Human cord blood CD4+CD25hi regulatory T cells suppress prenatally acquired T cell responses to Plasmodium falciparum antigens.

In malaria endemic regions, a fetus is often exposed in utero to Plasmodium falciparum blood-stage Ags. In some newborns, this can result in the induction of immune suppression. We have previously shown these modulated immune responses to persist postnatally, with a subsequent increase in a child's susceptibility to infection. To test the hypothesis that this immune suppression is partially mediated by malaria-specific regulatory T cells (T(regs)) in utero, cord blood mononuclear cells (CBMC) were obtained from 44 Kenyan newborns of women with and without malaria at delivery. CD4(+)CD25(lo) T cells and CD4(+)CD25(hi) FOXP3(+) cells (T(regs)) were enriched from CBMC. T(reg) frequency and HLA-DR expression on T(regs) were significantly greater for Kenyan as compared with North American CBMC (p < 0.01). CBMC/CD4(+) T cells cultured with P. falciparum blood-stage Ags induced production of IFN-γ, IL-13, IL-10, and/or IL-5 in 50% of samples. Partial depletion of CD25(hi) cells augmented the Ag-driven IFN-γ production in 69% of subjects with malaria-specific responses and revealed additional Ag-reactive lymphocytes in previously unresponsive individuals (n = 3). Addition of T(regs) to CD4(+)CD25(lo) cells suppressed spontaneous and malaria Ag-driven production of IFN-γ in a dose-dependent fashion, until production was completely inhibited in most subjects. In contrast, T(regs) only partially suppressed malaria-induced Th2 cytokines. IL-10 or TGF-ß did not mediate this suppression. Thus, prenatal exposure to malaria blood-stage Ags induces T(regs) that primarily suppress Th1-type recall responses to P. falciparum blood-stage Ags. Persistence of these T(regs) postnatally could modify a child's susceptibility to malaria infection and disease.

Fine specificity of neonatal lymphocytes to an abundant malaria blood-stage antigen: epitope mapping of Plasmodium falciparum MSP1(33).

Cord blood T cells have been reported to respond to a variety of exogenous Ags, including environmental allergens and various viruses and parasites, as demonstrated by enhanced proliferation and cytokine secretion. This finding is evidence that Ags in the maternal environment transplacentally prime and result in fetal development of memory T cells. Some studies suggest these neonatal T cell responses may arise by nonspecific activation of T cells that express TCRs with low binding affinity, thus lacking fine lymphocyte specificity. To address this question, we examined malaria Ag stimulation of human cord and adult blood mononuclear cells in samples from residents of a malaria endemic area in Kenya. We constructed overlapping 18-mer peptides derived from sequences contained in dimorphic alleles of the C-terminal 33-kDa fragment of Plasmodium falciparum merozoite protein 1. This study identified a dominant T cell epitope for one MSP1(33) allele (MAD20) and two T cell epitopes for the second allele (K1); these epitopes were nonoverlapping and allele specific. In a given donor, peptide-specific proliferation and IFN-gamma secretion were highly concordant. However, IL-10 and IL-13 secretion were not correlated. Importantly, the fine specificity of lymphocyte proliferation and cytokine secretion in cord and adult blood mononuclear cells was similar. Cord blood cells obtained from malaria-infected pregnant women were 4-fold more likely to acquire a peptide-specific immune response. We conclude that the fetal malaria response functions in a fully adaptive manner and that this response may serve to help protect the infant from severe malaria during infancy.

Late benefits 10-18 years after drug therapy for infection with Schistosoma haematobium in Kwale District, Coast Province, Kenya.

