Genetic localization of a locus controlling the intensity of infection by Schistosoma mansoni on chromosome 5q31-q33.

Three hundred million individuals are at risk of infection by schistosomes and around 200,000 die each year of this disease. Severe clinical disease in schistosomiasis is often the consequence of heavy infection which, in several endemic areas, are determined largely by the susceptibility/resistance of individuals. Previously, we reported evidence, based on a segregation analysis in Brazilian pedigrees, that intensity of infection by Schistosoma mansoni was influenced by a major gene, indicating that host genetic factors are probably critical in controlling schistosome infection and disease development. To localize this gene, referred to as SM1, we performed a genome-wide study on 142 Brazilian subjects belonging to 11 informative families Our results show a linkage to only one region, on chromosome 5q31-q33, with maximum two-point lod scores of +4.74 and +4.52 for D5S636 and the colony stimulating factor-1 receptor marker (CSF1R), respectively. This was corroborated by multipoint analysis, indicating a close proximity to CSF1R as the most likely location of SM1. This region contains several candidate genes encoding immunological molecules that were shown to play important roles in human protection against schistosomes.

The major parasite surface antigen associated with human resistance to schistosomiasis is a 37-kD glyceraldehyde-3P-dehydrogenase.

Schistosomiasis, due to Schistosoma mansoni, is a major health problem in many subtropical countries, and major efforts are being made to define a vaccine. In this regard, we have reported that sera from subjects with low susceptibility to infection by S. mansoni react with a major larval surface antigen (P-37), having an apparent molecular mass of 37 kD, against which sera of susceptible individuals show little reactivity. We have now cloned the cDNA for this antigen by screening a schistosome cDNA expression library with antibodies against the purified protein. The selected cDNAs encode a protein that is specifically identified by immune human sera containing antibodies against P-37, while sera exhibiting low or no reactivity toward P-37 fail to recognize the recombinant protein. The cloned cDNAs hybridize with a 1.2-kb RNA that is the transcript of a single copy gene. This RNA directs the synthesis of a 36.5-kD polypeptide that is precipitated by sera from the most resistant subjects. The amino acid sequence of the encoded polypeptide shows homology with the glycolytic enzyme Glyceraldehyde-3P-dehydrogenase (72.5% of positional identity with human Glyceraldehyde-3P-dehydrogenase). Antibodies against the recombinant protein identified P-37 on the larva. These findings, together with other reports, indicate that a number of conserved proteins may be major targets of host-protective immunity against S. mansoni. The hypothesis is discussed that genetic restriction of the immune response to these antigens may occur in heterogeneous human populations because of the limited number of T cell epitopes carried by these host-like proteins. Such genetic effects might allow parasite transmission through nonresponder (susceptible) individuals. This hypothesis and the protective properties of P-37 can now be tested using the recombinant protein and synthetic peptides derived from selected regions of the polypeptide chain.

Enhancement of human blood eosinophil cytotoxicity by semi-purified eosinophil colony-stimulating factor(s).

Purified human blood eosinophils, when incubated in human placental conditioned medium (a source of colony-stimulating factors) [CSF]) demonstrate an enhanced ability to damage antibody- or complement-coated schistosomula. This enhancement represents a 4- to 10-fold increase of eosinophil schistosomicidal ability and a 10-fold lowering of the threshold for antibody or complement required in the killing reaction. The activity that enhances eosinophil cytotoxicity and the eosinophil colony-stimulating activity in the placental conditioned medium are eluted in the same fraction (CSF-alpha) after chromatography on Sephadex G-100 and phenyl-Sepharose columns, suggesting that these two activities might be associated with the same molecule. CSF-alpha enhances the adherence step of the killing reaction: antibody-coated larvae were frequently found covered by several layers of eosinophils in tubes containing CSF-alpha. Such a degree of adherence was rarely seen in control tubes lacking CSF-alpha. This enhancement of the eosinophil adherence is detectable 45-60 min after addition of CSF-alpha to the culture. It is not affected by washing the cells after a short time of preincubation with CSF-alpha, and it occurs in the absence of protein synthesis, whereas colony-stimulating activity requires continuous protein synthesis and ceases when CSF is removed from the culture. Finally, CSF-alpha enhances the temperature-dependent reaction that insures the irreversibility of eosinophil attachment to schistosomula. These observations suggest that eosinopoietic factors could be responsible for some of the modified properties of blood eosinophils in eosinophilic individuals.

