Genome-wide significant linkage to IgG subclass responses against Plasmodium falciparum antigens on chromosomes 8p22-p21, 9q34 and 20q13.

A genome-wide scan was conducted for the levels of total immunoglobulin G (IgG) and IgG subclasses directed against Plasmodium falciparum antigens in an urban population living in Burkina Faso. Non-parametric multipoint linkage analysis provided three chromosomal regions with genome-wide significant evidence (logarithm of the odds (LOD) score >3.6), and five chromosomal regions with genome-wide suggestive evidence (LOD score >2.2). IgG3 levels were significantly linked to chromosomes 8p22-p21 and 20q13, whereas IgG4 levels were significantly linked to chromosome 9q34. In addition, we detected suggestive linkage of IgG1 levels to chromosomes 18p11-q12 and 18q12-q21, IgG4 levels to chromosomes 1p31 and 12q24 and IgG levels to chromosome 6p24-p21. Moreover, we genotyped genetic markers located within the regions of interest in a rural population living in Burkina Faso. We detected genome-wide significant and suggestive linkage results when combining the two study populations for chromosomes 1p31, 6p24-p21, 8p22-p21, 9q34, 12q24 and 20q13. Because high anti-parasite IgG3 and low anti-parasite IgG4 levels were associated with malaria resistance, the chromosomal regions linked to IgG3 and IgG4 levels are of special interest. Although the results should be confirmed in an independent population, they may provide new insights in understanding both the genetic control of IgG production and malaria resistance.

Evidence for epistasis between hemoglobin C and immune genes in human P. falciparum malaria: a family study in Burkina Faso.

Hemoglobin C (HbC) has been recently associated with protection against Plasmodium falciparum malaria. It is thought that HbC influences the development of immune responses against malaria, suggesting that the variation at the HbC locus (rs33930165) may interact with polymorphic sites in immune genes. We investigated, in 198 individuals belonging to 34 families living in Burkina Faso, statistical interactions between HbC and 11 polymorphisms within interleukin-4 (IL4), IL12B, NCR3, tumor necrosis factor (TNF) and lymphotoxin-α (LTA), which have been previously associated with malaria-related phenotypes. We searched for multilocus interactions by using the pedigree-based generalized multifactor dimensionality reduction approach. We detected 29 multilocus interactions for mild malaria, maximum parasitemia or asymptomatic parasitemia after correcting for multiple tests. All the single-nucleotide polymorphisms studied are included in several multilocus models. Nevertheless, most of the significant multilocus models included IL12B 3' untranslated region, IL12Bpro or LTA+80, suggesting that those polymorphisms play a particular role in the interactions detected. Moreover, we identified six multilocus models involving NCR3 that encodes the activating natural killer (NK) receptor NKp30, suggesting an interaction between HbC and genes involved in the activation of NK cells. More generally, our findings suggest an interaction between HbC and genes influencing the activation of effector cells for phenotypes related to mild malaria.

IL12B polymorphisms are linked but not associated with Plasmodium falciparum parasitemia: a familial study in Burkina Faso.

Chromosome 5q31-q33 has been linked to Plasmodium falciparum parasitemia in several independent studies. This chromosomal region contains numerous immunoregulatory genes. Among these, IL12B that encodes the p40 subunit of interleukin-12 (IL-12) appeared to be a promising functional candidate gene, and IL12Bpro, a promoter polymorphism, was associated with mortality from severe malaria in children. In this study, we characterized genetic variation in IL12B in 215 individuals belonging to 34 families and evaluated linkage and association of parasitemia with IL12B polymorphisms and haplotypes. We searched for IL12B polymorphisms in the coding regions and the corresponding intron-exon borders. We also examined IL12Bpro and IL12B 3'untranslated region (UTR) polymorphisms, which are thought to influence the production of IL-12. We showed a high level of conservation of IL12B-coding regions and identified five polymorphisms in introns and the two polymorphisms in the promoter and the 3'UTR regions. Although IL12B polymorphisms were linked to parasitemia, there was association of parasitemia with neither polymorphisms nor haplotypes. We cannot exclude that an unknown IL12B cis-regulatory element polymorphism affects both IL-12 production and parasitemia. However, our results suggest that genetic variation in IL12B does not explain differences in parasitemia in individuals living in an endemic area.

