University of Ghana http://ugspace.ug.edu.gh QR185.8 L9 F98 bite C.l G370404 University of Ghana http://ugspace.ug.edu.gh PHENOTYPIC CHARACTERIZATION AND IN VITRO RESPONSE OF LYMPHOCYTES OF GHANAIAN CHILDREN WITH BURKITT’S LYMPHOMA TO PLASMODIUM FALCIPARUM MALARIA ANTIGENS A Thesis subm itted to the Board o f G raduate studies, U niversity o f G hana, L egon, G hana. In partial fulfillm ent o f the requirem ents for the aw ard o f the M aster o f P hilosophy degree in Zoology (Applied Parasitology) By G O D FR ED F U T A G B I B. Sc. (H ons.) D epartm ent o f Zoology, U niversity o f G hana, Legon, A ccra Ghana. A ugust, 2002 University of Ghana http://ugspace.ug.edu.gh D E C L A R A T IO N The experim ental w ork described in this thesis w as done by m e, at the Im m unology Unit, N oguchi M em orial Institute for M edical R esearch, U niversity o f G hana under the supervision o f Prof. B. D. A kanm ori (Im m unology Unit, N M IM R ), Dr. D. A. Edoh (D epartm ent o f Zoology, U niversity o f Ghana). R eferences cited in this w ork have been fully acknow ledged. ............o ' .............. G O D FRED FU TA G B I (C A N D ID A TE) PROF. B. D. A K A N M O R I (SU PER V ISO R ) (C O -SU PER V ISO R ) ii University of Ghana http://ugspace.ug.edu.gh DEDICATION THIS W O R K IS D ED IC A TED TO M Y LO R D JESU S C H R IST IN W H O M I LIV E A N D M O V E AND H A V E M Y B EIN G A N D M Y FA M IL Y University of Ghana http://ugspace.ug.edu.gh A C K N O W L E D G E M E N T I am profoundly grateful to m y supervisors, Prof. B. D. A kanm ori and Dr. D. E. Edoh, for their encouragem ent, invaluable contributions and inspiration th a t m ade this w ork possible. M y debt to M r. A lfred D odoo o f E lectron M icroscopy U nit o f the N M IM R for allow ing me to use his sam ples and also helping w ith the laboratory work. I gratefully appreciate the help o f John A. Tetteh in the flow cytom etric analysis. I am thankful to the D irector o f the N M IM R for perm ission to w ork in the institute. M y thanks go to Dr. D aniel D odoo and Mr. M ichael F. O fori both o f Im m unology U nit o f the N M IM R, for their contribution to this work. I appreciate the help o f M r. Eric K yei- B aafour for assisting w ith the ELISA work. I am also indebted to Prof. Julius. A. M ingle o f the D epartm ent o f M icrobiology, U niversity o f G hana M edical School, who is in charge o f the B urk itt’s Tum our Project for perm ission to recruit the B L patients into this study. M y sincere thanks to M r. Isaac A nkrah also o f the D epartm ent o f M icrobiology, U niversity o f G hana M edical School, M rs. Em elia B oadu (social w orker w ith the B urk itt’s T um our Project), doctors and nurses o f D epartm ent o f C hild H ealth, K orle-Bu Teaching H ospital w ho assisted in the sam pling. M y thanks also go to the s ta ff o f Im m unology U nit, o f the N M IM R , nam ely W illiam V anderpuije, John A rko-M ensah, M ark Addae, A lex D anso-C offie, C aroline Danquah, Francis O w usu and Judith Antwi. M y debt to the guardians and parents for the perm ission to use the b lood sam ples from the children for the study, w ithout w hich this w ork have been im possible. University of Ghana http://ugspace.ug.edu.gh I salute m y colleagues and friends, Sam uel K w apong, N ancy D uah, G ideon H elegbe, G ordon A kanzuw ine A w andare, O sbourne Q uaye, R egina A ppiah-O ppong, Sena M atrevi, N icholas Israel N ii-T rebi, Stephen K yerem ateng, V icto r O w usu, Raphael N dondo A banja, U riel S. M cA kakpo and Selorm ey A dukpo. M y gratitude to the S taff o f D epartm ent o f Zoology, U niversity o f G hana for their contribution. v University of Ghana http://ugspace.ug.edu.gh TA B L E OF C O N TE N T S PA G E Title i D eclaration ii D edication iii A cknow ledgem ent iv Table o f contents vi List o f Tables x L ist o f F igures xi L ist o f A bbreviations xii A bstract xv C H A P T E R O N E IN T R O D U C T IO N 1 C H A P T E R T W O L IT E R A T U R E R E V IE W 6 2.1 M alaria as a disease 6 2.2 The parasite 6 2.2.1 Taxonom y. 6 2.3 Life Cycle o f Plasm odium. 9 2.3.1 Exo-erythrocytic stage 9 2.3.2 E rythrocytic schizogony 10 2.3.3 Sexual Stage 10 2.4 The V ector 12 University of Ghana http://ugspace.ug.edu.gh 2.5 Pathology o f M alaria 14 2.5.1 M alarial A naem ia 14 2.5.2 C erebral M alaria 14 2.6 B urk itt’s Lym phom a as a disease 15 2.6.1 The Epstein B arr V irus 16 2.6.2 Pathology o f B urk itt’s Lym phom a 17 2.7 Im m unity to M alaria 18 2.7.1 N on-specific (Innate) Im m unity to M alaria 18 2.7.2 A cquired Im m unity to M alaria 18 2.7.2.1 H um oral Im m unity to M alaria 19 2 .1 2 .2 C ellular Im m unity to M alaria 21 2.8 T-cells and M alaria 21 2.8.1 aP T -cells and M alaria 21 2.8.2 yST-cells and M alaria 23 2.9 Im m unity to BL 26 2.9.1 N on-specific (Innate) Im m unity to B L 26 2.9.2 Specific (Acquired) Im m unity to BL 26 2.9.2.1 H um oral Im m unity to BL 26 2.9.2.2 C ellular Im m unity to BL 27 2.10 The R ole o f M alaria in the pathogenesis o f eB L 30 C H A PTER TH REE M A T ER IA LS AND M E TH O DS 33 3.1 H um an Subjects, Sam ples and Study D esign 33 vii University of Ghana http://ugspace.ug.edu.gh 3.2 B lood C ollection 33 3.3 H aem atological analysis 33 3.4 Parasitology 34 3.5 Sam ple P rocessing 34 3.6 C ounting o f cells for v iability 35 3.7 Cell surface staining 35 3.8 Flow cytom etric analysis 38 3.9 S tim ulation o f peripheral b lood m onuclear cells (PB M C) 40 3.9.1 P reparation o f w hole P. fa lc iparum (LPA R ) 40 3.9.1.1 Parasite culture 40 3.9.1.2 Separation o f P. fa lc iparum schizonts 40 3.9.2 P reparation o f Red B lood Cells (LRBC) 41 3.9.3 P reparation o f M itogens 41 3.9.4 S tim ulation procedure 41 3.10 Cytokine A ssay by ELISA 42 3.11 Ethical C onsideration 43 3.12 Statistical analysis 43 C H A PTER FO U R R ESUL TS 44 4.1 Sum m ary 44 4. 2 C haracteristics o f Subjects 45 4. 3 F requency o f T cells is low er in BL patients than in healthy controls 46 viii University of Ghana http://ugspace.ug.edu.gh 4. 4 B-cell levels are elevated in BL and show activated phenotype 47 4 .5 M arked low level o f T cells expressing TCR-y5 in B L 49 4. 6 Lym phocytes in BL exhibit an activated phenotypic profile and express high level o f the apoptotic m arker (CD 95) 50 4. 7 y5+ T Cells are m ore activated than ap + T cells in B L 52 4. 8 The ratio o f CD4/CD8 in BL patients is h igher than in healthy children 55 4. 9 Percentages o f TCR-y5+ cells expressing the variable (V )-segm ents, V51 and Vy9, in BL patients and healthy controls 56 4.10 P lasm a levels o f cytokines 57 4 .10 .1 Tum our necrosis factor-alpha (T N F -a) 57 4.10.2 In terleukin-10 (IL-10) 58 4.11 K inetics o f T N F -a and IL-10 secretion by in vitro stim ulated PB M C 59 4.12 Cytokine levels in supernatants after in vitro stim ulation 61 4.12.1 T N F -a 61 4.12.2 IL-10 63 C H A P T E R F IV E D IS C U S SIO N AN D C O N C L U S IO N S 64 5.1 D IS C U S S IO N 64 5.2 C O N C L U S IO N S 72 References 73 A ppendix 102 University of Ghana http://ugspace.ug.edu.gh LIST OF T A B LES Page Table 1. Cell counts from the N eubauer cham ber haem atocytom eter 35 Table 2. A ntibody panel used for surface staining 37 Table 3. Functions o f T-cell m arkers 38 Table 4. Sites and distribution o f tum our in BL patients 45 x University of Ghana http://ugspace.ug.edu.gh LIST OF FIG U R E S Page Figure 1. A chart show ing the classification o f Plasm odium species 8 F igure 2. L ife C ycle o f Plasm odium fa lc iparum 13 Figure 3. F low cytom etric data show ing analysis o f lym phocyte surface m arker expression 39 Figure 4. F requencies o f CD 3+ and B cells in gated events 48 Figure 5. P roportion o f CD 3+ cells expressing T C R -gam m a delta in B L patients and H ealthy Controls. 49 F igure 6. F requencies o f CD 3+ T cells bearing various activation m arkers in BL Patients and H ealthy Controls 51 Figure 7. F requencies o f G am m a-D elta T Cells bearing various activation m arkers in B L Patients and H ealthy Controls 53 F igure 8. Percentages o f activation m arkers in G am m a-delta cells com pared to those in C D 3+ cells in B L patients 54 Figure 9. F requencies o f CD 4+ and CD8+ cells in patients and controls 55 F igure 10. F requecies o f expression o f T C R -gam m a-delta variable (V)- segm ents, V delta l and V gam m a9 in BL patients and H ealthy C ontrols 56 Figure 11. TN F-alpha levels in plasm a 57 Figure 12. IL-10 levels in plasm a 58 Figure 13. K inetics o f IL-10 production 60 Figure 14. TN F-alpha levels in supernatants 62 Figure 15. IL-10 levels in levels in supernatants 63 University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS A D CC A ntigen-dependent C ellular C ytotoxicity A ICD A ctivation-induced Cell D eath A ID S-B L A cquired im m unodeficiency syndrom e-related BL A PCs A ntigen P resenting Cells BL B u rk itt’s Lym phom a CD C luster o f D ifferentiation CM C erebral m alaria CPD C itrate-phosphate dextrose CPM C om plete Parasite M edium E B ER E pstein-B ar Early RN A eBL E ndem ic B urk itt’s Lym phom a EB N A Epstein- B ar V irus N uclear A ntigen ELISA Enzym e-L inked Im m unosorbent A ssay EBV E pstein- B ar V irus FasL Fas ligand FCS Foetal C a lf Serum FITC Fluorescein isothiocyanate FL Fluorescence channel FSC -H Forw ard Scatter H eight G -6PD G lucose-6-phosphate dehydrogenase deficiency HH V H um an herpesvirus HI H eat-inactivated University of Ghana http://ugspace.ug.edu.gh hIL H um an Interleukin HIV H um an Im m unodeficiency V irus H LA H um an Leukocyte A ntigen IAR C International A gency for R esearch on C ancer IFN Interferon Ig Im m unoglobulin IL In terleukin LA K L ym phokine-activacted K iller LCL L ym phoblasto id cell lines LM P L aten t m em brane protein LPA R Live parasite LRBC L ive R ed B lood Cells M A M em brane antigen M HC M ajo r H istocom pactibility Com plex NHS N orm al H um an Serum N K N atura l K iller N M IM R N oguchi M em orial Institute for M edical R esearch OD O ptical D ensity O PD O rtho-Phenylenediam ine PB M C Peripheral B lood M ononuclear Cells PBS Phosphate-buffered Saline PE Phycoerythrin PHA Phytohaem aglutin in University of Ghana http://ugspace.ug.edu.gh PPD Purified Protein D erivative RPE R - Phycoerythrin SSC-H Side Scatter Height TN F T um our N ecrosis Factor CTLs C ytotoxic T Lym phocytes TC R T Cell Receptor TG F Transform ing G row th Factor Th T helper V CA V iral C apsid antigen W BC W hite B lood Cells X I V University of Ghana http://ugspace.ug.edu.gh A bstract It has been show n in epidem iological studies that m alaria m ay p lay a role in the pathogenesis o f endem ic B urk itt’s Lym phom a (eBL). The contribution o f m alaria to the pathogenesis o f eBL is believed to be due to the im balances in the im m une regulation during m alaria infection. Studies have show n a loss o f C TL function due to a shift o f the im m une responses from T h l tow ards Th2 T-cell function during m alaria infection. This study sought to investigate the phenotypes o f peripheral b lood lym phocytes from eBL patients and their responses in vitro to m alaria antigens. L ym phocyte subset distributions and activation in the peripheral b lood w ere studied in 22 B L patients and 15 healthy G hanaian children by flow cytom etry. P lasm a and supernatant levels o f T N F -a and IL-10 w ere m easured by ELISA and com pared betw een the tw o groups. The results show that lym phocytes from B L patients have significantly low frequencies o f C D 3+ (p=0.003) and CD 8+CD 3+ (p=0.013) and both the frequency and the absolute counts o f y8+ T cells (p=0.005 and 0.007 respectively) com pared to the controls. The frequency o f V 51+ yd+ T cells w as significantly h igher in the patients com pared to the controls (p=0.047). The data also indicates that lym phocytes from BL patients w ere significantly m ore activated than those from the controls w ith regard to the expression o f the activation m arkers, CD95 and H L A -D R by CD 3T and y5+cells. Plasm a level o f T N F -a w as low er (p=0.002) w hereas that o f IL-10 w as h igher in BL patients than in controls (p=0.042). Peripheral blood m ononuclear cells (PBM C) from BL patients produced significantly less T N F-a com pared to the controls w hen stim ulated w ith Plasm odium fa lc ip a ru m schizonts (p=0.007) and phytohaem agglutinin (PHA) (p=0.050). S im ilarly, PB M C from BL patients secreted significantly less IL-10 in response to PH A than cells from controls x v University of Ghana http://ugspace.ug.edu.gh (p=0.016) but w ith regard to the cells stim ulated w ith P. fa lc ip a ru m schizonts there was no significant d ifference in secretion o f IL-10 betw een the two groups. Taken together, the data show that there are im balances in the im m une system o f BL patients sim ilar to those found in P. fa lc ip a ru m m alaria infection suggesting that recurren t P. fa lc iparum infection can be an additive risk factor for the developm ent and persistence o f eBL. xvi University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.1 INTRODUCTION M alaria is a m ajor childhood vector-transm itted parasitic disease that cause loss o f lives, high m edical bills and labour lost. It is estim ated that each year there are over three hundred (300) m illion clinical cases o f the disease w orld-w ide and in absolute num bers it kills three thousand (3,000) children under five years o f age p er day. T he causative agents in hum ans are four species o f a protozoan parasite know n as P lasm odium ; P. fa lc iparum , P.m alariae, P. vivax, and P. ovale . The dom inant and m ost lethal form o f m alaria is caused by P. fa lc iparum . (Sm yth, 1976; Roll