IgE antibody and resistance to infection. I. Selective suppression of the IgE antibody response in rats diminishes the resistance and the eosinophil response to Trichinella spiralis infection.

Selective suppression of the total IgE antibody response has been achieved in rats by injection of rabbit anti-rat epsilon-chain antibodies. This IgE-specific suppression was maintained during the course of a natural infection by the nematode Trichinella spiralis. Depletion of the IgE antibody response resulted in a marked reduction of the number of eosinophils attracted to the T. spiralis larvae encysted in striated muscle. Blood eosinophilia following T. spiralis infection, although reaching normal peak levels, was abbreviated in IgE-suppressed animals. Moreover, IgE-depleted animals were more susceptible to the infection; they harbored two to three times more larvae encysted in their muscles than their control litter mates.

The impact of host genetics on susceptibility to human infectious diseases.

The development of genetic epidemiology methods using recent human genetic mapping information, together with the growing availability of candidate genes, has led to major advances in the identification of host genes involved in human infectious diseases. Within the past year, highlights include the mapping of a locus controlling the intensity of infection by Schistosoma mansoni, the demonstration that mutations in the interferon-gamma receptor 1 gene are causative of disseminated infection due to weakly pathogenic mycobacteria, and the identification, in the CCR5 gene, of a deletion which provides high protection against HIV-1 infection. The impact of these findings on the understanding of infectious disease pathogenesis and on the design of future preventive and therapeutic strategies should be considerable.

Characterization of a schistosome T cell-stimulating antigen (Sm10) associated with protective immunity in humans.

Parasite antigens that are strong T cell immunogens represent potential candidates for vaccines against pathogens susceptible to T cell-mediated immunity. We have previously shown that chromatographic fractions of schistosomula extracts contain components that are major T cell immunogen(s) in natural schistosome infections in humans and might contribute to the induction of human protective immunity against this parasite. In the present study, we report on the molecular cloning and on the biochemical characterization of the active components of these fractions. The screening of a schistosomula cDNA expression library with antibodies raised against the fractions allowed the cloning of a cDNA that hybridized to a 0.56-kb mRNA of schistosomula and adult worms. This cDNA contains an open reading frame of 267 base pairs (bp) which encodes a 10-kDa polypeptide. The analysis of the cDNA sequence revealed 70% homology with the sequences of previously reported proteins of unknown function. The native molecules in the active fractions were analyzed by mass spectrometry after additional purification by reverse phase high-performance liquid chromatography (HPLC). This procedure revealed two components in the fractions of molecular mass 10383 +/- 2 Da and 10401 +/- 9 Da. Both polypeptides stimulated immune T cells and yielded tryptic peptides whose sequences matched the sequence of the cloned molecule. These two polypeptides probably correspond to different post-translationally modified forms of the polypeptide encoded by the cloned cDNA.

Contrasting cellular responses in Schistosoma haematobium infected and exposed individuals from areas of high and low transmission in Zimbabwe.

The study compared cytokine profiles of individuals from two areas with different transmission patterns for Schistosoma haematobium. One area was a high transmission (HT) while the other was a low transmission (LT) area for S. haematobium. Observations on cellular immune responses were made on stimulated peripheral blood mononuclear cells (PBMC), which were collected pre-treatment, then at 12 and 18 months post treatment. Stimulation was with schistosome worm and egg antigens and a mitogen, phaetohaemaglutinin (PHA). Observations were made on PBMC proliferation and the profiles of cytokine produced over a 5-day incubation period. The two distinct areas showed significant differences on both levels of proliferation and cytokine production for all the measured classes (IL-4, IL-5, IL-10 and IFN-gamma). PBMC from individuals from the LT area had high levels of proliferation but low cytokine production to both antigen stimulants while PBMC from individuals from the HT area showed low levels of proliferation but high cytokine production levels. Prior to treatment, individuals not excreting schistosome ova in the HT area had higher levels of proliferation to the stimulants, than the infected individuals. However, after treatment re-infected individuals showed high levels of proliferation. Before treatment, both infected and uninfected groups showed low and similar ratios, respectively, of IL-4:IFN-gamma, IL-5:IFN-gamma and IL-10:IFN-gamma, while IFN-gamma was high in the infected individuals. After treatment the non re-infected had higher levels of IL-4, IL-5 and IL-10, with the infected having high levels of IFN-gamma. Th1-like response dominated during infection with the Th2-like responses dominating post treatment and in uninfected individuals. The results indicated that the cytokine balance determines, in part, susceptibility or resistance to S. haematobium infection.

