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.

IGM kappa/lambda EBV human B cell clone: an early step of differentiation of fetal B cells or a distinct B lineage?

In agreement with the clonal theory, one B lymphocyte synthesizes one antibody due to allelic and isotypic exclusion. We analyzed an EBV B-cell clone, E29.1, derived from an 11 week-old embryo, and secreting both IgM kappa and IgM lambda. Structural analysis of produced IgM, indicated that lambda-containing pentamers could be considered hybrid molecules, expressing both the kappa and lambda. chains, with a kappa/lambda ratio between 5 and 10. It was also found that 60% of the lambda chains were secreted in free form, presumably as a result of a better affinity of mu chains for kappa chains. The sequence of the three transcripts had an entirely ORF (Open Reading Frame), and were very close to germline sequences, with, however, an additional codon between V kappa and J kappa gene which has never been described in adult myeloma protein or cDNA human sequence. This observation is suggestive of N diversity taking place in kappa chains. The possible role of Kde (kappa deleting element) recombination onto kappa/lambda locus activation was analyzed on a collection of 23 lambda clones. The status of rearrangement of kappa genes indicated that 35% of these clones had retained, at least, one kappa allele without the Kde recombination, four lambda clones had one kappa allele in germline configuration. Different hypotheses of maturation from pre-B cell to B cell with activation of light chain genes are discussed.

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.

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.

Responses of central neurones to piribedil and 2-bromo-alpha-ergocryptine: comparison with dopamine and apomorphine.

The effects of two putative dopamine receptor agonists, piribedil and 2-bromo-alpha-ergocryptine were studied by means of microiontophoresis. Their effects were compared with those of dopamine and apomorphine on neurones of the frontal cortex and of the caudate nucleus both of which are rich in dopaminergic terminals. Piribedil and S. 584, its dihydroxyphenyl derivative, displayed a potent dopamine-like inhibition in both these areas. 2-Bromo-alpha-ergocryptine exerted significant inhibition on cortical neurones which were spontaneously active but only weak or negligible inhibition on cortical and caudate glutamate-driven units. These observations raise the possibilities that 2-bromo-alpha-ergocryptine (1) mediates its inhibitory effect via a predominant presynaptic action; (2) acts preferentially on cortical neurones.

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.

High density lipoprotein levels in the serum of trypanosensitive and trypanoresistant cattle. Changes during Trypanosoma congolense infection.

Nonpermissiveness to trypanosome infection has been correlated in some instances with the presence of toxic serum factors, e.g. high density lipoproteins (HDL) of human serum can lyse T.b. brucei. The present study examines the possibility of a role for such factors in West African cattle that are resistant to trypanosomiasis. Cattle used in this study were previously selected as resistant or sensitive to trypanosomiasis under heavy natural Glossina challenge. - A comparison of the direct effect of serum from trypanoresistant and trypanosensitive Baoulé cattle on the development of pathogenic bloodstream or metacyclic forms of T. congolense, using modifications of the blood infectivity incubation test, failed to demonstrate a difference between these cattle. High density lipoproteins and cholesterol levels were compared in 115 cattle of known sensibility to trypanosomiasis. HDL-cholesterol formed 91% of the total plasma cholesterol. HDL-cholesterol levels in Zebu (mean of 111.8 mg/100 ml) were significantly higher than those in Baoulé cattle (86.2 mg/100 ml). There was no significant difference, however, in these levels between trypanoresistant (73.4 mg/100 ml) and trypanosensitive (84.5 mg/100 ml) Baoulé. Alterations in HDL-cholesterol levels were monitored during an experimental cyclic infection with T. congolense in 5 Zebu and 9 Baoulé. HDL-cholesterol levels decreased in all animals concomitantly with the appearance of trypanosomes in the blood and returned rapidly to their starting values after parasite elimination following drug treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of serum zinc levels with resistance of cattle to trypanosomiasis.