Back M alaria, 2001). The m ainstay o f com bating m alaria involves reducing hum an-infected-vector contact, chem oprophylaxis and chem otherapy. W hen D D T and chloroquine proved very potent in com bating m alaria, the form er by killing the vector and the la ter by elim inating the parasite, in 1955, the W orld H ealth O rganization began a m alaria eradication program in m any parts o f the w orld (W HO, 1955; Farid, 1980). The cam paign succeeded in w iping out m alaria from Europe, N orth Am erica and R ussia but failed in the tropics and subtropics, m ainly due to difficulty in reducing vector abundance sufficiently enough to 1 University of Ghana http://ugspace.ug.edu.gh decrease the transm ission potential below the critical level for sustained transm ission, and eventual developm ent o f insecticide resistance in A nopheles m osquitoes. The im proper use o f antim alarial drugs has also resulted in the em ergence o f drug resistant strains o f Plasm odium fa lc iparum in the tropics and subtropics. A s a result in 1976, the program w as officially declared a failure (Farid, 1980). T hus m alaria rem ains a public health problem in m any developing countries. In Ghana, m alaria is hyperendem ic w ith m ainly two species o f the parasite , P. fa lc iparum and P.ovale, involved. It accounts for at least 25% o f all clinical hea lth care attendance, w ith young children under 5 years o f age accounting for about 40% o f all cases. A ll over the country, it is the predom inant cause for seeking m edical care by all groups. The m ortality rate has been estim ated as 6.3 per 1000 in infants and about 10.7 per 1000 in children aged 1-4 years (M O H, 1991). A ttem pts by the M inistry o f H ealth to com bat m alaria has focused on prom pt m edical care w ith chem otherapy, m anpow er developm ent, research, surveillance, strengthening o f health care institutions for correct diagnosis and adequate treatm ent o f patients and referral o f severe disease to T eaching or R egional hospitals (M O H , 1991). The high m ortality and m orbidity caused by falciparum m alaria, particu larly in children, has m otivated m any researchers around the globe to find an effective antim alarial vaccine to com bat the disease. 2 University of Ghana http://ugspace.ug.edu.gh The pathogenesis o f m alaria involves invasion, alteration and destruction o f erythrocytes by m alaria parasites, local and system ic circulatory changes and im m une m echanism s. These m anifest c lin ically as severe anaem ia, cerebral m alaria, g lom erulonephritis and pulm onary oedem a am ong m any others. A lthough researchers have m ade m any strides to elucidate the phenom ena o f m alaria developm ent, there is still lack o f adequate inform ation on the developm ent o f the disease and its com plications. It is believed that severe m alaria m ay resu lt from im m une-m ediated dam age but the exact m echanism s are not fully grasped (G rau et a l , 1989; A bdalla and W eatherall, 1982). B urkitt's lym phom a (BL), on the other hand, is a m alignant m onoclonal B -cell tum our that has w orldw ide d istribution but w ith m uch h igher incidence in areas o f holoendem ic or hyperendem ic m alaria , especially, coastal and lakeside regions (A llen, 1999; Epstein and A chong, 1979; K afuko and Burkitt, 1970). BL is found to be strongly and consistently associated w ith Epstein-B arr virus (EBV); also know n as hum an herpesvirus 4 (HH V4), w hich is believed to be the m ain cause o f the disease. T he incidence rate o f the disease ranges from zero to 3.6% per year w orldw ide (C ook-M ozaffari e t al, 1998). In A frica, endem ic B L (eBL) occurs predom inantly in children below the age o f 16 years. A peak incidence is seen betw een five and ten years o f age (N krum ah, 1984). It is reported that eBL accounts for 30-70% o f childhood cancers in equatorial A frica. eBL also accounts for about 40% o f all childhood m alignancies in E ast A frica (A llen, 1999). M ale predom inance in the incidence has also been reported, w here boys are affected 2.5 tim es as often as girls (Em berg, 1999). In Ghana, 485 cases o f eBL w ere seen at Korle- 3 University of Ghana http://ugspace.ug.edu.gh B u teaching hospital over a period o f 15 years (1969-82). They w ere cases referred to the B urkitt's Tum our P ro ject at the hospital (N krum ah, 1984). W hereas the pathogenesis o f severe m alaria rem ains a m ystery, M orrow 's sum m ary o f epidem iological studies strongly suggest involvem ent o f m alaria in the pathogenesis o f B urkitt's lym phom a (M orrow , 1985). H ow ever, the m echanism s by w hich m alaria contributes to the developm ent o f BL tum our are not w ell understood. Som e, therefore, suggested that the established relationship betw een m alaria and BL is only because both happened to occur in the sam e geographical locations (A llen, 1999). T cells have been im plicated in im m unity to both m alaria and BL. R eports have show n increases in the frequencies o f y5T cells in individuals follow ing clin ical challenges o f Plasm odium fa lc ip a ru m m alaria (Carding et al, 1990; D e Paoli et al, 1990; H viid et al, 2001). It has also been show n that T cells keep surveillance on the expansion o f B cells (B iggar et al, 1981) and i f this is the case then eB L should no t be m entioned am ong people, especially children, from m alaria endem ic areas w here the proportion o f yST cells is found to be rela tively h igh (Hviid et al., 2000). On the contrary, reports have show n loss o f control o f E B V cells by T cells during m alaria (C asorati et al., 1989). H ow ever, the m echanism by w hich the effector functions o f T cells are inhibited during m alaria is yet to be understood. M oreover, m ost o f the review ed studies have been carried out on individuals w ith little or no challenges o f m alaria. 4 University of Ghana http://ugspace.ug.edu.gh This study, therefore, set out to investigate the effect o f m alaria on the effector functions o f peripheral blood lym phocytes in children suffering from eB L, w ho are also hardest hit by m alaria. The aim w as to phenotypically characterize peripheral b lood lym phocytes and look at their cytokine profile w hen stim ulated in vitro w ith P lasm odium fa lc iparum schizonts and to test the specific hypothesis that the response to P lasm odium fa lciparum , and pheno typ ic and fu n c tio n a l characteristics o f lym phocytes fro m children with eBL differ fro m those o f age- and sex -m atched healthy children in the sam e popula tion as a result o f m alaria induced im m une responses. Specific objectives were; * To phenotypically and functionally characterize lym phocytes from B L patients and age- and sex -m atched healthy controls. * To exam ine response o f lym phocytes from the sam e categories to P. fa lc iparum schizonts and m itogens. * To provide inform ation on the possible role o f m alaria in the developm ent o f B urk itt’s Lym phom a. 5 University of Ghana http://ugspace.ug.edu.gh CH A PTER TW O LITER A TU RE R EV IEW 2.1 M alaria as a disease M alaria is not a novel disease. It has been w ith hum anity since antiqu ity and was given nam es w ith respect to w hat w ere believed to be its cause. F or exam ple, it w as referred to as paludial derived from a Latin w ord palus, w hich m eans m arshy ground. Italian w riters believed m alaria w as caused by offensive vapours from m arshy areas. Thus the nam e “m alaria” w as coined from two Latin w ords m al and aria, which m eans “bad air”(Bruce- Chw att, 1988). The G reeks also recognized the association betw een periodic fevers and exposure to sw am ps, in the 4th century BC. In the 19th century, L averan first identified plasm odia as the causative agents o f m alaria. Ross then dem onstrated the role o f m osquitoes as vectors (Farid, 1980). The disease causes severe anaem ia, cerebral m alaria and m any other m alignancies in hum ans throughout the w orld w ith children and expectant m others being the m ost affected (Abdalla, et al, 1980; B erendt e t al., 1994; W H O , 1997). 2.2 The parasite 2.2.1 T axonom y. The m alaria parasite belongs to the fam ily P lasm odiidae o f the phylum A picom plexa. The italics in figure 1 traces the classification o f the parasite . The fam ily Plasm odiidae has only one genus although there are rem arkable differences betw een the various species. This is because the degree o f sim ilarity is so great that they could not be divided 6 University of Ghana http://ugspace.ug.edu.gh into separate genera w ithout difficulty. G am ham , (1966, 1980) realizing the problem , thought it is m ore appropriate to retain the genus P lasm odium and in troduce subgeneric nam es. He therefore d ivided them into ten subgenera based on ery throcytic stages, exo- erythrocytic stages, sporogonic stages and vertebrate host specificity . T hus we have; P lasm odium (prim ates), L averania (prim ates), V inckeia (non-prim ate m am m als), H aem am oeba (birds) G iovannolaia (birds), N ovyella (birds), H uffia (birds), Sauram oeba (lizards), C arinam oeba (lizards), and O phidiella (snakes). In this light, the correct nam es o f the four species that infect m an are Plasm odium (Plasm odium ) vivax, P. (P.) ovale, P. (P.) m alariae and P. (Laverania) fa lc iparum . H ow ever, the subgeneric nam es are not used in practice. It has been established that it is P. fa lc iparum m alaria tha t is dom inant in areas w here BL is endem ic (M orrow , 1985) 7 University of Ghana http://ugspace.ug.edu.gh Figure 1. A chart show ing the classification o f P lasm odium species Phylum : Sarcom astigophora Apicom plexa M icrospora A scetospora M yxozoa Ciliophora Class: Perkinsea Sporozoea Subclass: G regarin ia Coccidia P irop lasm ia Order: A gam ococcidiida P ro tococcidiida E ucoccidiida Suborder: A deleina E im eriina H aem osporina Fam ily: Plasm odiidae H aem oproteidae B abesiidae Genus: Plasm odium Species: Plasm odium vivax, P. ovale, P. fa lc iparum , P. m alariae, P. know lesi, P. berghei, P. yoelii, P. chabaudi, P. lophurae, etc. (Levine, et al., 1980; Levine, 1988) University of Ghana http://ugspace.ug.edu.gh 2.3 Life Cycle o f Plasm aodium . Infection com m ences w hen an infected fem ale anopheline m osquito inoculates plasm odial sporozoites into the hum an body w hen taking a b lood m eal. The life cycle com prises o f th ree phases o f developm ent: Exo-erythrocytic stage, E rythrocytic schizogony and Sexual stage 2.3.1 E xo-erythrocytic stage The num erous sporozoites that are injected into the body eventually en ter the b lood circulation. They rem ain in the blood stream for about 45 m inutes and then disappear. Their d isappearance is due to the fact that m any o f the sporozoites are destroyed by the im m une system w hile the rest invade the hepatic parenchym al cells. O nce inside the hepatocytes, the parasite m ultiplies rapidly by schizogony -a phase o f asexual reproduction referred to as pre-erythrocytic schizogony. This takes five to fifteen days in P. fa lc iparum , after w hich hepatic schizonts rupture to liberate m erozoites into the blood stream (G am ham , 1966). In P ovale and P. vivax infections, som e o f the sporozoites on invading the hepatocytes do not develop, instead rem ain dorm ant in the cells for som e tim e. A t this stage they are term ed as ‘hypnozoites’. They undergo schizogony later to cause relapse o f disease. 9 University of Ghana http://ugspace.ug.edu.gh 2.3.2 E rythrocytic schizogony The erythrocytic cycle begins w ith the invasion o f the erythrocytes by the m erozoites released from pre-erythrocytic schizogony. This involves attachm ent o f the m erozoites to the erythrocytes, a m echanism believed to be m ediated by a specific erythrocyte surface receptor (Hadley et al, 1986; B ruce-Chw att, 1988). The m erozoites are finally internalized by endocytosis (A ikaw a and Seed, 1980). P redilection o f m erozoites for erythrocytes o f a certain age is found in som e species: m erozoites o f P. vivax invade reticulocytes or young erythrocytes, those o f P m alariae attack older ones and P. fa lc iparum invades all ages o f erythrocytes indiscrim inately. In the erythrocytes, the parasite develops into a ring ‘fo rm ’ called ‘trophozo ite’. The trophozoite later d ivides to form the schizont, w hich m atures to form ‘m eron t’ w hich ruptures to release 6-36 m erozoites. This takes tw o days for P. fa lc iparum , P. ovale and P vivax and three days for P m alariae (W hite, 1996) R einvasion o f erythrocytes then follows. H ow ever, after a series o f asexual cycles som e o f the m erozoites p roceed to the sexual stage. 