Late benefits of remote antischistosomal therapy were estimated among long-term residents of an area with high transmission of Schistosoma haematobium (Msambweni, Kenya) by comparing infection and disease prevalence in two local adult cohorts. We compared 132 formerly treated adults (given treatment in childhood or adolescence > or = 10 years previously) compared with 132 age- and sex-matched adults from the same villages who had not received prior treatment. The prevalence of current infection, hematuria, and ultrasound bladder abnormalities were significantly lower among the previously treated group, who were found to be free of severe bladder disease. Nevertheless, heavy infection was equally prevalent (2-3%) in both study groups, and present rates of hydronephrosis were not significantly different. Therapy given in childhood or adolescence appears to improve risk for some but not all manifestations of S. haematobium infection in later adult life. Future prospective studies of continued treatment into adulthood will better define means to obtain optimal, community-based control of S. haematobium-related disease in high-risk locations.

Increased ratio of tumor necrosis factor-alpha to interleukin-10 production is associated with Schistosoma haematobium-induced urinary-tract morbidity.

Bladder and kidney disease, which affect approximately 25%-30% of subjects infected with Schistosoma haematobium, are mediated by T cell-dependent granulomatous responses to schistosome eggs. To determine why only some infected subjects develop disease, we examined the hypothesis that infected Kenyan subjects with ultrasound-detected urinary-tract morbidity (n=49) had dysregulated cytokine production leading to enhanced granulomatous responses, compared with subjects of similar age and intensity of infection without morbidity (n=100). Peripheral blood mononuclear cells from subjects with morbidity produced 8-fold greater levels of egg antigen-driven tumor necrosis factor (TNF)-alpha and had a 99-fold greater mean TNF-alpha:interleukin (IL)-10 ratio, compared with subjects without disease. No differences in cytokine response to non-egg-derived schistosome antigens were observed between groups. Subjects with morbidity had increased TNF-alpha production in response to endotoxin, suggesting an innate hyperresponsiveness. These results indicate that increased TNF-alpha production, relative to that of IL-10, is associated with developing bladder-wall morbidity with S. haematobium infection.

Mitochondrial DNA control region sequences from Nairobi (Kenya): inferring phylogenetic parameters for the establishment of a forensic database.

Large forensic mtDNA databases which adhere to strict guidelines for generation and maintenance, are not available for many populations outside of the United States and western Europe. We have established a high quality mtDNA control region sequence database for urban Nairobi as both a reference database for forensic investigations, and as a tool to examine the genetic variation of Kenyan sequences in the context of known African variation. The Nairobi sequences exhibited high variation and a low random match probability, indicating utility for forensic testing. Haplogroup identification and frequencies were compared with those reported from other published studies on African, or African-origin populations from Mozambique, Sierra Leone, and the United States, and suggest significant differences in the mtDNA compositions of the various populations. The quality of the sequence data in our study was investigated and supported using phylogenetic measures. Our data demonstrate the diversity and distinctiveness of African populations, and underline the importance of establishing additional forensic mtDNA databases of indigenous African populations.

Low heritable component of risk for infection intensity and infection-associated disease in urinary schistosomiasis among Wadigo village populations in Coast Province, Kenya.

To estimate their heritable component of risk for Schistosoma haematobium infection intensity and disease, we performed a community-based family study among an endemic population in coastal Kenya. Demography and family linkages were defined by house-to-house interviews, and infection prevalence and disease severity were assessed by standard parasitologic testing and by ultrasound. The total population was 4,408 among 912 households, with 241 identified pedigree-household groups. Although age- and sex-adjusted risk for greater infection intensity was clustered within households (odds ratio = 2.7), analysis of extended pedigree-household groups indicated a relatively low heritability score for this trait (h2 = 0.199), particularly after adjustment for common household exposure effects (adjusted h2 = 0.086). Statistical evidence was slightly stronger (h2 = 0.353) for familial clustering of bladder morbidity, with an adjusted h2 = 0.142 after accounting for household exposure factors. We conclude that among long-established populations of coastal Kenya, heritable variation in host susceptibility is low, and likely plays a minimal role in determining individual risk for infection or disease.

Large-scale, polymerase chain reaction-based surveillance of Schistosoma haematobium DNA in snails from transmission sites in coastal Kenya: a new tool for studying the dynamics of snail infection.