TNF as a malaria candidate gene: polymorphism-screening and family-based association analysis of mild malaria attack and parasitemia in Burkina Faso.

We have previously obtained strong evidence for linkage of mild malaria attack to the MHC region, with a peak close to the tumor necrosis factor (TNF) gene. We screened, for polymorphisms, the entire TNF gene in the same sample of 34 families comprising 197 individuals living in a Plasmodium falciparum endemic area and we found 17 polymorphisms. In a longitudinal study, we investigated whether the 11 most frequent and informative polymorphisms were associated with mild malaria attack and maximum parasitemia, which was the highest parasitemia in each individual over 2 years. Mild malaria attack and maximum parasitemia were positively correlated. Transmission disequilibrium tests showed nominal evidence for association between TNF-1031, TNF-308, TNF851 and TNF1304 polymorphisms, and mild malaria attack on the one hand, and between TNF-238, TNF851 and TNF1304 polymorphisms, and maximum parasitemia on the other hand. After accounting for multiple tests, we confirmed the association of TNF-238 with maximum parasitemia and the association of TNF1304 and TNF851 with maximum parasitemia and mild malaria attack. The association tests with mild malaria attack suggest a moderate effect of TNF-308 polymorphism. In conclusion, our study suggests that several TNF variants may be part of the genetic determinants for maximum parasitemia and/or mild malaria attack.

Ethnobotanical survey and in vitro antiplasmodial activity of plants used in traditional medicine in Burkina Faso.

In Burkina Faso, most people in particular, in rural areas, use traditional medicine and medicinal plants to treat usual diseases. In the course of new antimalarial compounds, an ethnobotanical survey has been conducted in different regions. Seven plants, often cited by traditional practitioners and not chemically investigated, have been selected for an antiplasmodial screening: Pavetta crassipes (K. Schum), Acanthospermum hispidum (DC), Terminalia macroptera (Guill. et Perr), Cassia siamea (Lam), Ficus sycomorus (L), Fadogia agrestis (Schweinf. Ex Hiern) and Crossopteryx febrifuga (AFZ. Ex G. Don) Benth. Basic, chloroform, methanol, water-methanol and aqueous crude extracts have been prepared and tested on Plasmodium falciparum chloroquine-resistant W2 strain. A significant activity has been observed with alkaloid extract of P. crassipes (IC(50)<4 microg/ml), of A. hispidum, C. febrifuga, and F. agrestis (4

Linkage and association between Plasmodium falciparum blood infection levels and chromosome 5q31-q33.

We have previously mapped a locus controlling Plasmodium falciparum blood infection levels (PFBI) to chromosome 5q31-q33. We genotyped 19 microsatellite markers on chromosome 5q31-q33 in a new sample of 44 pedigrees comprising 84 nuclear families and 292 individuals living in a P. falciparum endemic area. Using a nonparametric multipoint variance-component approach (by GENEHUNTER), we evidenced a peak of linkage close to D5S636 (P=0.0069), with a heritability of 0.46. Using a variance-component method for linkage-disequilibrium mapping of quantitative traits (by QTDT) and the Bonferroni correction for multiple testing, we further detected allelic association in the presence of linkage between blood infection levels and D5S487 (P=6 x 10(-5); P(c)=0.0011), which is located on the distal part of the peak. These results confirm the importance of chromosome 5q31-q33 in the genetic control of PFBI levels.

Familial correlation of immunoglobulin G subclass responses to Plasmodium falciparum antigens in Burkina Faso.

