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Immunologic characteristics of healthy and HIV-1-infected Ethiopians
Messele, T.
Publication date
2000
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Messele, T. (2000). Immunologic characteristics of healthy and HIV-1-infected Ethiopians.
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Immunologicc characteristics of healthy
andd HIV-1 infected Ethiopians
1011 102 103 104 CD38 8 1011 102 103 104 CD38 8 1011 102 103 104 C038 8
Tsehayneshh Messele
Immunologicc characteristics of healthy and
HIV-1-infectedd Ethiopians
Colophon n
©Tsehayneshh Messele, 2000. No part of this publication may be reproduced, storedd in a retrieval system, or transmitted, in any form or by any means, electronic,, mechanical, photocopying, recording, or otherwise, without prior permissionn of the author.
Thee studies described in this thesis were conducted at the Ethio-Netherlands AIDSS Research Project (ENARP) laboratory at the Ethiopian Health and Nutritionn Research Institute (EHNRl) in Addis Ababa, Ethiopia and at the Departmentt of Clinical Viro-lmmunology of CLB and the Laboratory for Experimentall and Clinical Immunology of the University of Amsterdam, Amsterdam,, The Netherlands.
Coverr illustration: Dot plots of activated and resting CD8+ T cell subsets, from too left to right, HIV-1 negative , HIV-1 positive asymptomatic as well as from an AIDSS patient as defined by CD38 and HLA-DR monoclonal antibody staining. Adaptedd from Chapter 3.
Printingg of this thesis was supported by: Universityy of Amsterdam
CLB B ENARP P
Immunologicc characteristics of healthy and
HIV-1-infectedd Ethiopians
ACADEMISCHH PROEFSCHRIFT
terr verkrijging van de graad van doctor
aann de Universiteit van Amsterdam
opp gezag van de Rector Magnificus
prof.. dr JJ.M. Franse
tenn overstaan van een door het college voor promoties
ingesteldee commissie, in het openbaar te verdedigen
inn de Aula der Universiteit
op p
dinsdagg 19 september 2000, te 10.00 uur
door r
Tsehayneshh Messele
Promotiecommissie: : promotor: : co-promotores: : leden: : prof.. dr F. Miedema drr D. Hamann drr T.F. RinkedeWit prof.. dr L.A. Aarden prof.. dr R.A. Coutinho prof.. dr J. Goudsmit prof.. dr J.M.A. Lange prof.. dr R.A.W. van Lier
r r
-CONTENTS S
Chapterr 1 General Introduction
Chapterr 2 Immunohaematological reference ranges for adult
Ethiopians s
Clin.Clin. Diag. Lab. Immunol., 1999, Vol. 6, 410-414.
29 9
Chapterr 3 Reduced naive and increased CD4 and CD8 cells in
healthyy adult Ethiopians compared to their Dutch counterparts s
Clin.Clin. Exp. Immunol., 1999, Vol.115, 443-450.
37 7
Chapterr 4 Plasma levels of viro-immunological markers as
surrogatee markers for HIV-1 disease progression in Ethiopians:: Correlation with cell surface activation markers
SubmittedSubmitted for publication
47 7
Chapterr 5 No difference in in-vitro susceptibility to HIV-1 between
high-riskk HIV-negative Ethiopian commercial sex workers andd low-risk controls
SubmittedSubmitted for publication
63 3
Chapterr 6 Reduced CCR5 expression in Ethiopian commercial
sexx workers with CCR2b-64l genotype
ManuscriptManuscript in preparation
83 3
Chapterr 7 Epilogue 93 3
Summary/Samenvatting g 103 3
Chapterr 1
Generall Introduction
Generall Introduction 11
HIV/AIDS S
Thee human immunodeficiency virus (HIV) which is the cause of acquired immuno-defficiencyy syndrome (AIDS) was first isolated in 19831"3. AIDS is the ultimatee clinical stage of infection by HIV and is characterized by opportunistic infectionss and specific malignant diseases in patients. HIV is an RNA virus and belongss to the family retroviridae, subfamily lentiviridae*. HIV-1 and HIV-2 are thee two known types of HIV with the former being distributed worldwide and the latterr found primarily in West Africa5"7. HIV-2 was found to be closely related to thee simian immunodeficiency virus (SIV)8,9. Although the degree of virulence mayy be less for HIV-2, both HIV-1 and HIV-2 are associated with AIDS2,3'10. Todayy most of the information on HIV has been obtained from studies on HIV-1.
