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QR185.8 L9 F98 
bite C.l 
G370404
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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
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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 )
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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
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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.
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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(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.
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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
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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.
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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-
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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.
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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.
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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
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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)
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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)
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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.
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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.
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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).
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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.
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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
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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
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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).
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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).
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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,
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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).
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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.,
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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
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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
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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.
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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
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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).
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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-
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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
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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
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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.
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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
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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
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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
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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.
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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.
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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.
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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).
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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).
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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.
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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).
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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
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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
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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.
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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
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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.
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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.
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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
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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}.
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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}.
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Figure 4. Frequencies of CD3+ and B cells In gated events
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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
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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}
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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
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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).
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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
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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
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Percentage
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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
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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
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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
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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
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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.
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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.
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Median concentration (pg/ml)
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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.
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Figure14. TNF-alpha levels in supernatants
Category
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TNF-alpha oncentration (pg/ml)
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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
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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;
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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.,
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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
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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+
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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
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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).
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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
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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.
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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.
These imbalances in the immune system observed in eBL share some similarities with P. 
falciparum  malaria in terms of T- and B-cell activation, T-cell lymphopenia, B-cell 
expansion and an elevated production of IL-10. This confirms our hypothesis that 
recurrent P. falciparum  infection is an additive risk factor for the development of eBL.
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APPENDIX
To prepare a litre o f phosphate-buffered saline (PBS), the following reagents were used.
N aC l.....................................................8.0g.
KH2P 0 4............................................. 0.2g.
N a2H P 0 4.12H 20 ............................. 2.9g.
KC1....................................................... 0.2g.
102