressive rise in anti-Plasmodial lgG1 from 256 to 640 to 1024 on days 3,5 and 7 respectively. This rose sharply to 16384 on day 14. With controls, average titres fluctuated from 1024 on day3 to 2080 on day 5 and then decreased to 1024 on day 7 and by day 14 it was 64 (Figure 7b). The picture painted by anti-Plasmodial lgG2a in rmlL-10 recipient was not anything different from the controls. The titre rose from 2080 on day 3 to 16384 on day 5 maintained till day 14 when it dropped to 4096. In controls, peak value 46 University of Ghana http://ugspace.ug.edu.gh Figure 7a showing IgG levels 3 5 7 14 Post-lnocu latio rt/days Figure 7b showing lgG1 levels 25000 20000 W£ 15000 l-F5 10000 ElCs HTs 5 7 Post-1 n oc u I at I o n/days 14 Figure 7c showing lgG2a levels 5 7 Post Inocutati on/days Figure 7e showing lgG3 levels M Cs H Ts | " X ! ? T I 0 r11! . - 1 L ^ r S . , t E S 5 7 P o st-Jnocu ia lio n /d ay s 14 Figure 7d showing lgG2b levels Post-lnoculation/days Figure 71 showing IgM levels 5 7 Post-lnoculation/days FIGURE 7: IFAT GRAPHS FOR rmlL-10 TREATED(Ts) AND CONTROL(Cs) MICE SHOWING THE VARIOUS IgG'S & IgM 47 University of Ghana http://ugspace.ug.edu.gh of 16384 was on day 5 only, which dropped quickly to 4096 on day 7 and a final drop to 64 by day 14 (Figure 7c). Anti-Plasmodial lgG2b fluctuated in the rmlL-10 recipients, which reached 544 on day 3 and rose to 10240 on day 5 and then dropped sharply to 2176 on day 7, this rose again to the highest value of 16384 by day 14. Comparatively, the values recorded by the controls were lower. It rose from 256 on day 3 to 1024 on days 5 and 7 and dropped to the titre value of 16 (Figure 7d). Anti-Plasmodial lgG3 recorded the minimum peak value of 8704, which occurred on day 5. By day 7, it had gone down to 160 and rose to 1024 by day 14. The control had its peak also on day 5 but with a lesser titre value of 2560, which dropped to 64 on day 7, and dropped further to 16 by day 14. These are as shown in Fig. 7e. (see appendix 3 for Table of Results) To summarize, ar\W-Plasmodial IgM had the highest peak on day 3. On day 5 it was anti-Plasmodial lgG2a which maintained the peak to day 7. However, day 14 saw lgG1 and lgG2b recording the maximum titre value of 16384 for the rmlL-10 recipient mice (Fig 9b). With the control, the anti-Plasmodial IgM recorded the highest peak (8704) on day 3 and on day 5 it was IgG and lgG2a. This was maintained on day 7 but with different peak value (4096). By day 14 all titre values had decreased to the minimum with IgG and IgM recording the highest of 256 (Fig 7). 48 University of Ghana http://ugspace.ug.edu.gh The splenic lymphocytes from the BALB/C mice were prepared with the following treatments: - (i) Ts- treated with IL-10 and challenged with parasites and sacrificed (ii) Cs- not treated with IL-10 but challenged with parasites and sacrificed (iii) Cw- not treated with IL-10 and not challenged with parasites but sacrificed were cultured in triplicate at 37°c in a C 02 incubator for 24hrs for cytokine release and was spiked with p-thymidine at a cell population of 105 for cell proliferation and incubated for a further 24 hours. With the rmlL-10 recipient mice (Ts), the mitogen-driven (phytohaemagglutinin- PHA-) culture recorded 690 counts (cpm) whilst the antigen-driven (Plasmodium infected RBCs-pRBCs-) culture was 216 cpm. However, the unstimulated cells had a count of 500cpm on day 3. On day 5, unstimulated cells count was 221 cpm and that for PHA dropped to 210 whilst pRBCs was 538cpm. Day 7 had the stimulated cell categories recording higher than unstimulated cell category. This was at 409cpm for unstimulated 426 cpm for PHA and 462 cpm for pRBCs. These values dropped to 213 for unstimulated, 189 cpm for PHA and 215cpm for pRBCs on day 14. (Details in Appendix 4). For the Cs-category, the unstimulated cells counts were 270, 189, 376 and 195 cpm for days 3,5,7 and 14, respectively. The mitogen-driven proliferative response was 674, 293, 359 and 266 cpm for days 3,5,7 and 14 respectively. 