Host genes are thought to determine the immune response to malaria infection and the outcome. Cytophilic antibodies have been associated with protection, whereas noncytophilic antibodies against the same epitopes may block the protective activity of the protective ones. To assess the contribution of genetic factors to immunoglobulin G (IgG) subclass responses against conserved epitopes and Plasmodium falciparum blood-stage extracts, we analyzed the isotypic distribution of the IgG responses in 366 individuals living in two differently exposed areas in Burkina Faso. We used one-way analysis of variance and pairwise estimators to calculate sib-sib and parent-offspring correlation coefficients, respectively. Familial patterns of inheritance of IgG subclass responses to defined antigens and P. falciparum extracts appear to be similar in the two areas. We observed a sibling correlation for the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against ring-infected-erythrocyte surface antigen, merozoite surface protein 1 (MSP-1), MSP-2, and P. falciparum extract. Moreover, a parent-offspring correlation was found for several IgG subclass responses, including the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against conserved MSP-2 epitopes. Our results indicated that the IgG subclass responses against P. falciparum blood-stage antigens are partly influenced by host genetic factors. The localization and identification of these genes may have implications for immunoepidemiology and vaccine development.

High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human resistance to Plasmodium falciparum malaria.

There is accumulating evidence for a role of immunoglobulin G (IgG) in protection against malarial infection and disease. Only IgG1 and IgG3 are considered cytophilic and protective against P. falciparum, whereas IgG2 and IgG4 were thought to be neither and even to block protective mechanisms. However, no clear pattern of association between isotypes and protection has so far emerged. We analyzed the isotypic distribution of the IgG response to conserved epitopes and P. falciparum blood-stage extract in 283 malaria-exposed individuals whose occurrence of infection and malaria attack had been monitored for about 1 year. Logistic regression analyses showed that, at the end of the season of transmission, high levels of IgG2 to RESA and to MSP2 epitopes were associated with low risk of infection. Indeed, IgG2 is able to bind FcgammaRIIA in individuals possessing the H131 allele, and we showed that 70% of the study subjects had this allele. Also, high specific IgG4 levels were associated with an enhanced risk of infection and with a high risk of malaria attack. Moreover, specific IgG2 and IgG3 levels, as well as the IgG2/IgG4 and IgG3/IgG4 ratios, increased with the age of subjects, in parallel with the protection against infection and disease. IgG4 likely competes with cytophilic antibodies for antigen recognition and may therefore block cytotoxicity mediated by antibody-activated effector cells. In conclusion, these results favor a protective role of IgG3 and IgG2, which may activate effector cells through FcgammaRIIA, and provide evidence for a blocking role of IgG4 in malarial infection and disease.

[Analysis of the genetic factors controlling malarial infection in man]. / Analyse des facteurs génétiques contrôlant l'infection palustre chez l'homme.

Genetic factors have clearly been shown to play a role in controlling malarial infection in animal models. There is now also increasing evidence for the genetic control of malaria in man. We carried out a segregation analysis based on blood parasite load phenotype for a population of the town of Bobo-Dioulasso (Burkina-Faso). This analysis demonstrated a strong genetic effect. Our results were not consistent with the segregation of a major gene and thus suggest that parasite load is under the control of minor genes. The genetic effect was stronger in children than in adults. We carried out a regression analysis in children and found that there was an association between the phenotype for blood parasite load and the q31-33 region of chromosome 5. We identified a gene in this region, Pfil1 (Plasmodium falciparum infection levels 1), which accounted for almost 50% of the variance in blood parasite load and which played a fundamental role in the control of infection. The 5q31-33 region contains several genes encoding cytokines that regulate T lymphocytes. The identification of genes controlling malarial infection opens up new possibilities for preventive and treatment strategies. It should be possible in the near future to identify individuals at risk of malaria, who would derive the greatest benefit from preventive and therapeutic measures. Finally, a deeper understanding of these genes controlling protective immune responses could be of value for the development of vaccines.

Relationship between interleukin-5 production and variations in eosinophil counts during HIV infection in West Africa: influence of Mycobacterium tuberculosis infection.

Eosinophils are important effectors of the non-specific immune response and we studied whether perturbations in the production of the type 2 cytokine, interleukin-5 (IL-5), could account for the variations in eosinophil counts observed in human immunodeficiency virus (HIV) infection. HIV-infected patients without helminthiasis were investigated in a cross-sectional study in West Africa. Eosinophil counts were significantly higher in CDC-B patients than in controls, but were dramatically decreased at the CDC-C stage. Phorbol 12-myristate 13-acetate (PMA)+ ionomycin-induced IL-5 production by peripheral blood mononuclear cells (PBMC) was decreased from the A stage of the disease, and significant correlations were observed between IL-5 production and eosinophil counts in tuberculosis (TB)-negative HIV-1-positive, TB-positive HIV-1-positive and TB-positive HIV-negative patient groups. Nevertheless, the production of IL-5 was not decreased in HIV-positive patients with TB, in contrast to HIV-positive patients without TB presenting with the same ranges of CD4+ counts. Our data suggest that, during HIV infection, the impairment in IL-5 production is one of the factors associated with the 'paradoxal' eosinopenia observed in tropical areas, but that IL-5 production during active TB is compensated by cellular subsets, yet to be identified.