Structurally,, HIV-1 is a spherical particle with a diameter of approximately 1000 nm. The outer membrane-protein of the virus covers the inner core, which containss the viral RNA, along with several copies of the enzyme reverse transcriptase11.. The envelope region of HIV-1 is highly variable. Based on the variationn in the larger outer membrane protein, gp120, HIV-1 at present is dividedd into 10 subtypes designated as subtypes A to J12,13. There is also a heterologouss group of viruses designated as subtype O, which do not match anyy of the subtypes described above14. The human CD4 molecule, which is a 55-kDD surface glycoprotein belonging to the immunoglobulin superfamilies, servess as the primary receptor for the virus to enter susceptible cells15"18. CD4 iss expressed primarily on helper T lymphocytes and also on cells of the monocyte/macrophagee lineage including dendritic cells, alveolar macrophages inn the lung and Langerhans cells in the skin19"24. It is now known that the CD4 moleculee is necessary, but not sufficient for HIV entry into target cells. Recently,, the chemokine receptors CCR5 and CXCR4 have been identified as thee major co-receptors of HIV-125"27. During infection, HIV first attaches to the CD44 molecule and the chemokine receptors on the cell surface28. This step is thenn followed by fusion of the viral and cellular membrane, resulting in the penetrationn of the virus core. Uncoating is followed releasing the virus RNA whichh is then reverse transcribed in to DNA by the viral reverse transcriptase. Thee DNA provirus is then transported to the nucleus and is integrated to the hostt cell genome with the help of virus-encoded enzyme, integrase29. The virus dependss on host transcription and translation factors for its replication. The synthesizedd virus genome and structural and regulatory proteins are transportedd and assembled at the cell membrane and progeny viruses bud fromm the cell membrane. A characteristic feature of HIV-1 infection is a highly variablee period between infection and development of AIDS. Although at the beginningg of the HIV epidemic it was thought that the viral load is low during the asymptomaticc period, it is now known that a large number of viruses are producedd per day throughout the infection period with continuous infection of neww cells resulting in gradual CD4 depletion and ultimately development of AIDS3031. .
122 Chapter 1
Thee global situation
Worldd wide, over 30 million people were infected with HIV by the beginningg of 1998 and 11.7 million people had already lost their lives to the disease.. HIV/ AIDS is among the top ten killers world wide32. Given the current speedd of the spread of infection, it is expected that it may even move to be amongg the top five killer diseases. Eighty nine percent of people with HiV live inn sub-saharan Africa and the developing countries of Asia. In Africa, nearly 21 millionn men, women and children are infected and AIDS has become one of the majorr causes of morbidity and mortality33.
Thee HIV problem in Ethiopia
Thee HIV/AIDS epidemic started relatively late in Ethiopia, the first HIV-1-positivee sera being detected in 1984 and the first AIDS patients reported in 198634.. However, since then the HIV-1/AIDS epidemic has spread to the entire countryy to reach a prevalence ranging 7-20% in the 15-50 year age groups in thee urban areas by 199835. The circulating subtype is C , which has been estimatedd to be the most prevalent (>48%) amongst HIV-1 -infected individuals inn the world 37.
HIVV infection and the immunological response
HIVV infection is characterized by dynamic and long-lasting interactions betweenn the virus and the immune system. Both humorai and cellular immune responsess are mounted to HIV infection.
Humorall immune response
Earlyy after infection, before seroconversion, there is high level viremia andd as this decreases a substantial antibody response, including an early and transientt IgM response and later a sustained IgG response specific for several proteinss of the infecting virus is seen38. The antibodies are directed to all the majorr antigens of the virus. As time goes on more broadly neutralizing antibodiess are produced. However, these antibodies do not prevent HIV diseasee progression and the generally accepted explanation for this phenomenonn has been the occurrence of antibody-escape mutant viruses39 partlyy because of the error-prone polymerase of the virus, which is thought to generatee an average of one point mutation in each genome copy40 and partly duee to mutations as a result of immune pressure.