3.4 SPLEEN CELL PROLIFERATION 49 University of Ghana http://ugspace.ug.edu.gh Figure 8a: control for the splenic lymphocyte proliferation 600 z i 500 K Ui 400 z O 200 U 100 0 700 IC s B T s I n t I 5 7 Post-tnoculation/days 14 Figure 8b: PHA-driven proliferation 900 800 IC s B T s M 5 7 Post -inoculation/days 14 Figure 8c : Prbc-driven proliferation 700 600 | 500 (u 400 | 300 § 200 100 E3Cs B T s r T 5 7 Post-lnoculation/days 14 FIGURE 8: Graph o f mean spleen lymphocytes proliferation o f IL-10 Treated (Ts) and Controls (Cs) 0 50 University of Ghana http://ugspace.ug.edu.gh That for antigen-driven was 228cpm on day 3 and recorded the highest of 510 cpm on day 5. This dropped to 396 cpm on day 7 and again to 222 cpm day 14. The proliferative response induced in cells from the Cw- category was higher for plasmodium-driven at 311 cpm followed by PHA- driven at 164 cpm and 139 cpm for unstimulated cells. These are shown in Figure 8. / Comparatively, induction of proliferative response was higher for plasmodium- driven than PHA-driven (fig. 7) because they had more ability to incorporate (3 Thymidine. In general, the proliferative induction of splenic lymphocytes of rmll_-10 treated BALB/c mice was not statistically different from that of the control (P> 0.01). Also, the PHA-driven response was highest on day 3 for both Ts and Cs categories with a value of 690 and 674cpm respectively. That of plasmodium- t driven occurred on day 5 for both Ts and Cs categories of 538 and 510cpm, respectively. 3.5 CYTOKINES MEASUREMENT 3.5.1 INTERLEUKIN-10 The serum IL-10 measured by means of ELISA was higher in rmlL-10 recipient mice than the non-recipient mice. Remarkably there was no detectable level of IL-10 in both groups of mice on day 3, but 48 hours later, the serum IL-10 was 16.38pg/ml in test mice and 12.48pg/ml in controls. This level rose drastically to 255pg/ml in test mice on day 7 whilst the level in control mice dropped to 51 University of Ghana http://ugspace.ug.edu.gh 10pg/ml. There was a further rise to 400pg/ml in test mice as against 64.04pg/ml in controls on day 14. This is as shown in Figure 9. With the supernates from culture, it showed that the cells were viable and had the capacity to induce IL-10 production. The response was more effective in the IL-10 non-recipient mice cells (both in mitogen-driven and plasmodium- driven culture). In order of days 3, 5, 7 and 14, the mitogen-driven IL-10 induction was 2.5/50, 90 and 40pg/ml for test mice and 1150, 170, 670 and 48.5pg/ml for controls, whilst, 333, 100, 36 and 773pg/ml for test in plasmodium culture and 340, 1150, 9.98 and 590pg/ml for controls were measured. Figure 10. (See tables in Appendix 4) 3.5.2 TUMOUR NECROSIS FACTOR-ALPHA (TNF-a) TNF -a levels in IL-10 recipient mice were almost the same as in the controls on days 3,5 and 7 However there was a drastic rise of TNF-a in the test mice to 625pg/ml as against 117pg/ml in controls on day 14. Mortality was observed after day 7 when there was appreciable detectable level of TNF-a in both test and control mice. The high level of serum TNF-a on day 14 in test mice may account for the mortality after day 14, though it appeared the controls were dying earlier than the test mice. This is shown in Fig. 11 52 University of Ghana http://ugspace.ug.edu.gh C O N C E N TR A TI O N /p g/ m l Fig. 9 IL-10 LEVELS IN SERA OF rmlL-10 TREATED (Ts) AND CONTROL (Cs) BALB/C MICE 53 University of Ghana http://ugspace.ug.edu.gh Figure 1Gb showing PHA-driven IL-10 levels LDO ZOo Post-lnoculation/days