PIP: Eosinophils are important effectors of nonspecific immune response, with eosinophilia being a classic sign of helminthic infection, allergies, and some inflammatory processes. The authors explored whether perturbations in the production of interleukin-5 (IL-5) could account for the variations in eosinophil counts seen in HIV infection. The 491 study subjects were recruited between 1993 and 1995 in Bobo-Dioulasso, Burkina Faso. Eosinophil counts were significantly higher in CDC-B AIDS patients than in controls, but were dramatically lower among CDC-C stage subjects. Phorbol 12-myristate 13-acetate (PMA)+ionomycin-induced IL-5 production by peripheral blood mononuclear cells (PBMC) was decreased from the A stage of the disease, and significant correlations were observed between IL-5 production and eosinophil counts in tuberculosis (TB)-negative HIV-1-positive, TB-positive HIV-1-positive, and TB-positive HIV-negative patient groups. The production of IL-5 was not decreased among HIV-positive patients with TB, in contrast to HIV-positive patients without TB presenting with the same ranges of CD4+ counts. These data suggest that during HIV infection, impairment in IL-5 production is one factor associated with the paradoxal eosinopenia observed in tropical areas, but that IL-5 production during active TB is compensated by as yet unidentified cellular subsets.

Genetic dissection of Plasmodium falciparum blood infection levels and other complex traits related to human malaria infection.

There is accumulating evidence of host genetic control in malaria infection and, in humans, some genes have been associated with severe malaria. Nevertheless, other important genes controlling blood infection levels, malarial disease and immune responses are likely to be identified. In this paper, we focus on segregation and linkage analyses of blood infection levels in an urban population living in Burkina Faso. We found evidence of a complex genetic control and a linkage to chromosome 5q31-q33. The identification of genes controlling complex traits related to malaria infection should be helpful in understanding protective mechanisms and the relationship between infection, malaria attacks and severe malaria.

Human malaria: segregation analysis of blood infection levels in a suburban area and a rural area in Burkina Faso.

The genetic control of blood infection levels in human malaria remains unclear. Case control studies have not demonstrated a strong association between candidate genes and blood parasite densities as opposed to surveys that have focused on severe malaria. As an alternative approach, we used segregation analyses to determine the genetic control of blood parasitemia. We surveyed 509 residents (53 pedigrees) in a rural area and 389 residents (41 pedigrees) in an urban area during 18 months. Each family was visited 20 times and 28 times in the urban area and in the rural area; the mean number of parasitemia measurements per subject was 12.1 in the town and 14.9 in the village. The intensity of transmission of Plasmodium falciparum was 8-fold higher in the rural area than in the urban area. Using the class D regressive model for both populations, we found that blood parasite densities were correlated between sibs. We obtained strong evidence for a major effect, but we found that the transmission of this major effect was not compatible with a simple Mendelian model, suggesting a more complex mode of inheritance. Moreover, there was a strong interaction between major effect and age, suggesting that the influence of the putative major gene may be more prominent in children than in adults. Further nonparametric linkage studies, such as sib pair analysis, that focus on children would help us better understand the genetic control of blood infection levels.

Malaria in humans: Plasmodium falciparum blood infection levels are linked to chromosome 5q31-q33.