2.3.3 Sexual Stage This stage is believed to be triggered by rising asexual parasitem ia, nu trien t depletion, effect o f drug suppression and /o r rising im m unity to asexual stage (S inden, 1983). The sexual stage begins in the hum an host but once the gam etocytes are form ed they are inactive and the sexual process cannot continue in the hum an host. 10 University of Ghana http://ugspace.ug.edu.gh W hen the gam etocytes are taken up by a fem ale anopheline m osquito , they becom e activated. (Sm ith and Sanford, 1988) In gam etogenesis, the fem ale gam etocyte undergoes only few structural changes to form a fem ale m icrogam ete. The m ale gam etocyte on the other hand, goes through elaborate alterations to give rise to eight m icrogam etes in a process referred to as ‘exflagellation’. In about 24 hours the zygote is form ed, w hen the gam etes fused and it is transform ed into a m otile ookinete. The ookinete penetrates the w all o f the m osquito’s gut and encysts as an oocyst. W hen m ature, the oocyst w ill burst releasing m yriads o f sporozoites, w hich then m igrate to the salivary g land ready to be inoculated into the nex t hum an host. Each stage in the parasite life cycle presents a d istinct surface antigen that the h o st’s im m une system has to react to. Sporozoites have a w ell-defined surface antigen called the circum sporozoite protein (CSP), w hich is found to trigger the production o f T cell- dependent antibodies (Zavala, et al, 1983. M erozoites and gam etocytes also have surface antigens; m erozoites surface proteins (M SPs) and gam tocyte antigen 11.1, repectively, that are im m unogenic (Holder, 1988; K oenen et al., 1984; Targett, 1990). H ow ever, it is the asexual b lood stages o f the parasite that are responsible for clinical m anifestations o f m alaria. It has been found that infected erythrocytes express varian t antigen called P lasm odium fa lc iparum erythrocyte m em brane protein 1 (P fE M P l) and that though each parasite genom e contains about forty (40) P fE M P l genes, only one P fE M P l gene is expressed at a given time. P fE M P l has been dem onstrated to be a key elem ent o f m alaria im m unity and found to elicit protective im m unity in children (D odoo et al., 2001; Bull et al., 2000; M arsh and H ow ard, 1986). 11 University of Ghana http://ugspace.ug.edu.gh 2.4 The V ector The vectors o f hum an m alaria are fem ale anopheline m osquitoes. Factors that explain their capability to transm it m alaria include their habit o f feeding on and attraction to hum an blood, w hich is necessary for m aturation o f eggs and com pletion o f the gonotrophic cycle, and ability o f the parasite to survive and com plete its life cycle in the vector. M ale anophelines do not feed on blood and therefore cannot transm it m alaria. A m ong about 400 species o f anopheline m osquitoes only about 105 species w ere naturally or experim entally found to habour sporozoites. O ut o f 67 species that are naturally infected w ith Plasm odium species only 27 species are estab lished to have significant degree o f transm ission o f m alaria. A nopheles gam biae (com plex) is a group o f anopheline m osquitoes that are m ost efficient in hum an m alaria transm ission and are associated w ith stable m alaria (W em sdorfer, 1980). The life cycle o f the P. fa lc iparum is illustrated in F igure 2. 12 University of Ghana http://ugspace.ug.edu.gh Figure 2. L ife C ycle o f Plasmodium falciparum (C ourtesy o f m alariatest.com ) MOSQUITOES € i , - 9 o c >'s t i Anopheles ' ^ j j E x / l a g e l l a l e d Female > fti'amclc Mosquito ) PEOPLE S e l l i 7 0 111 T r o p h n z o i t e 13 University of Ghana http://ugspace.ug.edu.gh 2.5 Pathology of Malaria The pathology o f malaria has been established to be due to enhanced clearance o f erythrocytes, the release o f erythrocyte and parasite materials into the circulation and the host’s response to these events. In absence o f other confirmed cause o f the clinical manifestations, any o f the symptoms and laboratory features that show that a patient is suffering from severe malaria includes impaired consciousness, respiratory distress, multiple convulsions and severe anaemia among others. The major clinical manifestations o f severe malarial pathology that are more likely to end fatally are severe anaemia and cerebral malaria (WHO, 2000). 2.5.1 Malarial Anaemia The pathogenesis o f malarial anaemia is believed to be multifactorial but the exact mechanisms are not fully grasped. However, it has been postulated that malarial anaemia may be caused by haemolysis due to rupture o f schizonts, immune-mediated clearance o f both infected and uninfected erythrocytes and suppresion o f erythropoiesis (Abdalla and Weatherall, 1982). Kurtzhals et al., (1997) have shown in their studies that P. falciparum infection indeed causes reduced response o f bone marrow to erythropoietin but it is reversible. Cytokine dysregulation has also been shown to contribute to severe malarial anaemia (Akanmori et al., 2000; Grau et al., 1989). 2.5.2 Cerebral Malaria Cerebral malaria (CM) is caused by P. falciparum infection and is one o f the most prominent manifestations of severe malaria in humans but its pathogenesis is not clearly understood (Berendt et al., 1994). However, CM is found to be associated with high plasma levels o f TNF (Grau et al., 1989) and Perlmann, et al., (1997) postulated that 14 University of Ghana http://ugspace.ug.edu.gh elevated IgE levels, leading to overproduction o f TNF, might be a contributor to the pathogenesis o f cerebral malaria. Another mechanism that is believed to contribute to the pathogenesis o f cerebral malaria is microvascular obstruction, with accompanying local hypoxia and nutrient depletion (i.e. ischaemia). Sequestration o f erythrocytes containing mature stages of parasites in the deep vascular beds o f vital organs including the brain, cytoadherence and rosette formation, and increased deformability o f the infected erythrocytes are suggested to be important in the sequence o f events that lead to the microvascular obstruction (Berendt et al., 1994; MacPhersen et al., 1985; Maguire et al., 1991). 2.6 Burkitt’s Lymphoma as a disease BL was identified, for the first time, by Dr Burkitt in 1957 while working in Uganda (Burkitt, 1958). It is a malignant lymphoma that affects, primarily, the upper and lower jaws, abdomen, bone marrow, central nervous system, salivary glands and thyroid (Aderele et al, 1975; Burkitt, 1958, 1970; Durodola, 1976; Magrath, 1991, 1997; Ziegler, 1970). The most common presenting features in BL patients from equatorial Africa are those involving the jaw and the abdomen with the jaw being the most frequently involved site (Burkitt, 1958,1970; Burkitt and Wright, 1963) There are two main types of BL, the African type, which is endemic (eBL) and the American type, which is non-endemic or sporadic (sBL). eBL tumour is found to be the fastest growing tumour known in history and the patient's death is as a result o f blocking of most o f the throat (Allen, 1999). Acquired immunodeficiency syndrome-related BL (AIDS-BL) has also been identified (Wright, 1999). 15 University of Ghana http://ugspace.ug.edu.gh 2.6.1 The Epstein Barr Virus EBV, also known as human herpesvirus 4, (HHV4), was for the first time isolated by Epstein and Barr in cultured BL cells (Epstein et al, 1964). It is virtually ubiquitous in the human population (=90% prevalence) and the vast majority o f individuals who harbour it show no apparent disease. However, EBV is consistently found to be strongly associated with human malignancies such as BL. In an endemic African region, in a total o f 191 BL cases, compiled from 10 different studies, 184 were EBV positive (96%) and also in 395 BL cases from non-endemic regions, 212 were positive (53.5%) (IARC, 1998). Other clinical manifestations o f EBV infection are a lymphoproliferative disease, infectious mononucleosis and undifferentiated form o f nasopharyngeal carcinoma (Epstein et al, 1964; Hanto,e? al., 1985; zur Hausen, et al, 1970). EBV is transmitted by saliva and from mother to child (Meyohas et al., 1996) and is acquired early in life. Just like HIV, EBV has evolved as its strategy the ability to live and persist in the lymphocytes o f the immune system itself. The virus is found to transform and ‘immortalize’ B-cells so that an infected individual carries B-cells containing EBV genome for life. EBV is the most potent growth-transforming agent known (Zerbini and Emberg, 1983) EBV has been found to develop a multiple strategy to perpetuate its existence in infected B-lymphocytes o f immunocompetent hosts. This involves establishment o f cell phenotype specific programs o f viral gene expression and the transduction o f cellular genes that modulates immune responses. Four o f such programs have been demonstrated in EBV1' cells, which are latently infected (Emberg, 1999). 16 University of Ghana http://ugspace.ug.edu.gh A type III program, also known as latency III has been demonstrated in lymphoblastoid cell lines (LCLs) obtained by in vitro immortalization o f normal B-cells and in immunoblastic lymphomas (Young et al., 1989). The cells at Latency III express all EBV proteins associated with latency: EBV nuclear antigens (EBNAs), EBNA1-6, and virus encoded latent membrane proteins (LMPs), LMP1, LMP2A and -2B and Epstein-Bar early ribonucleic acids (EBERs). In type II program, at least one, and possibly three o f the LMPs (LMP 1, LMP2A and -2B) are expressed in addition to EBNA1 and EBERs. This has been demonstrated only in in vitro system in transfected B cells (Rowe et al., 1992). However, it has been detected in vivo in other cell types (Pallesen, et al, 1993). The viral products that have been detected in type I latency are EBNA1, EBERs and LMP2A. The type I program is established in BL biopsies and some BL-derived cell lines (Rowe et al., 1987). Thus viral products that are expressed in all the three programs are EBNA1 and EBERs. It has now been shown that some o f the EBV infected B lymphocytes in blood express only EBNA1 (Chen et al., 1995). This may facilitate immune evasion, as there will be no alternative if EBNA1 is not immunogenic. 2.6.2. Pathology of Burkitt’s Lymphoma The tumorigenesis o f BL is not clear but it is believed that constitutive activation o f c-myc by translocations between chromosome 8 and chromosomes 14, 2 and 22 in BL tumour cells, (that is, transfer o f the c-myc oncogene to chromosomes bearing the immunoglobulin genes), may be involved (Adams, et al., 1983; Croce, et al., 1979; Dalla-Favera, et al, 1982; Manolov and Manolova, 1972; Taub, et al, 1982). These chromosomal translocations are found to result in increased B-cell proliferation (Baumforth et al., 1999) especially in lymphoid tissues, which are located in the upper and lower jaws, abdomen, 17 University of Ghana http://ugspace.ug.edu.gh bone marrow, central nervous system, salivary glands, thyroid, breast, and infrequently, cardiac muscles (Aderele et al., 1975; Burkitt, 1958, 1970; Durodola, 1976; Magrath 1991, 1997; Ziegler, 1970). 2.7 Immunity to Malaria 2.7.1 Non-specific (Innate) Immunity to Malaria Certain host factors are found to confer some resistance to malaria infection. Absence o f the Duffy blood group is known to protect against P. vivax infection. Genetic factors such as p-thalassaemia, which influences the rate o f haemoglobin synthesis; glucose-6- phosphate dehydrogenase (G-6-PD) deficiency, an important erythrocyte metabolic enzyme and sickle cell trait are also found to impair intraerythrocytic developmental stages o f the parasite. The reticulo-endothelial system in the liver and spleen assists in this regard by clearing parasitized cells from circulation through phagocytosis (Bruce-Chwatt, 1985; Friedman, 1978). However, it is believed that this clearance involves unparasitized erythrocytes as well, thus leading to severe anaemia (Dondorp et al., 1999). 2.7.2 Acquired Immunity to Malaria Epidemiological studies conducted in areas o f stable malaria transmission have shown an age-related increase in malaria specific antibodies and consequent decrease in morbidity. It has been established that repeated exposures to infection over the years leads to acquisition o f antimalarial antibodies, which can be lost if infection is not regular, due to loss o f immunological memory (Deloron and Chougnet, 1992; Egan et al, 1996; Sarthou et al., 1997). 18 University of Ghana http://ugspace.ug.edu.gh In malaria endemic regions, human foetuses and newborn babies are found to be protected from malaria attack by a substance believed to be an immune-mediator transferred from their immune mothers across the placenta (Bruce-Chwatt, 1952; Reinhardt et al., 1978). Studies have also established protection o f infants from malaria in early life through passive transfer o f antibodies from their immune mothers through breastfeeding (Akanmori eta l., 1995; McGregor, 1984). 