Figure 10c showing Prbc-driven IL-10 levels □ Cs HTs 5 7 Post-lnoculation/days 14 F IG U R E 10: Graph o f IL-10 LEVELS IN SPLEEN CELLCULTURE SUPERNATES FROM IL-10 TREATED (Ts) &CONTROLS (Cs) ' 54 University of Ghana http://ugspace.ug.edu.gh With the supernates it was observed that the plasmodium-driven culture had the capacity to induce TNF-a especially on days 3 and 5. However low levels of TNF-a were measured in the serum on days 3 and 5. Effective response was from plasmodium- driven culture. For both test and control mice, 3000pg/ml was measured on day 3 and 2525pg/ml on day 5 for control mice. The rest of the measurements were below 100pg/ml, except mitogen-driven induction of 230pg/ml for controls on day 3. This is shown in Fig. 12. 3.5.3 INTERFERON-GAMMA (IFN-y) The measured values of IFN-y in both test and control seemed to be very low; 2.21pg/ml for test mice and 5.25pg/ml for controls on day 3. However an increase to 25pg/ml for test mice and 6.64pg/ml for controls were observed on day 5. A sharp rise to 510pg/ml for the test mice whilst as low as 4.7pg/ml for controls on day 7 \A/ere measured. This figure dropped sharply to 9.68pg/ml in test mice with a slight increase to 28pg/ml in controls on day 14. Though IFN-y is known to induce TNF-a there seem not to be any correlation between the two cytokines(Figures 11 & 13). The inductive response was effective in both mitogen-driven and plasmodium- driven culture, but there was induction of IFN-y in the culture controls. Needless to say there was no appreciable detection of IFN-y in cells from non-inoculatedl mice. For the plasmodium- driven culture, it was 1075, 880, 1205 and 570pg/ml in test mice and 1205,725,50 and 880pg/ml for controls on days 3, 5, 7and 14 55 University of Ghana http://ugspace.ug.edu.gh respectively. For the mitogen-driven culture, the levels were 130, 925, 1205 and 410pg/ml for test mice and 405, 42, 133 and 880pg/ml for controls on days 3, 5, 7 and 14 respectively. Culture controls for rmlL-10 recipient were 305, 250, 552 and 12pg/ml and that for non-recipient were 434, 45,50 and 16pg/ml for days 3, 5, 7 and 14 respectively. This is shown in Fig. 14. 56 University of Ghana http://ugspace.ug.edu.gh C O N C E N TR A TI O N /p g/ m l Fig. 11: TNF-alpha LEVELS IN SERA OF rmlL-10 TREATED(Ts) AND CONTROL (Cs) BALB/C MICE 57 University of Ghana http://ugspace.ug.edu.gh 140 _ 120 8 100 | 80 £ 2 60 o z 40 OO 20 Figure 12a showing control for TNF- alpha levels HCs BTs 5 7 Post-Inoculation/days 14 300 250 £ 200 i l i s o2 S’'100 50 0 Figure 12b showing PHA-driven TNF-alpha levels T ■ Cs BTs ,r - r * i z t t r r E T 5 7 Post-lnoculation/days 14 F IGURE 12: TNF-alpha LEVELS IN SPLEEN CELL CULTURE SUPERNATES FROM rm lL-10 TREATED(Ts)AND CONTROLS (Cs) 3 58 University of Ghana http://ugspace.ug.edu.gh C on ce nt ra tio n/ pg /m l Fig. 13: IFN-gamma LEVELS IN SERA OF rmlL-10 TREATED (Ts) AND CONTROL (Cs) BALB/C MICE 700 STs PCs 600 500 400 300 200 100 UjiT - d A A i m i o V » v « w T O »3»3*5^VVV 5 7 Post-inoculation/days 14 59 University of Ghana http://ugspace.ug.edu.gh Figure 14a : control for IFN-gamma levels 3 5 7 14 Post-lnoculation/days Figure 14b showing PHA-driven IFN-gamma levels 1600 5 7 Post-lnoculation/days Figure14c showing Prbc-driven IFN-gamma levels 3 5 7 14 Post-lnoculation/days F IGURE 14: IFN-gamma IN SPLEEN CELL CULTURESUPERNATES FROM rm lL-10 TREATED (Ts) AND CONTROLS(Cs) 60 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4 4.0 DISCUSSION AND CONCLUSION 4.1 DISCUSSION Malaria has been the bane of developing countries in the tropics and in as much as scientists try to arrest it once and for all, it has rather developed uncompromising clout and delivered the deadliest blow cumulatively ever to have received from any other disease. One