Plasmodium falciparum malaria remains a major cause of morbidity and mortality in many tropical countries, especially those in sub-Saharan Africa. Human genetic control of malaria infection is poorly understood; in particular, genes controlling P. falciparum blood infection levels remain to be identified. We recently evidenced the existence of complex genetic factors controlling blood infection levels in an urban population living in Burkina Faso. We performed, on 153 sibs from 34 families, sib-pair linkage analyses between blood infection levels and chromosome 5q31-q33, which contains numerous candidate genes encoding immunological molecules. Our results, obtained by means of the two-point Haseman-Elston (HE) method and a nonparametric (NP) approach, show linkage of parasitemia to D5S393 (P=.002) and D5S658 (P=.0004). Multipoint analyses confirmed linkage, with a peak close to D5S658 (P=.0013 and P=.0007 with the HE and NP methods, respectively). The heritability of the locus was .48, according to the two-point results, and .43, according to the multipoint results; this indicates that its variation accounted for approximately 45% of the variance of blood infection levels and that the locus plays a central role in the control of parasitemia. The identification of the gene is, therefore, of major interest in understanding the mechanisms controlling P. falciparum parasitemia.

Evidence for high genetic diversity and long-term endemicity of hepatitis C virus genotypes 1 and 2 in West Africa.

During 1994 and 1995, the prevalence of hepatitis C virus (HCV) and its genotypes were studied in several rural and urban populations in three West African countries: Guinea, Burkina Faso, and Benin. The following groups were screened for antibodies to HCV (anti-HCV): 459 villagers in the forest region of Guinea; 965 individuals in urban, suburban, and rural populations of the Bobo Dioulasso area, Burkina Faso; and 582 blood donors in Cotonou, Benin. In Benin, 60 patients with sickle cell anemia (30 with and 30 without history of multiple transfusion) and 13 hospital patients with liver disease were also tested. RT-PCR detection of HCV-RNA was carried out on all anti-HCV positive samples, followed by genotyping and sequencing of unrecognized subtypes. The prevalence rates of anti-HCV were 1.1% in the Guinean population group, 1.4% among blood donors in Benin, and 4.9% in residents of Burkina Faso. In patients with sickle cell anemia, five of the 30 polytranfused patients (17%) had anti-HCV, whereas none of the patients without a history of blood transfusion had anti-HCV (P < 0.05). Among the 13 patients with liver disease, five had anti-HCV, of whom four had history of blood transfusion. HCV-RNA was detected in 41 anti-HCV positive sera. All belonged to genotypes 1 or 2, with a high genomic diversity; 18 different subtypes were identified, including 2c, 2d, and 16 new subtypes. Such genetic diversity poses a challenge for vaccine development and also implies that HCV infection is long-established in these West African regions.

Lack of direct correlation between CD4 T-lymphocyte counts and induration sizes of the tuberculin skin test in human immunodeficiency virus type 1 seropositive patients.

SETTING: The study was conducted in Bobo-Dioulasso, Burkina Faso, where Mycobacterium tuberculosis infection and human immunodeficiency virus type 1 (HIV-1) infection are prevalent. OBJECTIVE: To identify proportions of representative (test) populations who are reactive to the tuberculin skin test, and to study the relationship between CD4 T-lymphocyte counts and the induration size of the tuberculin skin test in these groups. DESIGN: A group of 435 healthy students was tuberculin skin tested in order to evaluate the intensity of skin testing in a 'normal' population. The study group consisted of 195 subjects with or without tuberculosis, and with or without HIV-1 infection, who received a tuberculin skin test and a CD4 T lymphocyte count on the same day. RESULTS: In total, 90% of the control (nontuberculous, HIV negative) subjects, 32% of the HIV-1 seropositive subjects, 76.5% of the tuberculous patients and 57% of the tuberculous HIV-1 seropositive patients were tuberculin positive. There was no direct correlation between the induration size of reactions to the tuberculin skin test and CD4 T-lymphocyte count in these study groups using linear regression analysis. CONCLUSION: In vivo skin testing using tuberculin yields clinically significant information on the degree of immunodeficiency which is different from that of CD4 T-lymphocyte counts. The tuberculin skin test should therefore be used as an independent marker of the weakened immunological status of HIV-1 seropositive subjects.

Immunoblot analysis for serodiagnosis of tuberculosis using a 45/47-kilodalton antigen complex of Mycobacterium tuberculosis.