Generall Introduction 13
Cell-mediatedd immune response
Cell-mediatedd immunity does play an important role in the immune defensess against intracellular pathogens such as viruses. Cytotoxic T lymphocytess (CTL) destroy infected cells after recognizing antigen presented by majorr histocompatibility (MHC) class- molecule on the surface of such cells. Highh frequencies of HIV-specific CTL have been detected in peripheral blood of HIV-infectedd patients41. The activity of CTL has been found to decline as HIV diseasee progresses42,43, and this has been also associated with impairment of IL-22 production and a reduced clonogenic potential of CD8+ T lymphocytes44,45. AA correlation has been observed between generation of strong CTL response andd persistence of an asymptomatic state in adults. Another study in infants bornn to HIV-positive mothers showed that seroconversion of the infants is associatedd with disappearance of HIV-specific CTL in peripheral blood46. Specificc CTL responses, but not humoral immune responses are detected in exposedd but uninfected individuals. Taken together, these observations suggestt that CTL responses play a significant role in protection against infection andd in the regulation of HIV disease. On the other hand, there are also a few studiess which suggested a negative role of CTL on the immune system during HIVV infection mediated by killing of infected cells and disruption of normal tissue architecture47"49.. Furthermore a non-Iytic antiviral response which is active againstt HIV-1, HIV-2 and SIV and which is not MHC-restricted was indicated in CD8++ T cells50,51. These cells are found at highest levels in asymptomatic individualss and which decreases with disease progression52. The non-cytolytic CD8++ T cells anti-HlV response, termed CD8+ T-cell anti-viral factor {CAF), was initiallyy identified to be composed of p-chemokines53. However, subsequent studiess have shown that the anti-viral response mediated by CAF can block bothh SI and NSI viruses54"56 and this effect cannot be suppressed by antibodiess directed to p-chemokines suggesting that CAF's activity is not due exclusivelyy to p-chemokines57"59.
Differentiatedd CD4 T-helper subsets, which are associated with distinct typess of immune response, have been identified. These T-helper subsets designatedd as T-helper 1(TH1) and T- helper 2 (TH2) were first described in mouse60.. A third subset designated as T-helper 0 (THO) with both Th1 and Th2 propertiess was also described in humans. Tn1 cells produce cytokines such as IFN-yy and IL-2 that increase cellular immune response, whereas Th2 cells producee cytokines such as IL-4, IL-5, IL-6 and IL-10 that enhance antibody production.. A change in the balance of production of especially IL-4 and IFN-y hass been observed in several disease conditions61"64. There are also reports suggestingg that a shift from Th1 to Th2 type of response contributes to the immunee dysregulation observed in HIV infection and progression to AIDS is dependentt on Th2 cytokine phenotype dominance65,66. In contrast, there is also aa study arguing for the absence of an in-vivo Th1 to Th2 phenotype shift with HIVV disease progression67.
144 Chapter 1
Viro-immunologicall markers of HIV disease progression
Thee course of HIV infection and development of disease is characterized byy a wide variation among infected individuals. Studies in the West indicated thatt approximately 5% of HIV-infected persons develop AIDS within three years, whereass approximately 12% of the HIV- infected persons are expected to remainn AlDS-free for more than twenty years68"71. In contrast, HIV disease progressionn in general is reported to be faster and the time of survival after the onsett of AIDS is shorter in African patients72,73. Identification of factors which aree changing with HIV infection and which predict and possibly contribute to the outcomee of the infection and which could be useful in designing therapeutic strategiess for appropriate patient care has been a major part of HIV research-Severall cellular and serologic markers have been seen to have a strong predictivee value for HIV disease progression. The level of HIV replication as expressedd by viral loads is believed to be important in the immunologic decline thatt characterize HIV disease and that have a major impact on the course of the disease74,75.. Some studies have suggested that plasma HIV-1 RNA levels are betterr predictors of progression to AIDS and patients with elevated plasma HIV-RNAA levels are at high risk of poor outcome76,77. Consistent with those findings,, it was reported that HIV disease progressors had significantly higher HIV-- RNA copy numbers than did either slow progressors or long-term asymptomaticc HIV-infected patients78. In contrast, a recent study by Rizzardini eff al on HIV-infected Africans shows that viral load is lower in the Africans comparedd to the Europeans, suggesting that HIV pathogenesis in this populationn is mainly immunopahtologically driven79. The emergence of more virulentt virus strains is also implicated to determine HIV disease progression80,81.. These strains have syncytium-inducing (SI) capacity are thoughtt to evolve from the non-syncytium-inducing (NSI) strains detected early att infection. As the disease progresses SI viruses appear in approximately 50%% of patients. The appearance of SI viruses seems, however, to be rare in HIV-11 subtype C infections and the majority of AIDS patients infected by these genotypee harbor only NSI viruses82.