Linkage analysis of blood Plasmodium falciparum levels: interest of the 5q31-q33 chromosome region.

There is accumulating evidence for the involvement of genetic factors in the human response to malaria infection, mostly based on results obtained in studies of severe clinical malaria. The role of major gene(s) controlling blood parasitemia levels in human malaria has also been detected by means of segregation analysis. To confirm and to localize such gene(s), we performed a sib-pair linkage analysis investigating the role of five candidate chromosomal regions: 6p21 (HLA-tumor necrosis factor region), 2q13-q21 (genes coding for interleukin-1 alpha and beta), 14q11 (locus coding for the alpha chain of T cell antigen receptor), 7q35 (gene cluster for the beta subunit of T cell receptor), and 5q31-q33, which includes several candidate genes and was recently linked to a locus controlling infection levels by Schistosoma mansoni, denoted as SM1. The analysis was carried out on nine families from a southern Cameroon village, and the phenotype under study was blood infection levels with Plasmodium falciparum. No linkage was found with any of the four markers outside the 5q31-q33 region. A trend in favor of linkage was observed in the distal part of the 5q31-q33 region, especially with the marker D5S636 (P < 0.05 using the Monte Carlo P value), which was the marker that provided the highest evidence for linkage with SM1. These results suggest that a locus influencing P. falciparum levels in malaria could be located in the same genetic region as that containing SM1, indicating that the 5q31-q33 region may be critical in the control of different parasite infections.

Antileishmanial antibodies in an outbreak of visceral leishmaniasis in eastern Sudan: high antibody responses occur in resistant subjects and are not predictive of disease.

A 3-year longitudinal survey was carried out from 1998 to 2000 in a village in eastern Sudan where a visceral leishmaniasis (VL) outbreak occurred. Leishmania-specific antibodies were analysed by enzyme-linked immunosorbent assay and immunoblotting. Immunoblot analysis detected antibodies to Leishmania in 80% of the healthy subjects and half of them harboured high immunoglobulin (Ig) G antibody levels, similar to those of VL patients. These antibodies belonged to the IgG1 and IgG3 subclasses but neither their respective levels nor the immunoblot recognition patterns were predictive of VL. During this epidemic, a large proportion of subjects had a high antileishmanial antibody response, indicating that they were infected by Leishmania though most of them remained healthy during the whole study period. These results obtained in the context of an outbreak contrast with those obtained from studies performed in endemic areas characterized by lower parasite transmission levels. Furthermore, the clinical and serological follow-up of our study subjects showed that VL occurred mainly in subjects who had been serologically positive for 5-24 months rather than resulting from primo infection by the parasite.

Identification of a novel G245R polymorphism in the IL-2 receptor beta membrane proximal domain associated with human visceral leishmaniasis.

Binding of the interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) triggers a series of intracellular events culminating in lymphocyte proliferation and differentiation. We report here the identification of a novel G245R polymorphism in the membrane proximal domain of the IL-2 receptor beta chain (IL-2Rbeta). Present at a frequency of 7.2%, the IL-2-Rbeta G245R was identified in a population of Eastern Sudan exposed to a severe outbreak of visceral leishmaniasis (VL), a disease associated with a marked depression of T-cell antigen-specific responses. The location of the G245R polymorphism next to the box1/box2 proximal cytokine receptor homology segment and suggestive genetic association with the development of disease (P=0.043), suggest that it may affect Janus kinase (JAK) association and impair growth signal transduction. However, additional genetic association with a synonymous single nucleotide polymorphism (IL2RB+8777T) suggests that other variations of IL2RB or nearby genes participate in the highly significant linkage with VL at 22q12 previously reported for this population.

Schistosoma-specific helper T cell clones from subjects resistant to infection by Schistosoma mansoni are Th0/2.