Zinc, copper and magnesium levels were determined by atomic absorption spectrophotometry in the serum of 32 cattle (Zebus and Baoulés) which were proven to be sensitive to African trypanosomiasis under field challenge and 45 cattle (Baoulés and Ndamas/Baoulés) which were proven to be resistant. Copper and magnesium levels were similar in all animals but zinc levels were higher in sensitive animals (1.50 ppm) than in resistant ones (1.10 ppm) (p less than 10(-5)); the reported normal levels of serum zinc is 1.00 ppm. These differences persisted on repeated measurements and whether individuals were infected with trypanosomes or not. Elevated levels of zinc depressed the stimulation of bovine T cells by trypanosomes in vitro and is reported to inhibit antigen presentation by macrophages. Zinc levels may be an influencial factor determining susceptibility or resistance of West African cattle to trypanosomiasis.

Identification and selection of cattle naturally resistant to African trypanosomiasis.

Cattle were exposed to natural trypanosome challenge in an area of high Glossina density (Samandéni, Burkina Faso) for various periods of time during 1982, 1983, 1984 and 1985. All of 30 Zebu proved to be sensitive to trypanosomiasis i.e. they died or were treated in extremis in 10 +/- 4 weeks. Twenty-one (31%) Baoulé were as sensitive as the Zebu while 47 (69%) were resistant i.e. they survived in good condition. Twenty Ndama/Baoulé crosses, indigenous to Samandéni were all resistant. Weekly blood samples were taken (2,317 in total) for the determination of parasitaemia and packed cell volume (PCV) as a measure of anaemia, the most important pathological feature of cattle trypanosomiasis. In both Zebu and sensitive Baoulé 59% of the blood samples showed positive parasitaemia, of which 38% and 52% respectively were T. congolense the major cattle pathogen in the area considered. In resistant Baoulé and Ndama/Baoulé 11% and 10% of the samples were positive for trypanosomes of which only 4% and 2% were T. congolense respectively. PCV decreased from 35 to 20 in Zebu, 39 to 20 in sensitive Baoulé and 40 to 34 in resistant Baoulé, there was no change in the indigenous Ndama/Baoulé. Six Ndama/Baoulé indigenous to Samandéni remained resistant to trypanosomiasis when moved to another area of high Glossina challenge. Seven Ndama/Baoulé calves, conceived in Samandéni but born and kept for 2 1/2 years in a Glossina-free area, also proved to be resistant to challenge. Twelve Baoulé calves, born from cattle selected under natural field challenge, and which had not come in contact with trypanosomes for the first 10 months of their life, proved to be resistant when exposed in the field. These observations show that some, but not all, cattle from the Baoulé breed are naturally resistant to African trypanosomiasis, that this resistance does not need repeated exposure to trypanosomes early in life but appears to be inherited and functional against many types of antigenically different trypanosomes. Thus, selective breeding of trypanoresistant animals and their successful introduction, without trypanocidal drug protection, into areas of high Glossina density appears feasible.

The role of antibody in natural resistance to African trypanosomiasis.

The nature and usefulness of trypanoresistance in West African Baoule cattle was studied by exposing animals in areas of high Glossina density. Under such conditions, Zebu and some Baoule died soon with high parasitaemia while resistant Baoule showed little patent parasitemia, almost no anaemia and thrived. Subsequently the role that specific antibody may play in such trypanoresistance was analyzed. In the first instance we examined protective antibody titres during T. congolense infection of inbred mice strains, comparing results obtained in trypanosome-mouse combinations where the host controls parasitemia and survives with those obtained when the host fails to control parasitemia and dies. We then attempted to extend these observations to cattle by following the disease course and appearance of neutralizing antibodies in animals of known sensitivity to natural Glossina challenges, following artificial challenge with T. congolense infected Glossina.

Immune responses of trypanoresistant and trypanosusceptible cattle after cyclic infection with Trypanosoma congolense.