2.7.2.1 Humoral Immunity to Malaria Humoral Immunity, also known as antibody-mediated immunity, functions primarily to control extra-cellular infectious agents. It is known to play a major role in acquired resistance to infections. Antibodies, specialized proteins, are the immune effectors in humoral immunity. The mechanism involves prevention o f attachment o f infectious agents to the host cells, triggering o f complement-mediated destruction, opsonization for enhanced uptake by phagocytes or neutralization o f toxins produced by the parasites. Antibodies are secreted by activated B-lymphocytes. Antibodies bind to malaria antigens on the surface o f parasitized erythrocytes resulting in destruction and/or enhanced phagocytosis o f those cells and the parasites in them (Jakobsen et al., 1997). The overall level o f antimalarial antibodies is found to have strong association with degree o f exposure to infection (Marsh, et al., 1989) and in areas o f persistent malaria transmission, it increases with age reaching a plateau during early adult and remains high for the rest o f life (McGregor et al., 1970). Thus general Ig and total antimalarial antibodies are found to be high in residents o f malaria endemic areas (Bolad and Berzins, 2000). However, it has been established that antibody responses o f children and adults differ regardless o f degree o f exposure (Baird, 1995). Antibody responses induced during malaria infection are, so far, found in immunoglobins (Ig); IgA, IgG, IgM (Collins et al., 19 University of Ghana http://ugspace.ug.edu.gh 1971; Targett, 1970;), and more recently, IgE (Perlmann et al., 1999). No antimalarial antibody has yet been demonstrated in IgD. Studies have shown that IgG is more persistent than other antimalarial immunoglobulins and has a strong correlation with malarial precipitins in plasma o f donors at all ages over a year (McGregor, 1970). Moreso, passive and artificial transfers o f IgG confer protection against P. falciparum infection (McGregor et al., 1963). The persistence and association of IgG and malaria antigens suggest that IgG may play an important role in immunity to malaria parasites. On the other hand, it has been found that IgM levels rose sharply in association with parasitemia but declined drastically when chemotherapy was completed, although malaria antigens were still in circulation (Targett, 1970). This may suggest that IgM response may be more to disease than to parasite. M alaria parasites have also evolved ways o f inducing immunosuppression and diverting immune responses to repeated regions o f surface antigens, eliciting production of redundant non-protective B-cell responses (Anders, 1986). It has also been reported that certain immunodominant epitopes divert responses away from more important targets in the antigenic variation (Howard, 1987). In children, antibodies to these critical antigenic targets are not fully developed making them more vulnerable to malaria attack (Baird, 1995). Recently, elevated levels o f both total IgE and antimalarial IgE antibodies have been shown in malaria patients (Perlmann et al, 1999) and its levels are found to be significantly higher in patients with cerebral malaria than those with uncomplicated falciparum malaria. This makes researchers believe that IgE may play a role in the pathogenesis o f cerebral 20 University of Ghana http://ugspace.ug.edu.gh malaria. Moreover, TN F-a, a cytokine found to correlate with severity o f P. falciparum malaria attack (Grau et al., 1989), is found to be associated with IgE. 2.7.2.2 Cellular Immunity to Malaria The immune system basically comprises o f a range o f cell types, which participate in direct effector functions, in immune regulatory mechanisms, antibody secretion, or antigen presentation. However in specific cellular immunity, T-lymphocytes are paramount. Lymphocytes are divided into two broad categories: B-lymphocytes, which are precursors o f antibody secreting cells, and T-lymphocytes, some o f which are mainly cytotoxic and others that regulate immune responses through production o f cytokines. Cytokines are regulatory proteins secreted by white blood cells and various cell types in the body. Cytokines are different from hormones in that a cytokine can be produced by more than one cell types and has a broad spectrum o f action but within a short range whereas hormones are secreted by one type o f specialized cells and have a specific action, which is at a distant site. 2.8 T-cells and Malaria T cells are divided into two groups. The first group that expresses y/5 receptor (ySTCR) is called y5T cell group. The second group that expresses a /p receptor (aPTC R) is known as aP T cell group. Majority o f peripheral blood lymphocytes (>90%) are aP T cells (Haas et al.. 1993). 2.8.1 apT-cells and Malaria Two main types of aPT cells are recognized. These are CD4+ T cells and CD8+ T cells, which recognize antigens, presented on major histocompactibility complex (MHC), MHC 21 University of Ghana http://ugspace.ug.edu.gh II and MHC I respectively of antigen presenting cells (APCs). When activated, CD4 T cells secrete cytokines that define their main function o f regulating the immune system (Janeway et al., 1988). Based on the type and function o f cytokines produced, CD4 cells can be categorized into two subsets, CD4+ T helper 1 (T h l) and CD4+ T helper 2 (Th2) cells. T hl cells are mediators in cellular immunity but also regulate certain B cell responses. They are known to produce predominantly the cytokines, interleukin-2 (IL-2), Tumour necrosis factor (TNF), gamma interferon (IFNy) and lymphotoxin, triggering expansion and maturation o f T cells, and hence cellular immunity. Th2 cells on the other hand produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13, promoting maturation o f B cells and antibody production (Mosmann et al., 1989). CD8+ T cell group comprises o f cytotoxic T cells (TCLs) that are able to destroy target cell through direct contact and/or through production o f toxic cytokines. Some suppressor CD8+ T cells have also been identified (Koide and Engleman, 1990). W hereas the cytotoxic activities o f CD8+ against blood stage o f the parasite seems to be non-existent, they appear to protect against pre-erythrocytic stage with their activities directed against infected hepatocytes (Hockmeyer and Ballou, 1988). T hl cells are found in some rodent malaria to produce IFNy and IL-2 and are important in controlling infection at its early stages. Th2 cells on the other hand, secrete IL-4 and IL-10 and by these cytokines, induce B-cells to produce antibodies. These Th2 responses are found to be vital for protection against malaria parasites in late phase o f infection (Troye-Blomberg et al., 1994). The balance between Thl and Th2 subsets o f the aPT-cells would determine the state o f the immune regulation. In a murine model it has been found that chronic malaria leads to a shift in helper T cell response towards Th2 cells (von der W eid and Langhome, (1993), which may lead to immuno-incompetence. 22 University of Ghana http://ugspace.ug.edu.gh 2.8.2 yST-cells and M alaria Studies have shown the main role o f the minority group o f peripheral blood T cells, y5T cells as a first line o f defense to infectious pathogens (Augustin et al., 1989; Bluestone and Matis, 1989; Bom 1990 Janeway, 1988), an involvement during infection with viruses (De Paulo 1990; Carding et al., 1990), parasites (Georlick et al., 1991) and y8T cells that produce Th 1 -like and Th2-like cytokines have also been demonstrated (Ferrick et al, 1995). Human y5T cells are divided into sub-groups depending on the subset o f TCR V- segments expressed. The majority sub-group in Caucasians (about 70 to 90%) expresses both TCR variable segments Vy9 and V52 and are called Vy9+V52+T cells. The second most frequent sub-group expresses V81TCR V-segment and is known as V81+T cell (Casorati et al., 1989). On the contrary, a number o f studies have shown significant increase in the levels o f y8T cells during P. falciparum infection in adults. However, most o f these studies were conducted on non-immune donors and in a study, the elevation o f y5T cells was found not to be associated with disease severity (Ho et al, 1990; 1994; Perera et al, 1994). But no significant increase was found in P vivax infection (Worku et al, 1997) indicating the role o f parasite-related factors. Hviid et al., (1996, 2001) observed increase in y5T cells in children from malaria endemic areas. It has been shown in a mouse model that yST cells proliferate in response to rises in parasitemia and play an important role in controlling it (Langhome 1996; Seixas and Langhome, 1999). Growth inhibition o f yST-cells in vitro has also been confirmed and their response has been found to be associated with products from schizont rupture (Elloso et al, 1994). It has been suggested that cytotoxic activities o f these cells may take place in 23 University of Ghana http://ugspace.ug.edu.gh the spleen, since they are found to be localized in the spleen (Troye-Blomberg et al, 1994; Langhome 1996). They were also shown to control liver stage o f the parasite in experimental mice (Langhome, 1996). Both in acute P. falciparum infection and in vitro system, the elevated subset o f yST-cells was Vy9+V82+T cells (Goodier et al., 1995; Langhome 1996;). Now, it has been established that V81+T cells also expand in response to malaria antigens in vitro as well as in acute infection (Ho et al, 1994; Schwartz et al, 1996). A recent studies has shown that the rise in y5T cells during P. falciparum malaria infection, in individuals from malaria endemic areas, is mainly due to increase in V51+T cells (Hviid et al, 2001) and in malaria endemic areas levels o f V81+T cells were higher than that o f Vy9+V82+T cells in healthy donors (Hviid et al, 2000). This suggests, at least, that Vy9+V82+T is not the only subset responding to malaria infection. It also implies that the immune status o f the host has a bearing on the response o f these subsets o f y8T cells. It has also been established that healthy donors from malaria endemic areas have higher levels ofy5+T cells (>10% o f T cells) compared to Caucasians (<5% o f T cells) mainly due to expansion o f V81+ cells. Since no significant association has been found between y5+ cells or V51+ cells and malaria antibodies or parasitemia, the role o f these cells in antimalaria response is not clear (Hviid et al, 2000). Lymphokines secreted by y S ^ cells are known to stimulate macrophages (Goodier et al, 1995), thus may assist in primary infection when there are no specific memory cells. However, y8+T cells are implicated in the pathogenesis o f malaria due to their stimulative response to a stage o f parasite associated with disease development (Goodier et al, 1995). 24 University of Ghana http://ugspace.ug.edu.gh Also, y5+T cells are implicated in the pathogenesis o f malaria because y5+T cells are pronounced during infection in non-immune donors who are susceptible to severe disease (Miossec et al, 1990; Perera et al, 1994). Moreover, cytokines produced by Vy9*V82+T cells have been associated with pro-inflammatory response and especially, T N F a has been associated with severe and cerebral malaria (Goodier et al., 1995; Grau et al., 1989). The y5T cell response is not MHC-restricted (Langhome 1996) and has been found to be dependent on CD4+a(3+ T cells (Elloso et al., 1994). It has also been established that the majority o f y8T cells are CD4'CD8' cells and as y5T cells increase, percentage o f CD4+ cells declines (Worku et al., 1997), a scenario suggested to be either due to proliferative response or selective recruitment o f y5T cells into the circulation (Ho et al., 1994). Cytotoxic activity o f natural killer (NK) cells against erythrocytic stage o f P. falciparum has also been reported (Phillips, 1994). It has been well established that acute P. falciparum malaria leads to lymphopenia before initiation o f chemotherapy (Hviid et al., 1997), a phenomenon that can adversely affect protective immunity to the disease. The mechanisms that result in this turn o f events have not been fully established. Some researchers have pointed to disease-induced reallocation o f T cells to sites o f inflammation (Elhassan et al., 1994). Others have experimental evidence that suggest FasL-mediated programmed cell death as the cause o f lymphopenia o f malaria but have failed to provide direct relationship between Fas expression and lymphopenia of malaria (Balde et al., 1995; Kem et al., 2000; Matsumoto, et al., 2000 Toure-Balde et al., 1996). Although activation o f lymphocytes is consistent with lymphopenia in non-immune, non-exposed or less exposed individuals (Chougnet et al., 1992; Elhassan et al., 1994; Worku et al., 1997), no report has pointed to activation- 25 University of Ghana http://ugspace.ug.edu.gh induced cell death (AICD) as the cause o f decreased lymphocyte numbers in acute P. falciparum malaria. 2.9 Immunity to BL 2.9.1 Non-specific (Innate) Immunity to BL Studies have convincingly established the involvement o f EBV in development o f BL. Elevated antibody titres to EBV coded antigens has been reported in several studies o f BL cases from endemic African regions (Magrath, 1990; Nkrumah and Perkins, 1976). Non specific, early immune responses to E B V immunoblasts involving Natural Killer cells (NK), lymphokine-activated killer (LAK), antibody dependent cellular cytotoxicity (ADCC) and macrophage-mediated components have also been identified. These are followed by a persistent specific T-cell immunity. 2.9.2 Specific (Acquired) Immunity to BL 2.9.2.1 Humoral Immunity to BL Specific antibody responses to EBV have been found to involve immunoglobulins (Igs); IgG, IgM and IgD. These antibodies are produced early during infection and whereas IgM and IgD are transient, IgG antibodies persist throughout life and are found to control recurrence o f EBV infection. Production of IgG and IgM antibodies to viral capsid antigen (VCA) has been demonstrated (Jones et al, 1985; Niederman and Evans, 1997). Also, some o f the Igs are neutralizing antibodies that recognize EBV membrane antigen (MA) (Errand, 1992). The last antibodies produced are against EBNAs, which may or may not be detected due to poor response by certain individuals (Jones et al., 1985). Whereas antibodies to viral envelope antigens (MA, VCA) are able to neutralize viral activity through ADCC, BL cells lack expression of VCA and other antigens except EBNA1 and 26 University of Ghana http://ugspace.ug.edu.gh therefore, are not affected by natural humoral responses. However, an elevated antibody titre against EBV (VCA) has been observed in BL patients (Evans and Mueller, 1997). 