Although T helper cells play a critical role in human immunity against schistosomes, the properties of the T lymphocytes that govern resistance and pathogenesis in human schistosomiasis are still poorly defined. This work addresses the question as to whether human resistance to Schistosoma mansoni is associated with a particular T helper subset. Twenty-eight CD3+, CD4+, CD8- parasite-specific T cell clones were isolated from three adults with high degree of resistance to infection by S. mansoni. The lymphokine secretion profiles of these clones were determined and compared to those of 21 CD3+, CD4+, CD8- clones with unknown specificity, established from these same subjects in the same cloning experiment. Almost all parasite-specific clones produced interleukin (IL)-4 and interferon (IFN)-gamma in large amounts. However, they generally produced more IL-4 than IFN-gamma; variations in IL-4/IFN-gamma ratios were accounted for by differences in IFN-gamma production since IL-4 levels were comparable for the clones from the three subjects. T cell clones of unknown specificity produced significantly less IL-4 and more IFN-gamma than parasite-specific T cell clones. Most clones produced IL-2, and IL-2 production did not differ between the two types of clones. Parasite-specific T cell clones from the resistant subjects were compared to specific T cell clones from a sensitized adult from a nonendemic area: T cell clones from this latter subject were the highest IFN-gamma and the lowest IL-4 producers, compared to those of resistant subjects. Thus, parasite-specific T cell clones isolated from adults resistant to S. mansoni belong to the Th0 subset and produced more IL-4 than IFN-gamma (Th0/2), whereas clones of a sensitized adult from a nonendemic area are also Th0, but produce more IFN-gamma than IL-4 (Th0/1). These results support previous conclusions on the role of IgE in protection against schistosomes in humans, and may indicate that IFN-gamma is required for full protection.

Genetic epidemiology of infectious diseases in humans: design of population-based studies.

The spread and clinical manifestations of an infection in human populations depend on a variety of factors, among them host genetics. Familial linkage studies used in genetic epidemiology to identify host genes test for nonrandom segregation of a trait with a few candidate chromosomal regions or any regions in the genome (genomewide search). When a clear major gene model can be inferred and reliable epidemiologic information is collected (e.g., in schistosomiasis), parametric linkage studies are used. When the genetic model cannot be defined (e.g., in leprosy and malaria), nonparametric linkage studies (e.g., sibling-pair studies) are recommended. Once evidence of linkage is obtained, the gene can be identified by polymorphisms strongly associated with the trait. When the tested polymorphism is in strong linkage disequilibrium with the disease allele or is the disease allele itself (e.g., in HIV infection and malaria), association studies can directly identify the disease gene. Finally, the role of the detected polymorphism in causing the trait is validated by functional studies.

Segregation analysis indicates a major gene in the control of interleukine-5 production in humans infected with Schistosoma mansoni.

The interleukine-5 (IL-5) is a hormone of the immune system that is the main regulator of eosinopoiesis, eosinophil maturation and activation, and immunoglobulin A production. Thus, IL-5 contributes in several ways to human immune defenses against various pathogens, including helminths and infectious agents of the digestive and respiratory tracts. On the other hand, the increase in eosinophil number and the activation of these cells, which both have been related to elevated IL-5 production, are the cause of severe pathological disorders, as in asthma or hypereosinophilic syndromes. Although the immunological pathways leading to IL-5 synthesis have been identified, the reasons for the large variability observed in IL-5 production among subjects exposed to comparable antigenic stimulation are unknown. To investigate the role of genetic factors in this variability, we conducted a segregation analysis in a Brazilian population infected by the helminth parasite Schistosoma mansoni. The analysis was performed on IL-5 levels produced by blood mononuclear cells of these subjects after in vitro restimulation with either parasite extracts (IL-5/schistosomula sonicates [SS] phenotype) or a T-lymphocyte mitogen (IL-5/phytohemagglutin [PHA]). The results provide clear evidence for the segregation of a codominant major gene controlling IL-5/SS and IL-5/PHA production and accounting for 70% and 73% of the phenotypic variance, respectively; the frequency of the allele predisposing to low IL-5 production was approximately .22 for both phenotypes. No significant relationship was found between these genes and the gene controlling infection intensities by S. mansoni detected in a previous study. Linkage studies are ongoing to locate those genes that would help to characterize the genetic factors involved in pathological conditions such as severe helminth infections and allergic diseases.