Levels of prepatent Schistosoma haematobium infection were monitored in intermediate host snails (Bulinus nasutus) collected from transmission sites in coastal Kenya, using a polymerase chain reaction (PCR) assay amplifying the Dra I repeated sequence of S. haematobium. The timing and number of prepatent and patent infections were determined for each site and, where the time of first appearance was clear, the minimal prepatent period was estimated to be five weeks. High, persistent, prepatency rates (range = 28-54%), indicated a significant degree of repeated area contamination with parasite ova. In contrast, rates of cercarial shedding proved locally variable, and were either low (range = 0.14-3.4%) or altogether absent, indicating that only a small proportion of infected snails reach the stage of cercarial shedding. Given the apparently strong focal effects of environmental conditions, implications of these new data are discussed regarding the estimation of local force of transmission and the design of control activities.

Associations between anti-Schistosoma mansoni and anti-Plasmodium falciparum antibody responses and hepatosplenomegaly, in Kenyan schoolchildren.

Schoolchildren from 2 areas of Kenya, Kangundo and Kambu, have contrasting prevalences of hepatosplenomegaly, despite having similar prevalences and intensities of Schistosoma mansoni infection. However, in individual children, S. mansoni infection intensity is positively correlated with organomegaly. In a previous study, hepatosplenomegaly was associated with Th1-type anti-schistosome cytokine responses. Although the high-morbidity Kambu area had higher malaria transmission than did low-morbidity Kangundo, hepatosplenomegaly was not associated with clinical malaria or with patent malarial parasitemia. However, chronic exposure to malaria might be involved. Here, retrospectively, we assayed plasma from this original study, for anti-Plasmodium falciparum and anti-S. mansoni antibodies, to test whether greater exposure to Plasmodium was a cofactor for hepatosplenomegaly. We found that hepatosplenic children had significantly higher levels of anti-P. falciparum antibodies, compared with nonhepatosplenic children, a finding that strongly suggests that some experience of P. falciparum influenced the development of hepatosplenomegaly in these S. mansoni-infected children.

Randomized comparison of low-dose versus standard-dose praziquantel therapy in treatment of urinary tract morbidity due to Schistosoma haema tobium infection.

At present, anthelmintic therapy with praziquantel at a dose of 40 mg/kg of body weight is the recommended treatment for control of urinary tract morbidity caused by Schistosoma haematobium. Although this standard regimen is effective, drug cost may represent a significant barrier to implementation of large-scale schistosomiasis control programs in developing areas. Previous comparison trials have established that low-dose (20-30 mg/kg) praziquantel regimens can effectively suppress the intensity of S. haematobium infection in endemic settings. However, the efficacy of these low-dose regimens in controlling infection-related morbidity has not been determined in a randomized field trial. The present random allocation study examined the relative efficacy of a 20 mg/kg dose versus a 40 mg/kg dose of praziquantel in control of hematuria and bladder and renal abnormalities associated with S. haematobium infection in an endemic area of Coast Province, Kenya. After a nine-month observation period, the results indicated an advantage to the standard 40 mg/kg praziquantel dose in terms of reduction of infection prevalence and hematuria after therapy (P < 0.01 and P < 0.005, respectively). However, the two treatment groups were equally effective in reducing structural urinary tract morbidity detected on ultrasound examination. We conclude that in certain settings, a 20 mg/kg dose of praziquantel may be sufficient in providing control of morbidity due to urinary schistosomiasis in population-based treatment programs.

Acquired immune responses to Plasmodium falciparum merozoite surface protein-1 in the human fetus.