CD44 T cells are the main targets of HIV infection and their depletion is thee hallmark of the deteriorating immune system83"85. The quantitative decline off CD4+ T cells expressed as an absolute number or percentage of total lymphocytess is frequently used for staging of patients. Apart from progressive declinee in the number of CD4+ T cells, HIV infection is also associated with changess in the representation of many different subset of T cells. Following seroconversion,, the number of CD4+ lymphocytes declines rapidly and less rapidlyy thereafter, while the number of CD8+ lymphocytes increases with similar kinetics86.. However, within the CD8 population there are also subsets which declinee in parallel with CD4+ subsets. Furthermore, expansion of both CD4+ andd CD8+ T-cell memory subsets, loss of naive CD4+ and CD8+ T cells 87,88, increasee of CD4 and CD8 subsets expressing certain cell surface antigens, especiallyy those reflecting immunologic activation have been implicated in HIV-infectedd persons from studies in Europe and North America89"92. The expressionn of activation associated cell surface antigens HLA-DR and CD38
Generall Introduction 15
especiallyy on CD8+ T cells increases dramatically with disease progression and havee been shown to have a prognostic value for AIDS93,94. In addition to the describedd quantitative changes, qualitative alterations of cells of the immune systemm are observed in HIV infection. A functional impairment of T cells from HIV-infectedd subjects can be detected early at infection. Loss or reduction of T-cel!! proliferative capacity to in-vitro stimulation is one of these qualitative changes95"97.. The loss of in-vitro recall antigen response is detected early at infection98.. Also, it has been reported that T-cell proliferation in response to stimulationn with CD3 and CD3+CD28 MoAbs decreases shortly after seroconversion,, before the decline in CD4+ T-cell number is observed99,100. The responsee to mitogens, such as phytohaemaglutinin (PHA), remains unaffected inn the early phases, but is significantly reduced later in infection101. It has also beenn demonstrated that loss of T-cell reactivity to CD3 and CD3+CD28 MoAbs
inin vitro is a strong predictive marker for progression to AIDS, independent of
decliningg CD4 counts100,102. Thus, T-cell proliferative capacity not only has beenn shown to be an important independent predictor of progression to HIV disease,, but also has been used to monitor immunological improvement after therapy103,104. .
Severall other potential factors, so called surrogate markers, including inflammatoryy cytokines have also been suggested for use in monitoring disease progressionn in HIV-infected patients. Neopterin, which is a metabolite of guanosinee triphosphate is produced by macrophages when they are stimulated byy interferon gamma from activated T cells1 ,106. The level of soluble interleukin-22 receptor reflects the activation of T cells and that of R2
-microglobulinn reflects lymphoid activation more generally107. The levels of B2
-microglobulinn and neopterin are elevated in HIV infection and strongly correlatedd with the risk of progression to AIDS86,108. Cytokines are integral componentss of the immune response and their role in HIV disease progression hass been extensively investigated109"112. Cytokines, such as IL-2 and IL-12, are cruciall for cell-mediated immunity113, whereas it is well documented that cytokines,, like tumor necrosis factor-a(TNF-a), upregulates HIV replication in bothh T lymphocytes and monocytes/macro-phages via activation of cellular transcriptionn factor N F - K B1 1 3
"1 1 6. Tumor necrosis factor a (TNF-a) has two specificc cell surface receptors and these two receptors, sTNFaRI and sTNFaRII aree released from cells as a result of high level of TNF-a and are detectable in solublee forms in body fluids117. Although sTNFaRII is not specific for HIV infection,, serum levels of sTNFaRII are a strong predictor for disease progressionn in asymptomatic HIV-positive persons118,11.