2.9.2.2 Cellular Immunity to BL The T-cell immunity has been found to be predominantly mediated through reactivation of cytotoxic T cell responses (Svedmyr, et al, 1984). The EBV-specific CTL memory is found to be mainly HLA class 1 restricted and is directed against viral products expressed at latency III program (Gavioli et al., 1992; Murray et al, 1992). Aside the expression o f these potential target antigens, the EBV 1’ B cells express lymphocyte activation markers such as CD23, CD30, CD39 (Gordon et al., 1984) and secrete lymphokines such as IL-10 (Burdin et al., 1993). When BL cell lines and Lymphoblastoid B cell lines expressing the III latency program established from peripheral blood o f normal donors were screened, the majority produced significantly, more o f human interleukin-10 (hIL-10) than mature normal human B- lymphocytes. hIL-10 is not only found to suppress lymphokine production by T hl T cells but also known to act in an autocrine fashion, enhancing the expansion o f B cells. This would invariably lead to increase in EBV transformed cell line in the B cell pool. IL-10 also down-regulates the activation o f CTLs. CTLs are found to keep surveillance on the reappearance o f transformed B-lymphocytes healthy virus carriers (Lin and Askonas, 1981) but this function appears to be suppressed once the tumour has set in. It has been found that although EBV specific CTLs are capable o f recognizing viral nuclear antigens EBNA3, 4, 6, and to some extent, EBNA2, 5, LMP1 and LMP2 (Brooks et al, 1993; Burrows et al., 1990), yet no cytotoxic response has been 27 University of Ghana http://ugspace.ug.edu.gh detected against EBNA1. EBNA1 has also been observed in an experimental mouse model to be non-immunogenic (Trivedi et al., 1994). The inability o f the CTLs to recognize the EBNA1 must be o f concern since EBNA1 appears to be the only viral antigen expressed in BL cells. This may explain why the CTLs lack the capability to check the abnormal expansion o f B-lymphocytes in BL. Results obtained from other experiments have suggested destruction or dysfunction o f a subset o f CD4+ T cells, which are responsible for the induction o f CD8+ CTLs (Whittle et al., 1990). However, normal levels o f EBV-specific CTL precursors were demonstrated in BL patients (Rooney et al., 1997). In a recent study, it has been demonstrated that CD4+ T lymphocytes from healthy adults respond to EBNA1 and that among the virus-encoded antigens that stimulate CD4+ T cells, EBNA1 is preferentially recognized. This response o f CD4+ cells is believed to be protective because o f secretion o f IFN-y and direct cytolysis after encounter with transformed B lymphocyte cell lines (M iinz et al., 2000). This implies that CD4+ dysfunction is likely to be one o f the main factors in lack o f B cell control in BL patients. However, a study has shown that CD4+ T cells can induce Fas- mediated apoptosis in BL B cells; especially B cells with CD40 ligation at their surfaces. But the persistence expansion o f the malignant cells suggested that this Fas-mediated apoptosis is not functioning. There is the suggestion that the Fas-mediated death signal might be modulated by some activation markers at the cell surface (Schattner et al., 1996). Other researchers have classified the six virus-encoded nuclear antigens (EBNAs) found in LCLs as EBNAs 1, 2, 3A, 3B, 3C and leader protein (EBNA LP) in addition to the two latent membrane proteins (LMPs 1 and 2) (Murray et al, 1992). It has been found that EBNA3A, 3B, 3C have epitopes that are immunodominant among the different latent proteins and CD8+ CTL responses were markedly skewed toward these epitopes. 28 University of Ghana http://ugspace.ug.edu.gh However, no responses to EBNA1, EBNA LP, or LMP1 were observed (M urray et al, 1992). Khanna and his colleages (1992), in a study to localize EBV CTLs epitopes established that epitopes for EBNA3A and EBNA3C were recognized more frequently than any other epitopes whilst no CTL epitopes were localized in EBNA1. The invisibility o f the EBNA 1 to CD8+ cytotoxic T lymphocytes is now known to be due to prevention of processing and presentation o f EBNA1 on MHC class I molecule by its Glu/Ala repeat domain. The result obtained by M unz and his colleagues (2000) shows that it is instead presented on MHC class II molecule. Thus, the subset o f T cells that may help in controlling B cells that express only EBNA1 are y8T cells, for they are not MHC- restricted. It has been demonstrated that when EBV-transformed B cell line were used as stimulating cells they caused a striking expansion o f only V51+T cells o f T cells obtained from healthy donors and patients suffering from a chronic HLA-B27+ mono-arthritis. And in absence of V52+ cells, proliferative response were enhanced (Hacker et a l , 1992). In in vitro system, EBV- BL cells also have the ability to stimulate V81+ cells and that this becomes enhanced in presence o f EBV. These findings indicate that VS I* cells may be responsible for controlling abnormal proliferation o f B cells and have a crucial role to play in protection against pathogenesis o f BL. However, an elevated levels o f Vy9+ cells in peripheral blood during EBV infection in humans has been reported instead (De Paoli, 1990). This makes the protective role o f V81+ cells in BL unclear. However, if yS+ or V51+ T cells control the expansion o f B cells, then eBL should not be mentioned among people, especially children, from malaria endemic areas where the proportion of y8+ or V81+ T cells is found to be relatively high. Reports have shown loss of 29 University of Ghana http://ugspace.ug.edu.gh control o f EBV+ cells by T cells during malaria (Dalldorf, 1962). This may be due to immunosuppression, which is characteristic o f malaria infection. However, the mechanism by which the effector functions o f T cells are inhibited during malaria is yet to be fully unraveled. There is therefore speculation that effector functions o f V51+ T cells might be lost during malaria thus, rise in EBV~B cells and hence development o f eBL in malaria endemic regions. 2.10 The Role of Malaria in the pathogenesis of eBL It has now been established beyond doubt that malaria is a cofactor in the pathogenesis o f eBL and there are speculations that suggest that one o f the major roles o f malaria and EBV infections may be to provide an additive risk for development o f B-cell clones with chromosome translocations leading to constitutive c-myc activation. This is based on the background that neither malaria alone nor EBV alone provides sufficient B-cell stimulation to result in a noticeable increased risk for BL. However, the existence o f E B V and non malaria related BLs (Adams, et al., 1983; Dalla-Favera, et al., 1982; IARC, 1998) suggest that each factor can be replaced by other mechanisms. The contribution o f malaria is believed to be due to the imbalances in the immune regulation during malaria infection but this is yet to be fully proven. Several studies have pointed to immunosuppression (Geser et al., 1989; W hittle et al., 1984, 1990), which is a common feature in acute P. falciparum infection, as an important factor that could lead to increased susceptibility to BL. Several factors may account for the immunosuppression observed in P. falciparum malaria. It has been found in a murine model that chronic malaria leads to a shift in helper T cell response towards Th2 cells (von der Weid and Langhome, (1993). It has also been shown in a study that in vitro stimulation o f lymphocytes with malaria antigens induces secretion o f cytokines with Th2 profile such 30 University of Ghana http://ugspace.ug.edu.gh as IL-10 and TGF(3 (W ahlgren et al., 1995). The cytokines secreted by T hl are very vital in mounting protective immunity especially, against intracellular infectious agents. Skewing o f the helper response towards Th2 implies a rise in IL-10 secretion by Th2 cells, and IL-10 is known to suppress the functions o fT cells, particularly CTL function. IL-10 is also found to act as an autocrine growth factor for B cell (Mosmann and Coffman, 1989). B-cell activation also occurs in malaria and the number o f B cells rises with the general number o f lymphocytes (Geser et al., 1989; Whittle et al, 1984, 1990). Thus everything is in favour o f expansion o f B cells. A study has also shown that hemozoin, the end-product o f haemoglobin metabolism by intraerythrocytic malaria parasites, is an important factor in malaria-associated immuno- incompetence. It is found to affect both antigen processing and immunomodulatory functions o f macrophages (Scorza et al., 1999). Plasmodial infection is associated with rise in the level o f IgE in the blood o f the majority o f people living in malaria endemic areas and only up to five percent (5%) are anti-malarial antibodies. Fc-IgE is known to interact with IgE receptor (CD23) and increases the expansion o f B cells. (Perlmann et al, 1999). Children with malaria are found to have very high serum levels o f IgG and IgM, most o f which are not anti-plasmodial antibodies. The levels plateau after the age o f five to six years (Me Gregor, 1970) coinciding with the peak age o f incidence o f BL in holoendemic malarious areas (Molineaux and Gramiccia, 1980) but how abnormal levels o f IgG and IgM could contribute to development o f BL is not clear. Therefore as a consequence o f all these, the number o f B-lymphocytes latently infected with EBV will increase while the ability of T cells to suppress the outgrowth o f EBV- infected lymphoblastoid cells is impaired. This implies that acute P falciparum malaria 31 University of Ghana http://ugspace.ug.edu.gh may amplify the pool o f EBV+ B cells prone to accumulate oncogenic changes and undergo transformation, which are major events in BL pathogenesis. The course o f the major events in BL pathogenesis in children, it is believed to be: EBV-infection early in life, followed by persistent exposure to malaria also in early life and then the oncogenic process. There is no explanation for the fact that about ninety percent (90%) o f the world population is latently and permanently infected with EBV (Magrath, 1990) and yet only a few children suffer from BL. This may be due to the fact that in healthy immunocompetent EBV-carrying host; there is an efficient immune surveillance o f EBV-carrying B-cells in place. During P. falciparum malaria the immune surveillance may be disturbed as a result o f imbalances in the immune regulation. Children already have underdeveloped immunity (Baird, 1995) and therefore their immune mechanisms can easily be derailed making them more vulnerable to BL. 32 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE MATERIALS AND METHODS 3.1 Human Subjects Samples and Study Design Study subjects comprised children with BL referred to the Burkitt’s Tumour Centre at the Korle-Bu Teaching Hospital from all parts o f Ghana. The patients were clinically examined by consultant paediatricians of the Department o f Child Health, Korle-Bu Teaching Hospital. Inclusion o f the patients in the study was based on clinical information as well as cytological examination o f tumour aspirates by a pathologist. Informed consent was obtained from all parents or guardians before the children were enrolled in the study. Healthy Ghanaian children with comparable age and sex were included as controls. A total o f 10ml o f blood was taken from each patient or subject. The study was therefore a case- control one in which BL patients were compared with age and sex-matched healthy non- BL Ghanaian children. 3.2 Blood Collection Blood samples were collected in sterile 10ml heparinized vacutainer tubes using sterile butterfly needles. The tubes were heparinized to prevent coagulation. The samples were immediately taken from the hospital to the Immunology Unit o f the Noguchi Institute for Medical Research where they were processed. All the samples were processed within six hours after collection. 3.3 Haematological analysis An automated haematology analyzer (Sysmex KX-21, Japan) was used to determine all the 21 haematological parameters of the patients and the subjects. The absolute counts o f lymphocytes were determined from this analysis. 33 University of Ghana http://ugspace.ug.edu.gh 3.4 Parasitology Each sample was examined for presence of malaria parasites. Thick and thin blood smears were prepared, dried and the thin smears fixed in methanol. The films were then stained with freshly prepared 10% Giemsa (Laboratory Supplies, Poole BH15 ITD, England), for 10 minutes, washed carefully and thoroughly under running tap water. The slides were dried and observed with immersion oil under a light microscope (Olympus BH2, Japan) at lOOOx magnification. 3.5 Sample Processing The blood samples were processed under sterile conditions. Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep (Nycomed Pharma, As, Oslo) density gradient centrifugation. A volume o f 5ml o f venous blood was carefully layered on top o f 2ml of Lymphoprep and centrifuged at 814xg for 30 minutes. The ring of white blood cells was carefully aspirated and washed (centrifugated at 814xg for lOminutes) three times in RPMI1640 containing 10% heat-inactivated foetal ca lf serum (FCS) supplemented with gentimycin, and L-glutamine. 25|J.I o f PBMC suspension was stained with leukocyte stain and cells counted using the Neubauer chamber haematocytometer. Table 1 shows how the cell counts were obtained. The PBMC were then aliquoted into four vials (cryotubes) and cryopreserved (frozen at -196C in liquid nitrogen) in RPMI1640 supplemented with gentimycin, L-glutamine dimethyl sulphoxide (10%) and FCS (25%) using a gradient freezing device which yields up to 95% cell viability upon thawing (Hviid et al., 1993). This has been established at Immunology Unit o f NMIMR and used over several years. The plasma obtained was stored at -40C. Before use, the cells were thawed quickly in a water bath at 37C and washed (once for surface staining or three times for stimulation) with washing buffer. 