Control of Leishmania infantum infection is associated with CD8(+) and gamma interferon- and interleukin-5-producing CD4(+) antigen-specific T cells.

Visceral leishmaniasis is a severe and lethal disease caused by the protozoan parasites of the genus Leishmania. In areas where leishmaniasis is endemic, most infected individuals control the infection and remain asymptomatic; chemotherapy of visceral leishmaniasis restores some immunity which protects against relapses. In the present study, Leishmania-specific T-cell clones were established from six asymptomatic and five cured patients. Cytokines production by these clones was analyzed. A large fraction of the parasite-specific T-cell clones from asymptomatic patients were CD8(+) and produced high amounts of gamma interferon (IFN-gamma). Most CD4(+) T-cell clones from two asymptomatic subjects exhibited an unusual phenotype: production of high levels of IFN-gamma low levels of interleukin-4, (IL-4), but high levels of IL-5. In contrast, only few parasite-specific CD8(+) T-cell clones were obtained from cured patients after chemotherapy; moreover, CD4(+) T-cell clones from these patients exhibited an heterogeneous profile of cytokines from Th1-like to Th2-like phenotypes. These results point to CD8(+) T cells and to IL-5- and IFN-gamma-producing CD4(+) T cells as possible contributors to human resistance to Leishmania infection. They should stimulate new immunological approaches in the control of this disease.

Selection of U937 histiocytic lymphoma cells highly responsive to phorbol ester-induced differentiation using monoclonal antibody to the eosinophil cytotoxicity-enhancing factor.

Monoclonal antibodies (MoAbs) to the eosinophil cytotoxicity-enhancing factor (ECEF) were used to detect ECEF in U937 cells before and after phorbol ester (PMA)-induced differentiation into ECEF-secreting macrophages. Membrane-associated ECEF (mECEF), apparently an integral membrane component, is found in U937 cells and in 70% of monocytes and, at lower levels, on blood T lymphocytes. Expression of mECEF in U937 cells is heterogeneous, as is responsiveness to PMA. In PMA-treated cultures, the strongest mECEF expression is on adherent, differentiated macrophages, rather than on activated, nonadherent cells. To study the relationship of mECEF to PMA responsiveness, we positively selected by "panning" a cell line (U937 P+) with significantly higher mECEF expression than that of U937. U937 P+ cells respond to PMA as a differentiation stimulus more effectively than do U937 cells, with a fourfold increase in the number of differentiated macrophages (P less than .001) and a faster rate of differentiation (a fourfold increase at t = 12 hours, P less than .001). U937 P+ cells also show a 7.4-fold increase in response to suboptimal doses of PMA (P less than .001). These findings suggest that mECEF expression correlates with responsiveness to a differentiation stimulus in a histiocytic lymphoma cell line that is widely used as a model of monocyte maturation.

Eosinophil cytotoxicity enhancing factor: purification, characterization and immunocytochemical localization on the monocyte surface.

The monokine eosinophil cytotoxicity enhancing factor (ECEF) increases antibody-dependent cytotoxicity of eosinophils towards helminth larvae. A monokine biochemically indistinguishable from ECEF increases the release of leukotriene C4 and other arachidonic acid metabolites by eosinophils. We have developed monoclonal antibodies (mAb) to these monokines by immunizing mice with ECEF made by the U-937 histiocytic lymphoma cell line. mAb 81.10.C9 (IgG2b) and 9A6G (IgG1) inhibit the effect of the monokine on release of AA products. Both mAb bind ECEF, which appears after affinity chromatography purification as a major 13-14-kDa and a minor 62-kDa component (13-14 kDa and 52 kDa after reduction) in silver-stained gels. An additional component of 30 kDa is detectable after radioiodination of the immunopurified material. The specificity of both mAb was studied in several ways. In immunoprecipitation, both recognize the 13-14-kDa and the 30-kDa components, while the 62-(52)-kDa protein is not significantly precipitated. Both mAb react in enzyme-linked immunosorbent assay with products secreted by peripheral blood mononuclear cells and monocytes, as well as with those secreted by phorbol 12-myristate 13-acetate and lipopolysaccharide-stimulated U-937 cells and with the immunopurified proteins. These were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroeluted and assayed for ECEF activity. Activity was associated with the 13-14-kDa and the 30-kDa fractions, as seen by increased eosinophil antibody-dependent adherence to schistosomula and cytotoxicity. Granulocyte-monocyte-colony-stimulating factor and interleukin 1, but not tumor necrosis factor, could be detected in crude U-937 supernatants. However, active immunopurified ECEF has no activity in assays for granulocyte-monocyte-colony-stimulating factor, interleukin 1 or tumor necrosis factor. Immunocytochemical localization of ECEF employing the mAb shows strong surface staining of viable monocytes and U-937 cells, suggesting that ECEF is associated to the cell surface. These properties distinguish ECEF from other monokines previously reported to activate eosinophils.