To study the mechanisms by which certain West African taurine cattle are able to resist trypanosomiasis, the disease course and several immune parameters were examined in eleven Baoulé and five Zebu cattle after infection with tsetse-transmitted T. congolense (clone 1180 of stock Serengeti/71/STIB/212). All animals showed a similar onset of parasitemia but subsequently a continuum of disease was observed ranging from four Baoulé which were mildly susceptible (controlled parasitemia by week 10 post-infection and had little anemia) to two Baoulé and two Zebu which were highly susceptible (unable to control parasitemia, severe anemia leading to death or drug treatment in extremis). The remaining five Baoulé and three Zebu showed intermediate susceptibility. Although the most resistant animals were of the Baoulé breed, there was a spectrum of susceptibility which crossed the two breeds. Neutralizing antibody to metacyclic trypanosome antigens was detectable by day 18 in four of the mildly susceptible and three of the highly susceptible individuals but such antibodies were delayed in the remaining one severely susceptible animal. Neutralizing antibodies to antigenic variants of the first peak of blood trypanosomes were of significantly higher titre and appeared earlier in the four mildly susceptible as opposed to the highly susceptible animals. No differences in lymphocyte in vitro mitogen responses were observed in these animals except shortly before death in those severely affected. In vitro lymphocyte responses to allogeneic cells did appear to correlate with disease severity, in that animals with mild disease showed little immunosuppression of this response whilst in severely affected individuals the response was almost ablated.

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

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.

[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.

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.

Mechanisms of self-cure from Trypanosoma congolense infection in mice.

The mechanism(s) of resistance to African trypanosomiasis caused by Trypanosoma congolense was investigated by using the Dinderesso/80/CRTA/3 isolate to which C57B1/6 are resistant (low parasitemia and self-cure) and BALB/c sensitive (high parasitemia and death). The resistance of C57B1/6 is similar to that found in some natural hosts of African trypanosomes such as certain indigenous West African cattle and wild Bovidae. The antibody response to epitopes exposed on the variant surface glycoprotein of a clone obtained from the Dinderesso/80/CRTA/3 isolate was measured by a complement-mediated lysis assay in C57B1/6 and BALB/c. After infections with 10(4), 10(5), or 10(7) motile organisms, antibody appeared in C57B1/6 4 to 8 days earlier than in BALB/c. Peak antibody titers were similar in both strains but were reached about 4 days earlier in C57B1/6. In this strain, antibody appeared during and controlled the first wave of parasitemia, whereas in BALB/c, parasitemia reached a plateau above 10(8) organisms per ml before antibody could be detected, and at this time the animals were dying. At peak antibody response, the proportion of immunoglobulin (Ig) M and IgG antibody was the same in both strains. The antibody response had the same kinetics in C57B1/6 and BALB/c after injection of 10(4), 10(5), and 10(7) lethally irradiated but intact parasites, but the peak titers were 10(3) to 10(4) times lower than after live challenge. The response to nonirradiated trypanosomes appeared to be T cell independent, because the antibody titers were the same in congenitally athymic nu/nu and normal C57B1/6, and no evidence for the induction of T cell activity could be demonstrated in the infected nude mice. A role for trypanolytic serum factors in resistance could not be demonstrated. The extent of immunosuppression after infection with nonirradiated organisms was compared in the two strains by measuring the in vitro response of their splenic lymphocytes to concanavalin A, pokeweed mitogen, and allogeneic cells and their ability to mount an in vivo response to an unrelated trypanosome challenge. Both strains were partially immunosuppressed during rising parasitemia, but as C57B1/6 controlled parasitemia, immunosuppression was gradually reversed, whereas in BALB/c it became worse. Several explanations might account for the resistance of C57B1/6 to the Dinderesso/80/CRTA/3 isolate of T. congolense. It appears that an early immune response is a decisive factor in this resistance.