34 University of Ghana http://ugspace.ug.edu.gh 3.6 Counting of cells for viability A volume of 25^1 o f cell suspension was stained with Trypan blue (instead o f leukocyte stain) to count and also ascertain cell viability after which the cell concentrations were adjusted appropriately. Table 1. Cell counts from the Neubauer chamber haematocytometer Each o f the four (4) squares (chambers) o f the haematocytometer is Ix lm m and the depth is 0 .1mm. '=> Volume o f cell suspension per square =1x1x0.1mm3 o rlO ^m l. :. No o f cells per millilitre = N x dilution factor io31 = N x dilution factor x 104 where N is the average count per square and the dilution factor depends on the amount o f stain, and volume of original cell suspension used. 3.7 Cell surface staining Briefly, the PBMC were directly stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, R-phycoerythrin (RPE)- and RPE-Cy5-conjugated antibodies for two or three-colour fluorescent analysis. The antibodies were directed against CD3 (UCHT1; DAKO, Glostrup, Denmark), CD4 (MT310; DAKO), CD8 (DK25; DAKO), CD25 (ACT- 1; DAKO), CD69 (L78, Becton Dickinson (BD) Biosciences), CD95 (DX2; BD Immunocytometry Systems), HLA-DR (L243; BD Immunocytometry Systems), TCR-y8 (11F2; BD PharMingen (PE) and 11F2; BD Immunocytometry Systems (FITC), V51 (TS8.2; Endogen), V52 (B6; BD PharMingen), V83 (ImmunoTech, Marseilles, France), Vy9 (B3; BD PharMingen) and B cell (FMC7; DAKO). 35 University of Ghana http://ugspace.ug.edu.gh The PBMC were stained in twelve tubes in combinations shown in table 2. Monoclonal antibodies; PE anti-human V82, PE anti-human y/5 TCR and FITC Vy9 TCR which were not o f working concentration, were diluted according to specification (1 in 4). Before flow cytometric the cells were washed once with washing buffer and re-suspended in PBS supplemented with 2%FCS (FACS buffer). After counting as described earlier, the cell concentrations were adjusted to l.OxlO6 cells/ml or more. A volume o f 1 Ojj.1 o f the antibodies were put at the bottom o f the FACS tubes, and then followed by 100|il o f cell suspension per tube. The mixture was stirred briefly using vortex and incubated at room temperature for 20 minutes. After incubation, the cells were spun in 3ml FACS buffer at 814xg for 8 minutes. After a second wash the cells were re-suspended in 200|il FACS buffer for acquisition on the same day or fixed in 200|il PBS + 0.5% paraformaldehyde and acquired within three days. 8000 to 20,000 gated lymphocytes were acquired and analysed. The samples were acquired and analysed on a FACScan flow cytometer (BD). 36 University of Ghana http://ugspace.ug.edu.gh Table 2: Antibody panel used for surface staining Tube# 1. CD8FITC CD4 PE CD3 Cy5 2. ySFITC CD69 PE CD3 Cy5 3. ySFITC HLA-DR PE CD3 Cy5 4. Y6FITC CD25 PE CD3 Cy5 5. ySFITC CD95 PE CD3 Cy5 6. B cells CD25 PE 7. Y5FITC V52 PE 8. ySFITC V53 PE 9. Vy9 FITC Y6 PE 10. CD8FITC Y5 PE 11. y s f it c CD4 PE 12. V51 FITC Y5 PE The functions of some o f the T-cell markers are listed in table 3. 37 University of Ghana http://ugspace.ug.edu.gh Table 3. Functions of T-cell markers M arker Functions CD 3 Specific T-cell receptor (TCR) important in signal transduction for T-cell activation. CD4 Expressed by T helper cells and acts as co-receptor with the TCR for MHC class II recognition. CD8 Expressed by cytotoxic cells, important in maturation and positive selection o f MHC class I restricted T cells. CD25 Also known as interleukin-2 receptor (IL-2R), is an activation marker associated with T-cell growth. CD69 An activation inducer molecule (therefore known as early activation marker). CD95(APO- An activation marker that transduces an apoptotic signal for clonal 1/Fas) deletion o f T-cells. HLA-DR An activation marker that is part o f MHC class II molecule, restricts and regulates the immune responses is a highly specific way. TCR-yd Anti-microbial and cytolytic functions. 3.8 Flow cytometric analysis Before sample acquisition, colour compensation optimisation was carried out. Data was acquired and analysed using CELLQuest Software (BD, San Jose, CA) after setting appropriate forward and side scatter gates around the lymphocyte population. Negative isotype control were stained with IgGl and used to draw the cut-off line in the histogram. Lymphocytes were first selected by electronic gating according to forward scatter and side scatter, and then by their expression o f surface markers. The proportions o f lymphocytes, which were positive for the various markers, were then obtained from histograms (Figure 3). 38 University of Ghana http://ugspace.ug.edu.gh F igure 3. Flow cytom etric data show ing analysis o f lym phocyte surface m arker expression. A) D ot p lo t show ing selection o f lym phocytes by electronic gating. B) and C) illustrate separation o f gated lym phocytes according to expression o f surface m arkers. Positive cells are separated from negative cells by quadrant m arkers. The d istribution and m ean fluorescence o f cells are show n in the quadrant statistics. U L =upper left, U R =upper right, LL=low er left and LR =low er right._________________ A. K1036 001 0g 1 f ! c © SS r§* o ................. 0 200 400 600 8U0 1000 FSC-H**ghv» B. Q uadrant Statistics W ’ File: K1048.005 K - Acquisition Date: 11-Dec-01 Gate: G1 X Parameter. FL1-H Anti-TCR-gamma-deita-1 FITC (Log) ' & V Parameter: FL3-H CD3 Cy5 (Log) .'•S' ■ Quad Events % Gated % Total 10 10‘ 10' UL 2114 56.45 13.91 An(l-TCR-4*irMn«-0«lt»-1 FITC UR 620 16.56 4.08 LL 983 26.25 6.47 LR 28 0.75 0.18 c. Quadrant Statistics File: K1048.006 Acquisition Date: 11-Dec-01 Gate: G1 X Parameter: FL2-H CD95 PE (Log) Y Parameter FL3-H CD3 Cy5 (Log) Quad Events % Gated % Total UL 321 8.24 2,08 UR 2462 63.23 15.92 LL 240 6.16 1.55 LR 871 22.37 5.63 39 University of Ghana http://ugspace.ug.edu.gh 3.9 Stimulation of peripheral blood monuclear cells (PBMC) 3.9.1 Preparation o f whole P. falciparum (LPAR) 3.9.1.1 Parasite culture Frozen chloroquine resistant strains o f P. falciparum malaria parasites (3D7) were taken from the liquid nitrogen tank, thawed quickly in a 37°C water bath, an equal volume of thawing mix (normal saline-0.9%NaCl) was added and span down at 415xg for 10 minutes. This was repeated twice with complete parasite medium (CPM). The parasites were then added to a 50ml culture flask containing 200(il o f washed A+ red blood in 5ml of CPM, gassed with a gas mixture (2% 0 2, + 5.5% C 0 2 balanced with N2) and. incubated in a CO2 incubator at 37°C. The culture medium was changed every day and each time, a thin smear was prepared and examined, as described previously, to determine parasitaemia, growth stage and viability o f parasites. When the parasitaemia was about 5% subcultures were made, using prepared uninfected red blood cells (RBCs). 3.9.1.2 Separation o f P. falciparum schizonts W hen the majority (75% or more) o f the parasites were at the schizonts stage and the parasitaemia was about 2% or higher, the parasites were separated for stimulation on the same day. Briefly, 7ml o f fresh isotonic percoll (percoll + 10% lOx PBS) diluted with 28% RPMI1640 was placed in a 15ml centrifuge tube (coming) and carefully layered with 3.5ml o f P. falciparum culture and span at lOOOxg for 25 minutes. Cells at the interface between the percoll solution and parasite medium, which were mainly late stages or schizonts, were withdrawn carefully, pooled and washed three times with stimulation medium. A smear was prepared and stained with Giemsa to determine the percentage of the infected cells harvested that were schizonts. They were then kept at 4°C and ready for 40 University of Ghana http://ugspace.ug.edu.gh 3.9.2 Preparation o f Red Blood Cells (LRBC) Blood from an A+ donor was buffered with CPD (citrate-phosphate dextrose) and kept overnight at 4°C. The plasma was then removed and an equal amount o f red blood cells (RBC) buffer was added and span at lOOOxg for 8 minutes. The medium and white blood cells (WBC) were then removed. This was repeated three more times and an equal amount o f RBC buffer was added and stored at 4°C for use in parasite culture and stimulation. For stimulation, the LRBC were washed twice with the stimulation medium to avoid contamination o f culture with the RBC wash. 3.9.3 Preparation o f Mitogens W orking concentrations o f phytohaematogglutinin (PHA) and purified protein derivative o f Mycobacterium tuberculosis (PPD) (10|xg/ml) were also prepared using RPMI1640. 3.9.4 Stimulation procedure Before stimulation, the PBMC were taken from liquid nitrogen tank, thawed quickly and washed as described previously except that here the culture (stimulation) medium (RPMI1640 supplemented with 10%NHS, L-Glutamine, Penicillin/Streptomycin and filtered) was used. The cells were counted, also, as described previously after which the concentrations were adjusted to 1.0 xlO6 cells/ml. LPAR and LRBC were also counted and their concentrations adjusted to 7 .5xl07 cells/ml. After adding a volume o f 60|iL per well o f parasites (LPAR), red blood cells (LRBC), purified protein derivative (PPD) or phytohaematogglutinin (PHA), 600|iL of PBMC suspension from patients and controls were added and incubated in a CO2 incubator at 37°C. Culture supernatants were harvested after 24 hours and also on days 3 and 6 for cytokine analysis. 41 University of Ghana http://ugspace.ug.edu.gh 3.10 Cytokine Assay by ELISA Levels o f the cytokines, tumour necrosis factor-alpha (TNF-a) and Interleukin-10 (IL-10) were determined in culture supernatants o f PBMC and plasma o f BL patients as well as their healthy counterparts, who served as controls. 96-well microtitre plates (Immulon 4 HBX, Dynex) were coated with 50|il/well o f anti-human TN F-a or anti-human IL-10 monoclonal antibody at 2|ig/m l (diluted with carbonate buffer: 0.1M NaHCCh, pH 8.2) and incubated overnight at 4°C. The plates were then washed four times with a washing buffer (0.05% Tween 20 in phosphate-buffered saline (PBS)) at 250^1/well. A blocking solution (10% heat inactivated FCS in PBS) was added at 150(j.l/well and the plates incubated at room temperature for 1 hour. After incubation the plates were washed twice using an automated plate washer (Wellwash AC, ThermoLabsystems, Finland). A standard (recombinant) human TNF-a or IL10 was added at serial dilutions (diluent: RPMI + 5% HI AB serum NHS) from 2000pg/ml to 31pg/ml in addition to undiluted plasma or culture supernatants at 50ul/well. The plates were then incubated at room temperature for 2 hour on a shaker. Following incubation, the plates were washed four times using the plate washer. A biotinylated anti-human TNF-a or IL10 was diluted (diluent: 5% FCS in PBS) to l(ig/ml and added to the plates at 50|il/ml. The plates were again incubated for 45minutes at room temperature and washed five times as previously described. An avidin peroxidase conjugate was then added at 2.5|ig/ml (diluent: 5% FCS in PBS) and 50ul/well and incubated for 30 minutes. The plates were again washed five times. This was followed by addition o f OPD substrate (0.4mg/ml in citrate-phosphate buffer +0.4mg/ml 42 University of Ghana http://ugspace.ug.edu.gh H2O2 added immediately prior to use) at 100|il/well. The plates were then developed in the dark for 30 minutes, stopped with 2.5N H2S 0 4 at 50ul/well and read using a microtiter plate reader (Multiskan Ascent V I.24, ThermoLabsystems, Finland) at 492 nm. The OD values of the standards were used to draw the appropriate curves using a statistical software (TBLCurves, Jandel Scientific) and the curves were used to transform the sample OD values to concentrations in pg/ml. 3.11 Ethical Consideration Ethical approval for this study was granted by the University of Ghana Medical School Scientific Research and Ethical Committee, and the Institutional Review Board of the NMIMR. Participation in the study was strictly voluntary and signed informed consent of parents and guardians was obtained. 3.12 Statistical analysis Comparison between groups and subsets were done using Student’s t-test (t), except when equal variance and normality tests failed, in which case the Mann-Whitney rank-sum test (7) was adopted. Confidence intervals for median differences were calculated as described by Conover (1980). Spearman’s rank correlation was used to establish association between different parameters. SigmaStat software (Jandel Scientific, San Rafael, CA) was used for all statistical calculations except correlations, SPSS software was used for correlations and Microsoft Excel (Microsoft Corporation) and SigmaPlot software (Jandel Scientific) were used for graphical presentations. P values less than or equal to 0.05 were considered significant. 43 University of Ghana http://ugspace.ug.edu.gh C H A PT E R FO U R RESULTS 4.1 Summary This study involved twenty-two (22) Burkitt’s Lymphoma patients and fifteen (15) age- and sex-matched healthy children. Lymphocytes from eBL patients showed high levels o f CD4+CD3+ (p=0.004), CD95+CD3+ (p=0.008)’ HLA-DR+CD3+ (p=0.013), CD95+y5+ (p<0.001), HLA-DR+y5+ (p<0.001), V 5 l+ y8+ (p=0.047), and B cells (p<0.001) but lower levels CD3+(p=0.003), y8+(p=0.007), CD8TCD3+(p=0.013) and Vy9 y5+(p=0.001) o f lymphocytes compared to the controls. Plasma level o f TNF-a was lower in patients compared to controls (p=0.002) and conversely, plasma level o f IL-10 was higher in patients than in controls (p=0.042). Stimulation o f PBMC with P. falciparum schizonts, PHA and PPD showed remarkable reduction in immune response with regard to production o f TN F-a and IL-10 in patients compared to controls. P. falciparum, schizonts seem to induce elevated production o f IL- 10 in both controls and patients. The following graphs; figures 4-15 and tables 4 illustrate the results o f the study. The whiskers o f the bar charts represent the standard errors and in the box plots, the box shows the interquartile range; the line through the box represents the median; the whiskers show 95% confidence interval and the outliers are indicated by individual symbols. 