HIV-11 co-receptors and chemokines
Chemokinee receptors, which belong to a family of seven transmembrane spanningg G-protein-coupled receptors also serve as co-receptors for HIV-1 entry26,22 . It is now known that HIV-1 uses a number of chemokine receptors for itss entry. The CC-chemokine receptor, CCR5, is used by macrophage-tropic primaryy isolates, the viruses that predominate early in infection and are thought too be important for transmission of HIV-1. The CXC-chemokine receptor,
166 Chapter 1
CXCR4,, is used by T-cell-tropic or SI viruses that occur late during disease progressionn to AIDS25,120. HIV can also use other chemokine receptors, CCR2b andd CCR3, albeit to a lesser extent. Both CCR3 and CCR5 are expressed on microgliaa and it is suggested that both receptors are involved in HIV infection of thee central nervous system121,122. Bleul et al have shown that HIV co-receptors aree differentially expressed on human T lymphocytes. CXCR4 is predominantly expressedd on naïve T cells and CCR5 is mainly expressed on previously activatedd memory cells123. The expression pattern of the HIV-1 co-receptors of thee surface of CD4+ T cells is believed to have an influence on susceptibility of CD4++ T cells to HIV-1 infection, viral tropism and rate of disease progression124. AA study by Zhang et al indicated that CCR5 and CXCR4 serve as the major co-receptorss for different HIV-1 subtypes and co-receptor usage is determined by virall phenotype irrespective of viral genotype125. However, less frequent use of CXCR44 is reported for subtype C virus82.
Thee natural ligands of the HIV-1 co-receptors are chemokines, which are solublee factors thought to direct the migration of different leukocyte subsets to sitess of inflammation126. They can be subdivided into two groups. The a-chemokiness also known as CXC chemokines include SDF-1 and the p-chemokiness or C-C-chemokines include RANTES, macrophage inhibitory protein-1aa (MIP-1a) and MIP-1p. The CC-chemokines were shown to block infectionn of susceptible cells into vitro by macrophage-tropic primary HIV isolatess and SDF-1 was shown to inhibit T-cell-tropic viruses " .
I m m u n ee activation and HIV pathogenesis
Activationn of all components of the immune system is the major feature off HIV infection130,131. The activated state of the immune system is reflected by increasedd expression of antigens on cells which are otherwise expressed at a reducedd level on resting cells and by increased level of soluble proteins which aree released from activated cells132,1 .
Severall in-vitro studies have established the role of cellular activation in thee propagation of HIV infection in CD4 cells134,135. However, it seems paradoxicall that on one hand immune activation is most probably involved in thee immune control of HIV-1 infection4243 and, on the other hand, several lines off evidence support that activation of immune cells may lead to enhanced HIV-1
replication136"138.. Activated CD4+ lymphocytes are found to be more susceptible too HIV infection compared to their resting state139140 and in-vitro activation of
latentlyy infected lymphocytes triggers active viral replication141,142, in vivo, CCR55 is mainly expressed on memory or primed (CD45RO*) T cells123 and thesee cells are indicated to be selectively infected by HIV-1142. There is evidencee that CCR5 expression on this subset of cells is associated with HLA-DRR expression and increases with disease progression143.The strong associationn observed between the decline of CD4+ T cells and increased levels off activation markers on CD4+ T cells further support the view that cellular activationn promotes HIV disease progression. Another study by Weissman et ai demonstratedd that 100 times less virus is required to initiate HIV infection in cell culturee from an individual after immunization than before immunization,
Generall Introduction 17
indicatingg the potential contribution of cellular activation associated with an ongoingg antigen-specific immune response to the pathogenesis of HIV disease144.. It is suggested that activation of CD4+ T cells facilitates HIV infectionn in a number of ways either by enhancing HIV entry into the cell, triggeringg the completion of reverse transcription and viral integration or by stimulatingg viral transcription from provira! DNA145. In light of these observations,, chronic immune activation due to, among others, highly prevalent parasiticc infections has been suggested to explain, at least in part, the reported acceleratedd HIV disease progression in African patients146,147.