of its hallmarks is anaemia. The pathogenesis of this form of severe malaria is not fully understood. A suitable model of severe anaemia is associated with BALB/c mice infected with / P. berghei Anka. The mice develop a rapid increase in parasitaemia and die off by the 2nd or 3rd week (Schneider, 1968; Lucas et al., 1997). This situation was shown repeatedly in the present study (figure 1) before the final experiment was carried out. The progressive anaemia associated with malaria in mice begins on day 7 (Roth and Herman, 1979). This is detectable by the measurement of these haematological parameters: reticulocytes, haemoglobin levels and haematocrit (Roth and Herman, 1979; McGuire Jr. and Haines, 1985) in addition to parasitaemia. This is consistent with the present work, where the parasitaemia was 10.5% in th,e controls and 7% in the IL-10 recipient BALB/C mice on day 7, after this there was a sharp rise to 29.7% and 21% for IL-10 recipient and controls respectively on day 12. This occurred with a corresponding sharp rise in 61 University of Ghana http://ugspace.ug.edu.gh reticulocytes and sharp falls in PCV and haemoglobin levels (Figures, 2, 3, 4 & 5). Generally, the anemia has a positive correlation with the parasite count and reticulocytosis, but a negative correlation with haemoglobin and the haematocrit levels, as was observed in the study. However with the injection of rmlL-10, an anti-inflammatory cytokine, it was expected that a significantly delayed mortality would occur in these mice, as a result of suppressed parasitaemia, enhanced antibody mediation, stable levels of haemoglobin, haematocrit and low levels of the pro-inflammatory cytokines implicated in the pathology of malaria. Nevertheless, there appeared to be no significant change of the parameters measured between the IL-10 recipient and the control mice. It may be that rmlL- 10 injected into the mice was not enough to cause a significant observable change in the parameters measured between IL-10 recipient and control mice. The sharp rise of' the mean parasite count and reticulocytosis with their corresponding sharp fall in haemoglobin levels and that of haematocrit indicate that P. berghei has a predilection for immature RBCs. The appreciable rise of parasitaemia on day 5 compelled the bone marrow to respond (Rencricca et at., 1974; Maggio-Price et al., 1985) to the destruction of infected erythrocytes in the spleen (splenic phagocytosis). Therefore the sharp rise of the reticulocytes with a cumulative rise in the parasite count could be because of the affinity of P berghei for immature Rbcs, with the ripple effect in the other parameters (that is the sharp rise in reticulocytes which accompanied the losses in PCV and haemoglobin). This is supported by the observation that TNF-a caused an 62 University of Ghana http://ugspace.ug.edu.gh increase in immature erythroid cells (BFU-E), but inhibited later-stage of / erythropoiesis (CFU-E in the marrow) (Johnson et al., 1988). The decreases in PCV and haemoglobin levels seem to follow increases in TNF-a. This cytokine has been shown to cause dyserythropoiesis (Clark and Chaudhiri 1988). But it appeared there was no bone marrow suppression because the reticulocyte population did not fall. Of course one would have to measure the erythropoietin levels to confirm this. Erythropoietin is produced by the kidney in response to anaemia and induces bone marrow response (Erslev et al, 1989). The occurrence of a clearly excessive anemia as seen early in P. berghei infection is t not observed in /nice with P chabaudi infection (Akanmori etal., 1994). Such an early onset of anemia associated with P berghei could be an adaptive phenomenon. This phenomenon is one of the strain specific characteristics, which can be understood