44 University of Ghana http://ugspace.ug.edu.gh 4. 2 Characteristics of Subjects Twenty-two (22) BL patients were recruited {13 males and 9 females; Mean age (95%CI): 7.0 (5.5 to 8.0)} and out o f this seven died. The sites o f involvement o f the tumour and their combinations are shown in Table 4. Most o f the patients had abdominal (-77% ) and jaw(~55% ) masses. Fifteen healthy Ghanaian children {9 male and 6 female; M ean age (95%CI): 6.5 (5.0 to 7.5)} were included as controls. Table 4. Sites and distribution o f tumours in BL patients Sites and their com binations Number o f patients Abdomen 17 Jaw 12 Eye 3 Neck 1 Jaw & Eye 1 Abdomen & Jaw 7 Abdomen, Jaw &Eye 2 45 University of Ghana http://ugspace.ug.edu.gh 4. 3 Frequency of T cells is lower in BL patients than in healthy controls The mean frequency o f peripheral blood CD3+ cells was significantly lower in BL patients than in healthy controls {Mean difference (95%CI): 15.64(5.75 to 25.56); p(0=0.003 }(figure 4). The mean absolute number o f CD3+ cells was also lower in BL patients than in the controls but this was not significant {Mean difference (95%CI): 1.16x10s (-14.23 xlO5 to 11.91xl05)/m l;p(0=0.851}. 46 University of Ghana http://ugspace.ug.edu.gh 4. 4 B-cell levels are elevated in BL and show activated phenotype BL patients showed elevated levels o f B cells as compared to age-matched controls in terms of both frequency {Mean difference (95%CI): 4.96 (3.17 to 6.76); p(t)<0.001 } and absolute counts {Mean difference (95%CI): 0.11 xlO5 ( 0.02 xlO 5 to 2.21 x l0 5)/ml; p(0=0.047} (figure 4). Moreso, higher counts o f B cells in BL expressed the activation marker, CD25 than in controls {Mean (95%CI): 0.29(-0.83 to 1.41) x l0 5/ml, n=3; 0.033 (0.01 to 0.05) x l0 5/ml, n=13, respectively}. 47 University of Ghana http://ugspace.ug.edu.gh Figure 4. Frequencies of CD3+ and B cells In gated events 48 University of Ghana http://ugspace.ug.edu.gh 4. 5 M arked low level o f T cells expressing TCR-v5 in BL The lymphocytes bearing TCR-y5 were significantly lower in terms o f both frequency {Median difference (95%CI): 5.33 (1.62 to 8.47); p(7)=0.005 } and absolute numbers {Median difference (95%CI): 1.82xl05 (0.29xl05 to 5 .22xl05 )/ml; p(7)=0.007 } in BL patients as compared to age-matched healthy children (figure 5). Figure 5. Proportion of CD3+ cells expressing TCR-gamma delta In BL patients and Healthy Controls. 14 - 12 ■ 10 ' o “ Qn 8 O 5o 6 5? 4 2 0 - BL patients Healthy children Category 49 University of Ghana http://ugspace.ug.edu.gh 4. 6 Lymphocytes in BL exhibit an activated phenotypic profile and express high level o f the apoptotic marker (CD95) The proportions o f peripheral blood CD3T T cells expressing the activation markers, CD95 (apoptotic marker) and HLA-DR (late activation marker), were significantly higher in BL patients than in healthy children {Mean difference (95%CI): 14.11 (3.97 to 24.26); p (t)=0.008; Mean difference (95%CI): 13.08 (2.98 to 23.19) p (t)=0.013 respectively}. Frequencies o f CD3+ cells expressing CD25 (interleukin-2 receptor, IL-2R) and CD69 (early activation marker) were also higher in BL patients than in controls, though the differences were not statistically significant {Mean difference (95%CI): 3.28 (-0.72 to 7.27); (p(t)=0.104,1.26 (-0.23 to 2.74); p(t)=0.094 respectively} (figure 6). When the absolute numbers o f CD31" cells expressing the same activation markers were compared, the result was similar. The BL patients had significantly higher median number o f CD3+ cells expressing CD95 and HLA-DR {Median difference (95%CI): I2 .95x l05 (-l.0 8 x l0 5 to 27.83xl05), p(T)=0.035; 2.28xl05 ( I . l7 x l0 5 to I2 .30x l05); p(T)=0.026, respectively}than in healthy children. Based on comparisons o f the absolute number of cells, again, there were no statistically significant differences in CD25 and CD69 expression by the T-cells between the two groups. {CD25: Median difference (95%CI): 0 .49xl05 (-8.76105 to 3 .83xl05); higher in BL patients, p(T)= 0.805 and CD69: 0 .44xl05 (-0 .65xl05 to l.53x 105); lower inB L patients p(T)=0.40l} 50 University of Ghana http://ugspace.ug.edu.gh Figure 6. Frequencies of CD3+ T cells bearing various activation markers in BL Patients and Healthy Controls 60 □ BL Patients □ Healthy Controls 50 40 - 30 - 20 10 _ __T_■l ___ O h ____11 CD25 *CD69 CD95 H L A D R Activation Marker *Median values were used 51 % of all CD3+ Cells University of Ghana http://ugspace.ug.edu.gh 4. 7 y5+ T Cells are more activated than aB+ T cells in BL CD95iy8+and HLA-DR+y8+ T-cell frequencies were higher in BL patients than in controls {Mean difference (95%CI): 32.95 (17.04 to 48.86), p(/)<0.001; 30.42 (20.97 to 39.87), p(r )<0.001, respectively) } CD69+y5+ and CD25+y8+ T-cell frequencies were also higher in BL patients than in controls, though not significant {Mean difference (95%CI): 4.48 (-2.95 to 11.92), p(* )=0.221; 1.56 (-3.63 to 13.97) p(7)=0.431, respectively) } This shows that y8+ T were more activated in BL patients than in controls (Figure 7). Comparing the frequencies o f the activation markers (CD95, HLA-DR, CD69 and CD25) in CD3+ cells with those o f y5+ cells also showed that y8+ T cells were more activated than a(3+ T cells in BL patients but not so in the controls (figure 8). 52 University of Ghana http://ugspace.ug.edu.gh Figure 7. Frequencies of Gamma-Delta T Cells bearing various activation markers In BL Patients and Healthy Controls □ BL Patients ■ Healthy Controls 70 60 50 «0 30 20 10 zw CD25 CD69 CD95 HLA-DR Activation Marker 53 % of gamma-delta cells University of Ghana http://ugspace.ug.edu.gh Figure 8. Percentages of activation markers in Gam m a-delta cells com pared to those in CD3+ cells in BL patients □ Gamma-delta cells GI A ll CD3+ Cells CD25 CD69 CD95 HLA-DR Activation Marker 54 Percentage University of Ghana http://ugspace.ug.edu.gh 4.8 The ratio of CD4/CD8 in BL patients is higher than in healthy children The mean percentage o f CD4+CD3+ was significantly higher in BL patients than in controls {Mean difference (95%CI): 9.17 (3.21 to 15.12); p(t )=0.004} whereas the mean value o f CD8+CD3+ T cells was lower in patients compared to controls {Mean difference (95%CI): 7.49 (1.73 to 13.25); p(/ )=0.013}. Consequently, the mean o f CD4/CD8 ratio in terms o f percentages was significantly higher in patients compared to controls {Mean difference (95%CI): 1.30 (0.40 to 2.20); p(? )=0.006}. This trend is illustrated in figure 9 below. Figure 9. Frequencies of CD4+ and CD8+ cells in patients and controls 55 University of Ghana http://ugspace.ug.edu.gh 4. 9 Percentages of TCR-y5+ cells expressing the variable (W segm ents, V51 and Vy9, in BL patients and healthy controls The percentage o f V51+ y5+ T cells was higher in BL patients compared to controls {Mean difference (95%CI): 15.80 (0.22 to 31.38); p(f)=0.047} and conversely, the percentage o f Vy9+ y5+ T cells was lower in BL patients compared to controls { Median difference (95%CI): 36.34 (16.11 to 49.17); p(7)<0.001}. Figure 10 illustrates this. Figure 10. Frequecies of expression of TCR-gamma-delta variable (V)-segments, Vdeltal and Vgamma9 in BL patients and Healthy Controls □ BL ■ CONTROLS 60 50 0"5 S 40 © T J n E e1> 30 o s* 20 10 0 Vdeltal Vgamma9 TCR-gamma-delta chain 56 University of Ghana http://ugspace.ug.edu.gh 4.10 Plasma levels of cytokines 4.10.1 Tumour necrosis factor-abha (TNF-a) The median level o f T N F-a in peripheral blood as measured in the plasma by ELISA was significantly lower in BL patients compared to healthy controls {Median difference (95%CI): 101 (24 to 198) pg/ml, p(7)=0.002}. The distributions o f the plasma levels of T N F-a in study subjects are shown in the box plot (figure 11). Figure 11. TNF-alpha levels in plasma of BL patients and heathy controls Category 57 University of Ghana http://ugspace.ug.edu.gh 4.10.2 Interleukin-10 (IL-10') Plasma IL-10 was significantly higher in BL patients compared to healthy controls {Median difference (95%CI): 48(52 to 123) pg/ml, p(7)=0.042}. This is illustrated in figure 12. Figure 12. IL-10 levels in plasma of BL patients and healthy controls Category 58 University of Ghana http://ugspace.ug.edu.gh 4.11 Kinetics o f T N F-a and IL-10 secretion bv in vitro stimulated PBMC. To ascertain the best time point for measurement o f T N F -a and IL-10 in culture supernatants PBMC were cultured for Day 1, Day 3 and Day 6 in the presence o f malaria parasites and mitogens. In both BL patients and controls, peak T N F -a was produced within twenty-four (24) hours. No detectable levels o f T N F-a were found on Day 3. Similarly in, both BL patients and controls, IL-10 secretion generally declined from Day 1 to Day 6. The decline was very profound in PBMC from controls that were stimulated with phytohaematogglutinin (PHA) and purified protein derivative (PPD). Based on this, Day 1 measurements o f IL-10 were used. Figures 13 shows the trend o f IL-10 secretion. 59 University of Ghana http://ugspace.ug.edu.gh Figure 13. Kinetics of IL-10 production of lymphocytes of BL patients and healthy controls when stimulated with P. fa lc iparum m alaria parasites LPARB and UNSTIMBL are PBMC from BL patients stimulated with LPAR, and the unstimulated cells respectively, likewise LPAilCON and UNSTIMCON are PBMC from controls stimulated with LPAJR and the unstimulated cells respectively. 60 Median concentration (pg/ml) University of Ghana http://ugspace.ug.edu.gh 4.12 Cytokine levels in supernatants after in vitro stimulation. 4.12.1 TN F-a PBMC from BL patients secreted significantly much less T N F -a in response to LPAR, PHA and PPD compared to controls as measured in the supernatants {Median difference (95%CI): 500(88 to 1443) pg/ml, p(7)=0.007 for live parasites (LPAR); 429 (-271 to 1878) pg/ml, p(7)=0.050 for PHA and 1739 (598 to 2013) pg/ml, p(7)=0.007 for PPD}. There was no significant difference between the two groups with regard to secretion o f T N F-a by the unstimulated cells (UNSTIM). However, looking at the spread o f the box plot, it is obvious that unstimulated cells o f healthy children produce more T N F-a than cells from the BL patients. Figure 14 shows the distributions o f supernatant levels o f T N F-a inBL patients and their healthy counterparts. 61 University of Ghana http://ugspace.ug.edu.gh Figure14. TNF-alpha levels in supernatants Category 62 TNF-alpha oncentration (pg/ml) University of Ghana http://ugspace.ug.edu.gh 4.12.2 IL-10 PBMC from BL patients secreted significantly less IL-10 in response to PHA and PPD than cells from controls {Median difference (95%CI): 5961 (-) pg/ml, p (7)=0.016 and 1250 (-338 to 4883) pg/ml, p (r)=0.009, respectively }. With regard to the cells stimulated with LPAR and the unstimulated, there were no significant differences in secretion o f IL-10 between the two groups. The supernatant levels o f IL-10 in BL patients and their healthy counterparts are shown in figure 15. Figure 15. IL-10 levels in supernatants Category 63 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE DISCUSSION AND CONCLUSIONS 5.1 DISCUSSION The link between malaria and endemic (eBL) remains obscure, even though both diseases occur in the same areas o f the world. This study therefore sought to find out the role o f malaria in the pathogenesis o f eBL by comparing, with reference to controls, the characteristics o f the lymphocytes from eBL patients with regard to the proportions of lymphocyte sub-groups, expression o f lymphocyte surface and activation markers, and pro- and anti-inflammatory responses to Plasmodium falciparum malaria parasites, to the already established characteristics o f lymphocytes in P. falciparum m alaria in the same population. The distribution o f the tumour in the patients was as typical o f BL, affecting many organs o f the body. The jaw s and the abdomen are still the most frequently involved sites o f eBL (Burkitt, 1958, 1970; Burkitt and Wright, 1963). Although T cells play a central role in acquired cellular immunity, decreased levels o f T cells in malaria have been reported in several studies (Elhassan et al., 1994; Hviid et al., 1997; W orku et al., 1997). In the present study, there was a lower frequency o f T cells in BL patients as compared to controls. The absolute count of T cells in BL patients was also lower compared to the controls, although this was not statistically significant. This low frequency o f T cells may be partly due to increased frequency o f B cells in the BL patients. However, the fact that the absolute count o f the T cells in BL patients was also low implies that the low frequency o f T cells in BL patients may not be due to the high frequency o f B cells alone; 64 University of Ghana http://ugspace.ug.edu.gh other factors such as disease-induced reallocation and/or programmed cell death (apoptosis) o f T cells may be involved. Apoptosis may involve a variety o f mechanisms, including CD95 (APO-l/Fas)-mediated activation-induced cell death (Alderson et al, 1995; Dhein et al, 1995), Fas-independent activation-induced peripheral deletion as described in HIV'*' individuals (Katsikis et al., 1996), TN F-a-m ediated activation as found in glioma cells (Chen et al., 2002) or antibody ligation o f the TCR on activated T cells as observed in mice (Kishimoto and Sprent, 1999). The relatively high general activation o f T cells and high expression o f CD95 (apoptotic marker), both in frequency and absolute numbers in BL patients observed