FinallyFinally there is evidence to support that there is persistent CD4+ and CD8++ T-cell activation in HIV-infected individuals, which provides an optimal environmentt for continuous HIV replication. CD4 and CD8 lymphocytes are cruciall for the maintenance of appropriate immunological response against a widee range of pathogens. In addition, CD4 cells secrete factors that affect the growthh and differentiation of lymphoid cells and haematopoietic cells and the functionn of non-lymphoid cells as well. Therefore, it is clear that quantitative or qualitativee abnormalities of the CD4 and CD8 populations, as a resuit of HiV diseasee and abnormal immune activation, can have profound effects on the immunee system function.
HIVV infection susceptibility/resistance
Fromm the observation that some individuals remain uninfected despite high-riskk exposure, it has been concluded that host factors exist that determine susceptibilityy to HIV infection. Studies conducted in various population groups, includingg commercial sex workers, discordant couples, infants born to HIV-infectedd mothers and exposed health care workers, have described several host factorss which possibly contribute to HIV infection resistance148"151.
CCR55 and CXCR4 are the main co-receptors used by HiV25120. Polymorphismm of the gene encoding CCR5 is one of the factors found to be associatedd with resistance150. The A32 base pair deletion mutation in the CCR5 genee is shown to result in a premature stop codon and loss of HIV-1 co-receptor activity.. However, some individuals with this mutation were found to be HIV-infected,, indicating that the protection conferred by this mutation is not absolute152,153.. In addition, HIV-1-infected individuals, heterozygous for the A32bpp deletion mutation, have been shown to have slower rates of CD4 decline,, have lower viral loads and survive longer compared to individuals with thee wild genotype154,155. Moreover, reduced cell surface expression of CCR5 hass been reported in individuals with heterozygous deletion compared to the wildd genotype group156. The A32bp deletion is common in Caucasians but rare inn Africans. Other mutations on the CCR5 than the A32bp deletion have also beenn reported. A mutation in the promoter region of the CCR5, CCR5P1, has beenn found to have a negative effect, in that infected individuals homozygous forr this mutation progress rapidly to AIDS157,158. In contrast, polymorphism of thee coding region of CCR2b has been suggested to be associated with a delay inn disease progression but not with reduced transmission risk159,160. This mutation,, CCR2-64I, changes valine to isoleucine and is linked to a CCR5
188 Chapter 1
promotorr polymorphisms (CCR5-59653T). It is shown that CCR2-64I effect on AIDSS progression is not mediated by a negative effect on the CCR5 co-receptor function,, although a slightly reduced expression of CCR5 is reported in individualss with CCR2-64) mutation161. Furthermore, a mutation on the 3 untranslatedd region of the SDF-1 chemokine gene (SDF1-3A) is indicated to be associatedd with rapid disease progression162,163. No polymorphism is reported forr CXCR4, which is mainly expressed on naive cells and is used by SI viruses.
Thee role of the cell surface expression levels of the co-receptors on cellularr susceptibility and tropism of the virus is also implicated . A correlation betweenn low expression of CCR5 and reduced infectability of T cells in vitro is alsoo reported164'165.
In-vitroIn-vitro studies have shown that the CC-chemokines RANTES, MIP-1a
andd MIP-1p can block the entry of macrophage-tropic viruses into susceptible cells127,128.. This inhibition is thought to be mediated by blocking env-driven HIV fusionn through competition for the chemokine receptors or receptor downregulation.. In relation to this, high level of chemokines was detected in exposedd but uninfected individuals149,1. Other host genetic factors have also beenn implicated to play a role and increased frequencies of certain HLA alleles weree detected in exposed but uninfected individuals166. Furthermore, acquired protectivee immunity after exposure to HIV was also suggested to play a role in HIV-11 infection resistance. HIV-1-specific cytotoxic T lymphocytes are detected inn exposed health workers, commercial sex workers and was indicated as one off the mechanisms of natural protective immunity167,168. From studies on commerciall sex workers in Kenya and Thailand mucosal immunity was also shownn to be highly associated with HIV-infection resistance168,169. HlV-specific mucosall IgA, in the absence of systemic IgG response, was detected in mucosall sites of high proportions of HIV-1 exposed but uninfected women. Thiss response was found rarely in HIV-infected women it's involvement in mediatingg protection against HIV infection. Recently, another study by Mazzoli
etet ai reported HlV-specific IgA in the serum of exposed seronegative partners of
HIV-seropositivee persons, implying the involvement of not only mucosal but also systemicc IgA-mediated immunity to HIV170.