when they are looked at concurrently in relation to the internal milieu of cytokines. Plasma IL-10 was higher in the recipient than the control, however there seem not to be any significant difference in the mortality between the two groups although it appeared mortality was delayed in the IL-10 recipients. IL-10, an anti­ inflammatory cytokine is known to have a positive feedback down regulation on the pro-inflammatory cytokine TNF-a (Hyde, 1992; Ho and Moore, 1994). Yet a high TNF-a was measured in the IL-10 recipient. This supports the observation that TNF-a increases in production during malaria infection (Miller et al., 1994) and it could not be regulated by IL-10. Speculatively, IL-10 secretion is relatively late compared with other cytokines (Oppenheim, 1994), therefore before effective 63 University of Ghana http://ugspace.ug.edu.gh regulation could be done, the mice died. Also, another possible reason was that it was not secreted near the cells producing TNF-a since IL-10 acts locally. Moreover, TNF-a is known to be protective when an appreciable level is detected (Clark and Chaudhri, 1988). This was the case in the first 7 days of inoculation but the situation changed when large amount of TNF-a was produced far exceeding the anti-inflammatory IL-10. This was when the mice started dying. This is in line with Clark and his colleagues’ observation (Clark and Chaudhri, 1988). Whether TNF -a has a positive association with the severity of malarial anaemia (Miller et al., 1989a) could not be substantiated in this study. Erythropoietin (EPO) has been reported to prevent the decrease in hematocrit observed after TNF -a treatment of mice for five days (Johnson et al., 1990). The negative correlation of TNF-a with hematocrit in this work supports that (figure 5 & 11). It appears therefore that for a well-defined mechanism of anemia in malaria, a spectrum of parameters should be determined simultaneously. The role of immunoglobulin (constitutive and/or induced) in erythrophagocytosis was suggested as cause of anaemia during malaria infection (Roth and Herman, 1979). It was obvious that enhanced erythrophagocytosis occurred between the 7th and 12th day because Ig coated parasite-infected erythrocytes are readily ingested by activated macrophages (Shear et al., 1979; Tosta & Wedderburn, 1980). That was when lgG2a recorded the highest peak value both in the test 64 University of Ghana http://ugspace.ug.edu.gh and control mice. This is consistent with work done by Waki (1994); lgG2a was detected on the surface membrane of P. berghei-lnfected erythrocytes after incubation with hyperimmune serum. Though, parasitaemia suppression by anti- Plasmodial IgG subtypes has been studied it is contrary to our finding. Whilst, lgG2a is reported to play a protective role in Plasmodium berghei NK65 infection in CBA mice (Akanmori et al., 1994), its involvement in pathology of P. berghei Anka infection in BALB/c mice cannot be overlooked. The interest is centered between day 7 and 12 because that was the period appreciable mortality was observed (Fig. 6). In addition, Bouharoun-Tayoun and Druilhe reported on the protective nature of lgG1 and lgG3, the two cytophilic isotypes and the non protective role of lgG2 and IgM in children and adults who have sustained a primary malarial attack (Bouharoun-Tayoun and Druilhe, 1992). In the rat, lgG1 is most efficient for opsonisation of-erythrocytes for phagocytosis by macrophages, whilst lgG2a is more efficient in mediating antibody-dependent cytotoxicity (Miklos et al., 1993) Nevertheless, in the mouse, the profile seems not to be clear as different isotypes of antibody are involved in the process of phagocytosis (Ralph et al., 1980). lgG3 was significant in the protection of BALB/c mice infected with 17X strain of P. yoelii (Majarian et al., 1984). Hence the low level of lgG3 measured between day 7 and ,12 with a significant increase in TNF-a in the BALB/c mice in both test and cohtrol tend to give credence to that work. This is consistent with a reported case in West Africa patients with severe malaria (Sarthou et al., 1997). 