Activation of human eosinophils by monokines and lymphokines: source and biochemical characteristics of the eosinophil cytotoxicity-enhancing activity produced by blood mononuclear cells.

We and others have previously reported that human blood mononuclear cells release in culture certain substances that enhance the capacity of purified human blood eosinophils to kill the antibody-coated larvae of Schistosoma mansoni. The present study shows that this eosinophil cytotoxicity-enhancing activity (ECEA) is released by monocytes and T lymphocytes. Monocytes produce ECEA in resting and in LPS-stimulated cultures; T lymphocytes release such activity when stimulated by mitogens such as concanavalin A. Furthermore, the human monocytic line U-937 also releases ECEA-like activity when stimulated by LPS. The enhancing activity produced by monocytes has been partially characterized: it is sensitive to proteolysis by trypsin, relatively heat stable, and associated with molecules that have an apparent molecular weight of 14,000 to 65,000 daltons and isoelectric points of 3.8-3.9, 4.2, 4.5, 4.8-4.9. This shows that while ECEA produced by monocytes is heterogeneous in size and charge, it is probably different from interleukin 1.

Resistance to Schistosoma mansoni in humans: influence of the IgE/IgG4 balance and IgG2 in immunity to reinfection after chemotherapy.

The hypothesis of an association between human resistance to reinfection by the parasite Schistosoma mansoni and anti-larval immunoglobulin isotypes was tested by logistic regression in the presence of the explicative variables water contact, age, and sex. Of the seven isotypes tested (IgM, IgG1, IgG2, IgG3, IgG4, IgA, and IgE), only IgE, IgG4, and IgG2 showed an association (positive for IgE and negative for IgG2 and IgG4) with resistance to reinfection after chemotherapy. The opposite effects of IgE and IgG4 were undissociable in the analysis, indicating that these isotypes probably antagonize each other in protection. The negative association of IgG2 with resistance is consistent with the view that anti-carbohydrate antibodies may facilitate reinfection. Finally, epidemiologic and immunologic studies support the view that there is a progressive but slow development of acquired immunity in children and adolescents.

Evidence for an association between human resistance to Schistosoma mansoni and high anti-larval IgE levels.

The anti-larval IgE antibody response of adolescents with high or low resistance to infection by Schistosoma mansoni was evaluated before parasitological cure with oxamniquine and over an extended post-treatment period during which the least resistant subjects regained high infections. IgE from most sera, taken at several bleeding times before and after treatment, reacted, on immunoblots, with a large number of antigens (Ag) in schistosomular tegument extract. A family of 120-165-kDa cross-reacting molecules and a 85-kDa Ag were the most prominent Ag. Some of these determinants were shown to be located on the outer tegumental membrane and to be accessible to IgE on living larvae. The comparison of IgE between the two study groups showed that IgE levels were on average six-to eightfold higher (p less than 0.01) in the sera of the most resistant adolescents whereas there was no difference in patterns of Ag recognition between study groups. In contrast to IgE, anti-larval IgG and IgM levels were either similar in both groups or higher in the least resistant subjects when these exhibited high reinfection levels. IgG that competed for the binding of IgE to larval Ag were detected in most sera and their levels were higher in the least resistant group after reinfection. Finally, the treatment had no observable long-lasting effects on the levels and on the specificity of the anti-larval IgE. Altogether, these observations can be taken as evidence supporting a role of IgE in human resistance to infection by S. mansoni.