in the present study may suggest CD95 (APO-l/Fas)-m ediated activation-induced cell death (AICD) in T cells in acute eBL, though one cannot completely exclude disease-induced reallocation o f the cells away from the peripheral circulation. AICD is normal and natural because protective cellular immune response does not only involve activation and expansion o f cells but also apoptosis, during activation and effector activity, a phenomenon which is important in regulating cell numbers and ensuring homeostasis (Liu and Janeway, 1990). When cell numbers are reduced at the acute stage o f the disease, it may impair protective cellular immunity to the disease. TCR- y5+ cells, which in this study were found to have highly activated phenotype and expressed the highest frequency o f CD95, also showed the most dramatic reduction in frequency and absolute numbers even during acute stage o f the disease, further suggesting the involvement o f CD95 in T cell apoptosis in eBL. Although activation-induced cell death (AICD), involving CD95 as the cause o f decreased lymphocyte numbers in acute malaria has not been fully established, there is evidence to show that, at least, increases in peripheral CD95-induced apoptosis occur (Balde et al., 65 University of Ghana http://ugspace.ug.edu.gh 1995; Kem et al., 2000; Matsumoto et al., 2000; Toure-Balde et al., 1996). Thus the role o f malaria in the pathogenesis of eBL might be to complement the activation of lymphocytes due to EBV-infection and hence an elevation in the expression o f CD95 by T-lymphocytes, consequent deletion o f CD95+ T cells and reduction in lymphocyte numbers in individuals with underdeveloped immunity to P. falciparum malaria such as children. The y5+ cells are found to respond early and rapidly to certain bacterial and parasitic infections (Bom et al., 1999; Halary et al., 1999) and respond to various promiscuous and self-antigens (Hayday, 2000). Studies have also suggested their role as keeping surveillance on expansion o f B cells (Biggar et al., 1981). Based on these findings, it is believed that y5+ cells play an immuno-regulatory role in immune responses. The decrease in y5T cells in eBL observed in the present study may therefore adversely affect their role o f immune surveillance and regulation in eBL because the fast-growing tumour cells might overwhelm them. This may imply that not only is there a lack o f an effective control o f the abnormal proliferation o f the tumour cells but also protection against other infectious agents. The mean percentage o f CD4+CD3+ was significantly higher in BL patients than in controls whereas the mean value o f CD8+CD3+ T cells was lower in patients compared to controls. As a result the mean ratio of CD4 to CD8 was significantly higher in patients compared to controls. The rise in the CD4/CD8 ratio therefore was not only due to selective increases in CD4+ cells but also an accompanying decrease in CD8+ cells. The 66 University of Ghana http://ugspace.ug.edu.gh decrease in CD8+ cells may be due to an apoptotic deletion. A study has demonstrated elevated apoptosis in CD8+ cells upon recognition o f self-antigens presented on activated B cells (Bennett, et al., 1998), and the present study has also confirmed activation o f B cells in BL patients. B cell activation is also a characteristic o f m alaria and therefore recurrent malaria may speed-up the removal o f the CD8+ cells in children making them more vulnerable to eBL, since CD8+ cells or cytotoxic T lymphocytes (CTLs) are very vital in controlling diseases caused by intracellular infectious agents such as EBV. The low frequency o f CD8+ cells may also be due to loss o f CD8+ yS+ cells from the peripheral circulation as yS+ cell numbers declined, because about 30% o f the y8+ cells in BL patients were CD8+. This study has also shown that in eBL, majority and significantly higher (compared with controls) proportion o f y8+ cells are V81+ cells contrary to observed elevation of Vy9+ cells during EBV infection in humans in a population non-endemic for m alaria (De Paoli, 1990). This may imply that the higher proportion o f V81+ cells observed instead o f Vy9+ cells is due to malaria. Elevated level o f V51+ cells is known to be associated with endemicity and severity o f P. falciparum malaria (Hviid et al., 2000, 2001). None of the study subjects had clinical malaria, so the high proportion o f VS1+ cells observed may just be a confirmation o f the reported high proportion o f V81+ cells in healthy children from Ghana. V81+ cell expansion has also been reported in HIV infection (Autran, et al., 1989). However the prevalent rate o f HIV infection in Ghana and especially our study age group is negligible (<4%) (Dr. B. Q. Goka, pers. com.), and as such cannot be responsible for the difference. Activated B cells are found to be antigenic target o f V81+ 67 University of Ghana http://ugspace.ug.edu.gh cells (Halary et al., 1999). Dominance o f V51+ cells may therefore imply protective immune response to the tumour cells in eBL but in the face o f low levels o f y8+ cells their absolute numbers are much lower and they might be overwhelmed by any fast-growing tumour such as BL. Our stimulation assay shows that response o f PBMC from BL patients was remarkably low compared to the controls with regard to secretion o f TNF-a to LPAR and PHA. Cytokine secretion to LPAR represents malaria-specific response while PHA represents non-specific stimulation. This implies that in BL patients, both malaria-specific and non specific responses were low with respect to TNF-a production. Similarly, PBMC from BL patients produced significantly less IL-10 than controls when stimulated with PHA, again indicating low non-specific response in BL patients. The generally low levels o f TN F-a and IL-10 production PBMC o f BL patients may be due to many factors. But two important factors that cannot be overlooked are the observed low frequency o f T and/or low absolute numbers o f y8+ T cells in BL patients compared to controls and the fact these cells have a phenotype which indicates that they are poised to undergo AICD (Alderson et al., 1995). PBMC from BL patients were over-activated and expressed high levels o f the apoptotic marker (CD95), which may have affected their cytokine production upon stimulation. Various cell types are known to produce TNF-a but mainly monocytes/macrophages are responsible for TNF-a production. However, monocytes/macrophages require cytokine stimulation from T cells, which also secrete substantial amounts o f TNF-a. Therefore the 68 University of Ghana http://ugspace.ug.edu.gh low proportion o f T cells in PBMC from BL patients may account for the low level of TN F-a measured in the supernatants. A study has shown that lymphokines secreted by y8 T cells activate macrophages, the main producers o f TNF-a (Goodier et al., 1995). The reduction in the number of y8+ T cells observed in the present study may therefore reduce the function o f macrophages and TNF-a production in cells from eBL patients. On the other hand, it would be expected that cells from BL patients should produce more IL- 10 than those from healthy children. This is because BL is a B-cell tumour and as the present data have shown, with high levels of activated B cells, it is expected that there will be an increase in production of IL-10, B cells being a major source o f the cytokine. The low supernatant level o f IL-10 in eBL patients is therefore not clear but it may be accounted for by the same factors that explain the low level o f TNF-a such as low levels o f y8+ T cells numbers. There was slightly higher production o f IL-10 in response to stimulation with LPAR in both patients and controls compared to the unstimulated PBMC (Figure 16). Stimulation with P. falciparum schizonts-infected erythrocytes, therefore, seems to elicit production o f IL-10 in both groups. The elevated level o f IL-10 in response to malaria parasites is consistent with what other researchers have found. Studies have shown that stimulation o f lymphocytes with malaria antigens induces secretion of cytokines with Th 2 profile such as IL-10 (W ahlgren et al., 1995). This was also found in vivo (von der Weid and Langhome, 1993). A recent study has shown that malarial antigens stimulated PBMC, obtained from malaria patients at acute infection, to produce IL-10. When recombinant human IL-10 was added in vitro production o f TNF-a was completely abolished in response to malarial antigens (Ho et al., 1995). 69 University of Ghana http://ugspace.ug.edu.gh W hereas PBMC from patients produced significantly less amount o f TNF-a (even slightly lower than unstimulated PBMC from eBL patients) when stimulated with LPAR, with regards to the production o f IL-10 cells from the BL patients produced an amount sim ilar to that o f the controls. This implies that the capacity o f PBM C from eBL patients to produce IL-10 when challenged by P. falciparum m alaria parasites is not reduced significantly due to the disease. If the secretion o f TN F-a is significantly reduced in BL patients, as the present data suggests, that the shift o f the immune response towards the production o f anti-inflammatory cytokines (such as IL-10) at the expense o f pro- inflammatory cytokines (such as TNF-a) during P. falciparum m alaria may be more serious in eBL patients than the controls. Plasma levels o f IL-10 were significantly high in BL patients compared to controls. Conversely, plasma levels o f TNF-a were significantly low in eBL patients compared to controls. The higher plasma level of IL-10 supports the in vitro studies that seem to suggest that elevated level o f IL-10 is a characteristic o f eBL (Burdin e t al., 1993). An elevated production of IL-10 is not an anti-tumour response, rather IL-10 is known to enhance the growth o f B cells and hence the tumour cells. Any factor that contributes to elevation o f IL-10 level would also contribute to the growth and persistence o f the tumour. The high plasma level o f IL-10 and the response o f PBMC from the BL patients to P. falciparum malaria parasites indicate that whereas IL-10 level is already high, during P. falciparum malaria the level may be elevated. In this light, another contribution o f P falciparum malaria to the pathogenesis o f eBL aside activation o f lymphocytes and 70 University of Ghana http://ugspace.ug.edu.gh lymphopenia, may be skewing o f the immune responses toward production o f anti inflammatory cytokines through recurrent infection with P. falciparum malaria parasites. The low plasma levels o f TNF-a also indicate that the reduced number o f T cells observed is not due to TNF-a-mediated apoptosis. The cause o f low plasma level o f TNF- a may be multifactorial but the main factors could be downregulation o f its secretion by the high level o f IL-10, low T-cell frequency and absolute numbers o f y5+ T cells in particular. The hope o f controlling the expanding B cells and the fast growing tumour rests mainly with the activities o f CTLs (CD8+ cell), y5+ T cells (Biggar et al., 1981) and o f course TNF-a, a pro-inflammatory cytokine that is very vital for protective cellular immunity as it activates other cells o f the cellular immune system essentially CTLs. Unfortunately, as discussed earlier, there is reduction not only in T cell frequency but also in CD 8+ and y5+ T cells numbers in addition to low plasma levels o f TNF-a. Moreover, IL-10 is found to suppress the capacity o f CTLs in clearing EBV-infected and cancer cells (Chouaib et al., 1997; von der Weid and Langhome, 1993). This implies that the T- and y8-cell lymphopenia, low level o f TNF-a and high level o f IL-10 observed in BL patients have serious adverse effect on the cellular immune system as a whole with serious implications not only for the ability to mount protective response against BL but also other infectious agents. This turn o f events suggest that BL patients at acute stage cannot overcome the disease without medical intervention. This may account for the high death rate among the BL patients recruited for this study. 71 University of Ghana http://ugspace.ug.edu.gh 5.2 CONCLUSIONS The findings from the present study show that there is remarkable general activation of lymphocytes and high level o f circulating lymphocytes that express the apoptotic marker, CD95 in BL patients. The high expression o f CD95 is believed to be caused, at least partly, by P. falciparum malaria. The elevated expression o f the CD95 would lead to AICD, which may account for the low peripheral levels o f T cells and y5+ T cells in particular. This needs to be further investigated by looking at the expression o f the activation markers o f the lymphocytes from BL patients when they have malaria. These results also suggest that the shift o f the immune response towards production o f anti inflammatory cytokines is a characteristic o f eBL and that any factor that shifts the immune responses toward production o f anti-inflammatory cytokines such as P. fa lciparum malaria, will contribute to the development and persistence o f eBL. Our data also suggest that during P. falciparum malaria, the cells from BL patients are likely to produce, at least, as much IL-10 as cells from their healthy counterparts thereby contributing to the already high levels o f circulating IL-10 in the BL patients. This would adversely affect the capability o f the already beleaguered T cells to mount protective immune responses in the patients. However to fully unravel the trend o f the events mentioned, a longitudinal study is also recommended. 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