65 University of Ghana http://ugspace.ug.edu.gh Likewise, IgM measured in both test and control, though higher in test animals, seems to follow the normal pattern: highest on day 3 and lowest on day 14. This was in agreement with Eling, (1980) that the spleen-dependent immuno- pathological response was involved in the mortality and morbidity of a P. berghei infection in mice. These thymus-dependent host responses to infection have been demonstrated by other authors, when blood monocyte response was depressed in infected T-cell depleted mice. They suggested that activation of T- cells during infection may enhance the differentiation of stem cells into monocytes which are then recruited by the spleen (Jayawardena, 1977). Again, Wyler and Gallin (1977) have demonstrated the presence of a monocyte- chemotactic factor in the spleens of immunologically intact mice infected with P. berghei. Though the spleen plays a vital role in malaria by contributing to innate resistance and acquired immunity yet both test and control mice died off almost at the same period regardless of the presence of IL-10. i Moreover, spleen cells were cultured to see the extent of proliferation. The proliferative response initially was higher for mitogen-driven culture than the antigen-driven culture. However, on day 3 the control response was higher than antigen-driven response. It suggests that the cultured spleen cells might have encountered the malarial antigen in vivo, so alone in in vitro culture they induced response. With time, antigen-driven gave a response equaling that of the mitogen-driven response. This reiterates the fact that there was development of memory T cells, which are important in subsequent infections. Also, the results 66 University of Ghana http://ugspace.ug.edu.gh showed that the T cells were viable because of their ability to incorporate 13- thymidine and underscores the importance of the spleen in the induction of antimalarial immunity (Figure 8). The IFN-y measured in both test and control mice, seemed to be higher in test than control. This observation agrees with the TNF-a concentration measured. It means the relationship between antibodies (immunoglobulins) and cytokines should be well exploited. It would have been useful to measure levels of other anti-inflammatory cytokines in order to verify the effect of exogenous IL-10 on these anti-inflammatory cytokines. Again, the erythrophagocytosis with the exogenous IL-10 or combination with other cytokine should be looked at again. It is likely that the severe malarial anaemia might have a specific pathology as suggested (Kurtzhals etal., 1997). 4.2 CONCLUSION • The effect of injections of rmlL-10 on survival, course of parasitaemia, haematological and immunological changes in BALB/c mice infected with P. berghei provided the following observations: • IL-10 was higher in the recipient than the control mice • There was no difference in the parasitaemia or hematological parameters measured in the two groups’ (reticulocyte counts, haemoglobin, PCV). • There was no difference in the mortality between the two groups though it appeared to be delayed in the rmlL-10 recipient mice than control. 67 University of Ghana http://ugspace.ug.edu.gh • IL-10 is known to have a positive feedback down regulation on TNF-a but a high TNF-a was measured in the rmlL-10 recipient mice. • High measurement in rmlL-10 recipient confirms that TNF-a increases in production during malaria infection. • This confirms the initial hypothesis that inadequate IL-10 an anti-/ inflammatory cytokine is responsible for the anaemia in acute murine malaria. 