We evaluated the immunoglobulin G (IgG) antibody response to the 45/47-kDa secreted protein of Mycobacterium tuberculosis by immunoblot assay, to assess its potential value for serological diagnosis. Control subjects consisted of healthy volunteers with negative or positive tuberculin skin tests. Most (>98%) scored negative in an immunoblot test when the sera were analyzed at a 1:400 dilution. Approximately 40% of sera (diluted 1 in 400) from tuberculous patients (positive smears) recognized the antigen complex. The sensitivity of the test for patients suffering from extrapulmonary tuberculosis was similar to that for patients suffering from pulmonary tuberculosis but who had negative smears. The frequency of positive reactions among the patients suffering from other pulmonary diseases was similar to that among the control subjects. In tuberculous patients infected with human immunodeficiency virus, the sensitivity of the immunoblot test was significantly lower. Thus, this test based on an antigen complex used in an immunoblot assay to detect the presence of IgG antibody has a specificity of 98% and a sensitivity of 40%. The simultaneous use of different purified antigens, selected at the same high specificity level, may improve the sensitivity of such an assay.

The human pre-B cell receptor: structural constraints for a tentative model of the pseudo-light (psi L) chain.

In human pre-B cells, the mu chain is associated with a surrogate light chain composed of the lambda-like and Vpre-B gene products. This pre-B cell receptor presumably triggers early steps of B cell differentiation, We have determined the NH2-terminal amino acid sequence of the lambda-like chain, showing that the mature chain results from the cleavage of a leader segment of 44 residues, leaving a polypeptide of 169 amino acids having partial features of the Ig light chain domains, with the exception of the first 50 amino acid NH2-terminal region. We have completed the nucleotide sequence of the Vpre-B gene, which appears to contain 126 residues in its mature form of which the 24 COOH-terminal portion was not Ig-related. Analysis of transfectants has provided direct evidence that lambda-like and Vpre-B chains assemble together even in the absence of heavy chain, prompting the search for a structural basis of this interaction. Comparison with the domain organization of the regular Ig lambda chain suggests that most of the psi L chain can be accommodated within a CL-VL-like structure, with an extra "subdomain" contributed by the non-Ig-like portions of both the lambda-like and Vpre-B polypeptides.

Discrete early pro-B and pre-B stages in normal human bone marrow as defined by surface pseudo-light chain expression.

Vpre-B and lambda-like genes are selectively expressed in B cell precursors and encode polypeptide chains associated in a mu-pseudo light chain (mu-psi L) complex which is thought to regulate some early steps of B cell differentiation. We have generated anti-Vpre-B monoclonal antibodies which allowed us to identify different steps of differentiation from the pro-B to the immature B cells by following surface expression of Vpre-B, mu and light chains in normal adult human bone marrow. Already present at the surface of a small fraction of B cell progenitors (CD34+/CD19+) the Vpre-B molecule was consistently found coexpressed with CD19 and was also found with the sequentially occurring CD10, CD20, CD21, CD22 and CD5 markers. Three discrete cell types were identified: (i) a subpopulation expressing Vpre-B without mu and which represented an early stage of differentiation, (ii) a minor subpopulation co-expressing Vpre-B and mu without the conventional light chains and (iii) a major subpopulation co-expressing Vpre-B, mu and kappa or lambda chains, considered an intermediate pre-B/B stage. The presence of the psi L chain in various cell subpopulations, in possible association with discrete molecules and/or different contexts, suggests its involvement at several steps of early B cell differentiation.

Reconstitution of human immunoglobulin VH repertoire after bone marrow transplantation mimics B-cell ontogeny.

Reconstitution of the human Ig repertoire after allogeneic and autologous bone marrow transplantation (BMT) was followed by analysis of the expression of the six VH families using in situ hybridization on BM cells at 30, 60, and 90 days postgraft. Our results indicate that during the early post-transplantation period, the expressed repertoire of VH is dramatically affected: VH3, the major expressed family in adult B cells, is decreased two- to threefold and is compensated by transient overexpression of the other families, especially VH4, VH5, and VH6. Similar results were observed in allogeneic and autologous grafted patients. Kinetics of reconstitution mimics normal repertoire development described in ontogeny, although normalization, as compared with the adult pattern, may take as long as 1 year.