E N A R PP a n d scope of this thesis
Thee Ethio-Netherlands AIDS Research Project (ENARP) is a collaborativee project between the governments of Ethiopia and the Netherlands. Itt started in 1994 with three main objectives:
i.. training of Ethiopian scientists ii.. capacity development and iii.conductingg research on HIV.
Thee project is based at the Ethiopian Health and Nutrition Research Institute (EHNRl)) in Addis Ababa and is supported by three research groups in Amsterdam,, the Netherlands: 1) The Division of Public Health and Environment, Municipall Health Service (Prof Roel Coutinho, Epidemiology); 2) Department of Viro-lmmunolgyy at CLB (Prof Frank Miedema, Immunology) and 3) The
Generall Introduction 19
Departmentt of Human Retrovirology, Acadamic Medical Center {Prof Jaap Goudsmit,, Virology). The project has established a well-equipped laboratory. ENARPP has also started a cohort study on HIV infection progression in two factories,, Akaki and Wonji, 20 km and 100 km away from Addis Ababa, respectively. .
Researchh for this thesis was conducted as part of the immunology research programm of ENARP.
Scopee of this thesis
HIVV infection is associated with several changes of the immune system. Inn chapter 2 CD4 and CD8 values as well as haematological parameters are comparedd between Ethiopian and other populations. Since the base line values off some of these parameters in Ethiopians were different in proportions and numberss compared to Dutch and other published values for Africans, immunohaematologicall reference ranges established for adult Ethiopians is shownn also in chapter 2. The representation of several T-cell subsets are studiedd in HIV-negative Ethiopians and compared to HIV-negative Dutch subjectss in Chapter 3. Furthermore, HIV- associated changes in these subsets aree analyzed in Ethiopians who are HIV-negative, HIV- positive with and without AIDSS in chapter 3. Chapter 4 levels of soluble viro-immunological markers in
HIV-infectedd and -non-infected Ethiopians are presented. In addition, the prognosticc value of these markers for HIV disease progression in Ethiopians is discussed. .
AA study on the existence of possible host factors associated to HIV infectionn resistance in high-risk HIV-1-negative Ethiopian commercial sex workerss is presented in chapter 5. Chapter 6 shows the expression levels of
co-receptorss in different CD4 and CD8 T-cell subsets of HIV-infected and -non-infectedd Ethiopian commercial sex workers. The co-receptor expression is correlatedd to a polymorphism in the CCR2b gene. In chapter 7, the work includedd in this thesis is discussed in relation to current literature.
References s
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200 Chapter 1
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Immunohaematologicall reference ranges for Ethiopians 31
Immunohematologicall Reference Ranges for Adult Ethiopians
ASTERR T S E G A Y E , ' TSEHAYNESH MESSELE. TESFAYE TILAHUN, ERMIAS HAILU, T E F E R AA S A H L U , R O N A N D O O R L Y , A R N A U D L. F O N T A N E T ,
ANDD TOBIAS F. RINKE DE WIT
Ethiopian-NetherlandsEthiopian-Netherlands AIDS Research Project, Ethiopian Health and NutritionNutrition Research Institute, Addis Ababa, Ethiopia
Receivedd 10 August 1998 Returned for modification 25 September 1998/Accepted 59 January 1999 AA cross-sectional survey was carried out with 485 healthy working adult Ethiopians who are participating in aa cohort study on the progression or human immunodeficiency virus type 1 (HIV-1) infection to establish hema-tologicall reference ranges fur adult HIV-negative Ethiopians. In addition, enumeration of absolute numbers andd percentages of leukocyte subsets was performed for 142 randomly selected HIV-negative individuals. Immu-nologicall results were compared to those of 1,356 healthy HI\-negative Dutch blood donor controls. Immuno-hematologicall mean values, medians, and 95th percentile reference ranges were established. Mean values were ass follows: leukocyte (WBC) counts, 6.1 * lO'/liter (both genders); erythrocyte counts, 5.1 x 1012
/liter (males) andd 4.5 x 10'Miter (females); hemoglobin, 16.1 (male) and 14.3 (female) g/dl; hematocrit. 4 8 3 % (male) and 42.0%% (female); platelets, 205 x 10'liter (both genders); monocytes, 343/u.l; granulocytes, 3,057/u.l; lympho-cytes,, 1,857/u.l; CD4 T cells, 775/(*l; CD8 T cells, 747/u.l; CD4/CD8 T-cell ratio, 1.2; T cells. 1,555/jil; B cells, 191/u.l;; and NK cells, 250/fj.l. The major conclusions follow, (i) The VVBC and platelet values of healthy HIV-negativee Ethiopians are lower than the adopted reference values of Ethiopia, (ii) The absolute CD4 T-cell counts of healthyy HIV-negative Ethiopians are considerably lower than those of the Dutch controls, while the opposite iss true for the absolute CDS T-cell counts. This results in a significantly reduced CD4/CDS T-cell ratio for healthyy Ethiopians, compared to the ratio for Dutch controls.