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N.Y pp79- 115. 79 University of Ghana http://ugspace.ug.edu.gh APPENDIX 1 TABLE 1 | | | | | | AVERAGE HAEMATOLOGICAL DATA FOR THE IL-10 INJECTED MICE AND CONTROL I.D DayPost Inocu la ­ t io n Paras ita ­ em ia % Hb m g/l Reties % PCV % Tf-210800 0 0 10.25 2.4 52 Tf-240803 3 0 '10.976 1.86 50.2 Tf-260805 5 0.178 9.852 1.54 49.4 Tf-280807 7 6.998 9.754 3.76 49.2 Tf-020912 12 29.73 5.876 26.2 36.33 Tf-040914 14 32.465 6.19 27.8 37 Cf-210800 0 0 10.576 3.22 49.8 Cf-240803 3 0 10.226 1.62 48.6 Cf-260805 5 0.182 10.45 1.66 49.2 Cf-280807 7 10.552 9.954 7.16 44.6 Cf-020912 12 21.025 6.437 25.3 41 Cf-040914 14 31.365 6.31 27.1 36 TABLE 2 PERCENTAGE SURVIVAL FOR IL-10 TREATED AND CONTROL MICE % Post Inoc. 0 3 5 7 12 14 18 19 Sur.Time(Cf) 100 100 100 100 40 20 20 0 Sur.Time(Tf) 100 100 100 100 60 40 20 0 KEY: ' Sur Time (Cf) - Survival time for Control Mice Sur Time (Tf) - Survival time for Treated Mice 80 University of Ghana http://ugspace.ug.edu.gh APPENDIX 2 TA B LE 3 | M ean s p le n ic lym p ho c y te s p ro li fe ra t io n /# o f ce lls w ith Be ta up ta ke c o u n te d pe r m in Days I.D C o n tro l PHA Prbc 3 Ts-3 503 690 216 5 Ts-5 221 210 538 7 Ts-7 409 426 462 14 ' Ts-14 213 189 225 3 Cs-3 270 674 228 5 Cs-5 189 293 510 7 Cs-7 376 359 396 14 Cs-14 195 266 222 Cw- 139 164 311 81 University of Ghana http://ugspace.ug.edu.gh APPENDIX 3 TABLE 4 | | | ' MEAN IFAT TITRE VALUES FOR IL-10 TREATED AND CONTROL MICE I.D code igG IgG l lgG2a lgG2b lgG3 IgM P. I/DAYS Ts-240803 Ts-3 160 256 2080 544 160 12384 3 Ts-260805 Ts-5 10240 640 16384 10240 8704 8704 5 Ts-280807 Ts-7 10240 1024 16384 160 160 1024 7 TS040914 Ts-14 4096 16384 4096 16384 1024 1024 14 Cs-240803 Cs-3 256 1024 2080 256 544 8704 3 Cs-260805 Cs-5 16384 2080 16384 1024 2560 4096 5 Cs-280807 Cs-7 4096 1024 4096 1024 640 160 7 Cs-040914 Cs-14 256 64 64 16 4 256 14 I.D code IgG lgG1 lgG2a lgG2b lgG3 IgM P.I/DAYS Ts-240803 Ts-3 160 256 2080 544 160 12384 3 Cs-240803 Cs-3 256 1024 2080 256 544 8704 3 Ts-260805 Ts-5 10240 640 16384 10240 8704 8704 5 Cs-260805 Cs-5 16384 2080 16384 1024 2560 4096 5 Ts-280807 Ts-7 10240 1024 16384 160 160 1024 7 Cs-280807 Cs-7 4096 1024 4096 1024 640 160 7 TS040914 Ts-14 4096 . 16384 4096 16384 1024 1024 14 Cs-040914 Cs-14 256 64 64 16 4 256 14 82 University of Ghana http://ugspace.ug.edu.gh APPENDIX 4 E L IS A DATA (pg/ml) TABLE 5 I CYTOKINE LEVELS IN BOTH SERA AND CELL CULTURE SUPERNATANT SAMPLES SERA SAMPLES DAY(P.I) I.D IL-10 TNF-alpha IFN -gamm a 3 Ts-1 0 46.8 2.21 5 Ts-3 16.38 23.4 25 7 Ts-5 254.76 70.2 510 14 Ts-7 400 625 9.68 DAY(P.I) I.D IL-10 TNF-alpha IFN -gamm a 3 Cs-1 0 60 5.25 5 Cs-3 12.48 60 6.64 7 Cs-5 10 76.9 4.7 14 Cs-7 64.06 117 28 0 igative cont 0 10.05 3.87 CELL CULTURE SUP IL-10 LEVELS(pg/ml) I.D CONTROL PHA Prbcs Ts-1 0 2.5 333 Ts-3 0 , 50 100 Ts-5 0 90 36.06 Ts-7 0 - 40 773 Cs-1 0 1150 340 Cs-3 0 170 1150 Cs-5 0 670 9.98 Cs-7 0 48.5 590 CTL 0 1.66 0 CELL CULTURE SUPERNATES TNF-alpha LEVELS(p<3/ml) I.D CONTROL PHA Prbcs Ts-1 11.37 60 3000 Ts-3 10.7 55 150 Ts-5 17.38 9.36 22.73 Ts-7 4.68 10 18.72 Cs-1 20.73 230 3000 Cs-3 20.73 90 2525 Cs-5 100 3.34 22.06 Cs-7 5.35 ' 2.67 13.4 Control 35 4.68 19.4 SELL CULTURE SUPERNATES IFN-gamma LEVELS(pg/ml) I.D C O N TR O L PHA Prbcs Ts-1 305 130 1075 Ts-3 250 925 880 Ts-5 552 1205 1205 Ts-7 12 410 570 Cs-1 434 405 1205 Cs-3 45 42 725 Cs-5 50 133 50 Cs-7 16 880 880 Control 3.32 1.4 0 APPENDIX 4 83 University of Ghana http://ugspace.ug.edu.gh Contd APPENDIX 4 TABLE 6 THE VARIOUS STANDARD CURVE VALUES IL-10 STANDARD VALUES Absorb, at 450nm Cone. pg/m l 1st reading 2nd reaam mean mean 0 -0.004 0 0 0 0.035 0.04 0.0375 0.0375 15.6 0.075 0.064 0.0695 0.0695 31.2 0.13 0.13 0.13 0.13 62.5 0.252 0.273 0.2625 0.2625 125 0.489 0.475 0.482 0.482 250 0.957 1 0.9785 0.9785 500 1.648 1.786 1.717 1.717 1000 TNF-a lpha STANDARD VALUES Absorb, at 450nm Cone. pg/ml 1st reading 2nd readin mean mean 0 0.004 0.002 0.002 0 0.035 0.035 0.035 0.035 23.4 0.063 0.065 0.064 0.064 46.9 0.124 0.12 0.122 0.122 93.8 0.23 0.22 0.225 0.225 187.5 0.449 0.439 0.444 0.444 375 0.781 0.812 0.7965 0.7965 750 1.436 1.414 1,425 1.425 1500 IF N -g am m a STANDARD VALUES Cone. pg/ml FOR SERUM FOR SUPERNATANTS 1st readinc 2nd readin mean 1st readinc 2nd readin mean -0.001 -0.01 0 -0.002 -0.003 0 0 0.039 0.03 0.0345 0.017 0.017 0.017 9.4 0.071 0.073 0.072 0.048 0.052 0.05 18.8 0.131 0.132 0.1315 0.104 0.104 0.104 37.5 0.236 0.221 0.2285 0.204 0.203 0.2035 75 0.394 0.4 0.397 0.404 0.412 0.408 150 0.724 0.755 0.7395 0.789 0.794 0.7915 300 1.381 1.362 1.3715 1.519 1.468 1.4935 600 84 University of Ghana http://ugspace.ug.edu.gh Appendix 5 3. The male and tamale cells become mature, fuse, and migrate to. the stomach wall 2 Which are sucked up the tamaleAnopheles Where a cys' is formed mutupfy and burst out into the body VJ cavity iHsday* ^esajj^a law ls ■“ i^sporowifes 1. Immature male and female cells areCTucedmthe an bloodstream © 10. Become full grown in3days person when (he mosquito ✓ ( 11. Burst out and Invade other red cells injected into a repeating this cycle \9. Then invade the / ' red blood cells K — / [ \ \ 8. The parasites multiply in the 12. Some parasites continue slowv / '- ‘ -S ,.~~7 *iv0r cells for about 11-12 days development in tissue cells, * ' '» / Asexual Cycle occasionally invade red blood / \ in Humana cefls. and start the infection all 14 days or more over again as a relapse (average 30 days) Credit: www .m edscape.com Life Cycle of Plasmodium species 85 University of Ghana http://ugspace.ug.edu.gh Appendix 6: . Developmental Cycle of P. berghei in Anopheles stephensi The developmental cycle ol Plasmodium Oergtwt tn Anopheles s*ophansJ** (1) Snofftly after the mosqutto >ngestt> on mtocJod Wood meal, mate and lemate gametocytes ere liberated from the red blood cells rt>k/stmn occurs ap*tf# geoeratr'c a zygote 11 -5 h) (31 The zygote then elongates to became an ookinete ( ID 24 h). (4) The motile ookmetr penetrates between or wthm rrwdgut eprtheltai ceas (24­ 40 M an j movea to the space between opitheiu ceHs and the basal Lamina where it transforms into « young oocyst undergoes nuclear ct vision while its plasma membrane retracts liom the capsu e and gve t. rise to mvagwtations and suDcapsufar vacuoles (61 v«*c jo te s coalesce into twge ctefts tt\at penetrate and divide ffie cvt>plas>m. loftrung the sporoblast (7) Soo'oraties evag«naie atorvg me surface o l me CS-Dea*ftg memOfane& tr«J imei o ' me spofoMast (beginning on day tot. a nucleus and venous cytoplasmic components of tt>e sporobtast a r* rar.aferrpd into the spcToroite during ttie budding process (8) The mature oocyst ♦riled w tlfi ne**«e-*t'»ped spororoites releases spomrones from day i t (9) Soorozofles migrate to and invaoe saiivar* gianos ioeginrnng on day 1?) 110> SprnrwoitPs contained w rtfiir me arm ar cells o* me giancs are .r^ectod into the venebrate host duong -ut'&'K juer.t feedings (tjegirvncg on day 13), M l , m d 'ju t kitiwn M, hae f^voe t, S tl, salivary glands ISM mterspc.rts70He matti* Credit: www.lifesci.ucla.edu/hhmi 86 University of Ghana http://ugspace.ug.edu.gh APPENDIX 7: Blood smear of P. berghei % r * . _^ ' a X ' t A Credit: w w w .ncb i.n lm .n ih .gov /m a la ria /roden t Ring, trophozoite and schizont stages of P. berghei in mouse host normocytes and reticulocytes (darkly stained) 87 University of Ghana http://ugspace.ug.edu.gh