Hematologicall reference values for Ethiopians have never beenn established, although a few attempts at determining he-moglobinn and hematocrit levels in some populations have been madee ( 1 . 15, 22). T h e values which are currently used in the countryy are adopted from textbooks which refer mainly to Caucasiann subjects (24).
Similarly,, the immunological reference values used in Ethi-opiaa are derived from non-Ethiopian subjects. T h e need to estimatee Ethiopian immunological reference values, like those forr total lymphocytes and their subpopulations. has increased, especiallyy due to the importance of CD4 T cells in monitoring humann immunodeficiency virus (HIV) infection progression (8,, 10, 20). At the end of 1997, an estimated 2.5 x 10" Ethio-pianss were HIV infected, including 150.000 children (Ethiopi-ann Ministry of Health. 1998).
Severall factors, including genetics, dietary patterns, sex, age, andd altitude, affect immunohematological parameters (11, 24). Sincee these factors differ depending on the populations and geographicall areas studied, it is not surprising that sometimes radicall differences have been reported for i m m u n o h e m a t o -logicall parameters worldwide. For example, low CD4 T-cell countss in Asians (13) and Chinese (5. 6), low CD4/CD8 T-cell ratioss in Saudi Arabians (19), and leucopenia in Sierra Leo-neanss (18) have been observed. A recent study, though the sub-jectss were few, indicated low percentages of CD4 T cells and highh percentages of CD8 T cells in Ethiopians (25). Also, low CD44 T-cell counts in Ethiopian Jews in Israel were reported (16).. In contrast, the hemoglobin and hematocrit levels in Ethiopianss are reportedly high (I, 15, 22), most likely due to thee fact that the studied populations are living in the Ethiopian
TABLEE 1. Means, medians, and 95 ih percentile reference ranges of hematological parameters for 4S5 HIV-negative adult Ethiopians
Subjectt group {tj} aa nd parameter Malee (280) Meann Ï SD Median n 45rrr range Femalee COS) Meann SD Median n 955 ^e range "" All values in pareri theses s
WBCC count 11 in" liter) 6.00 t 1.8 5.9 9 3.0-9.S S 6,22 2.2 5.99 (0.99)" 3.0-12.2 2 aree f' values (Mann Whitncv v
RBCC cpunt (l')'-.lner) ) 5.11 ~ 0.4 5.0 0 4.3-5.9 9 4.55 * 0.4 4,5(0.000!) ) 3.7-5.2 2 UU test) for c o m p a r i s o n H e m o g l o b i n n le^ell (g'dl) 16.11 - 1.1 16.1 1 13.9-18.3 3 14.33 ~ 1.2 I4.4(0.<XX)1) ) 12.2-16.6 6
ill medians l o r male a ndd female
Hematocrit t I'VI I 48.33 i 3.4 48.2 2 41.6-55.1 1 42.00 3.2 42.11 (0.000!) 35.3-48.8 8 subjects. . Platelet t ( l l l " l i i 2077 i 203 3 97-3 3 ouni i erl l 62 2 4 4 2022 67 193(0.22) ) 98-352 2
** Cur responding author. Mailing address: Ethiopian Health and Nutritionn Research Institute (F.HSR1). P.O. Box 1242, Addis Ahaha, Ethiopia.. Phone: 251-1-757751, 251-1-130642, or 251-1-753330. Fax: 251-1-756329.. F.-mail: cnarp(«telecom.net.ct.