Infants born in areas of stable malaria transmission are relatively protected against severe morbidity and high density Plasmodium falciparum blood-stage infection. This protection may involve prenatal sensitization and immunologic reactivity to malaria surface ligands that participate in invasion of red cells. We examined cord blood T and B cell immunity to P. falciparum merozoite surface protein-1 (MSP-1) in infants born in an area of stable malaria transmission in Kenya. T cell cytokine responses to the C-terminal 19-kDa fragment of MSP-1 (MSP-1(19)) were detected in 24 of 92 (26%) newborns (4-192 IFN-gamma and 3-88 IL-4-secreting cells per 10(6)/cord blood lymphocytes). Peptide epitopes in the N-terminal block 3 region of MSP-1 also drove IFN-gamma and/or IL-13 production. There was no evidence of prenatal T cell sensitization to liver-stage Ag-1. A total of 5 of 86 (6%) newborns had cord blood anti-MSP-1(19) IgM Abs, an Ig isotype that does not cross the placenta and is therefore of fetal origin. The frequency of neonatal B cell sensitization was higher than that indicated by serology alone, as 5 of 27 (18%) cord blood samples contained B cells that produced IgG when stimulated with MSP-1(19) in vitro. Neonatal B cell IgG responses were restricted to the Q-KNG allele of MSP-1(19), the major variant in this endemic area, whereas T cells responded to all four MSP-1(19) alleles evaluated. In utero sensitization to MSP-1 correlated with the presence of malaria parasites in cord blood (chi(2) = 20, p < 0.0001). These data indicate that prenatal sensitization to blood-stage Ags occurs in infants born in malaria endemic areas.

Isolation and characterization of flagellates from rodents and canids in Masinga, Machakos District, Kenya.

A total of 728 animals comprising of 633 rodents and 95 canids were examined for leishmanial parasites. Flagellates were isolated from 67 out of 111 (60.4%) Acomys subspinosus (spiny mouse), 12 out of 143 (8.4% ) Mastomys natalensis (multimammate rat), 2 out of 50 (4.0%) Lemniscomys striatus (striped mouse), 2 out of 6 (33.3%) Herpestes sanguineus (slender mongoose), 1 of 1 Helogale parvula (dwarf mongoose) and 1 out of 84 Canis familiaris (domestic dog). All isolates were characterized by Isoenzyme analysis using nine enzymes, namely, malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucose phosphate isomerase (GPI), isocitrate dehydrogenase (ICD), nucleoside hydrolase (NH), glucose 6-phosphate dehydrogenase (G6PD), malic enzyme (ME), 6-phosphogluconate dehydrogenase (GPGD) and mannose phosphate isomerase (MPI). Enzyme profiles of these isolates were compared with those of five WHO Leishmania reference strains and five well characterized rodent trypanosomes of the subgenus Herpetosoma. The profiles of the isolates were found to be different from those of the Leishmania and Trypanosoma reference strains but the parasites were morphologically similar to rodent trypanosomes. These results suggest that Leishmania parasites were not among the isolates. The enzymes profiles of the three mongoose isolates were identical but differed from profiles of isolates from rodents and dog. This is the first time in Kenya that a high prevalence of nonpathogenic trypanosomes is reported in rodents and canids. From the epidemiological point of view, these trypanosomes must be differentiated from the pathogenic species of trypanosomes and Leishmania that infect man and other animals. The results of this study suggest that rodents do not seem to play a role as reservoirs of Leishmania parasites in Masinga Location, Kenya.

Visceral leishmaniasis : activation of human T-lymphocytes by two specific Leishmania antigens.

T cell responses to a specific Leishmania antigens, 70kD and 116kD were investigated in individuals from an endemic area for leishmaniasis. Peripheral blood mononuclear cells were obtained from 38 individuals (24 test an d14 controls) and T cell responses to phytohaemaglutinin (PHA), Concanavalin A (ConA) and the specific Leishmania antigens were examined. PHA and Con A-induced responses were all positive in both the test and control groups. Eighteen out of 24 individuals with previous history of visceral leishmaniasis and 12 out of 14 normal individuals residing in the endemic areas responded to the 70kD antigen. In contrast, all the eight normal individuals all the eight normal individuals from a non-endemic area for leishmaniasis did not responded, indicating that cellular responses to the 70kD antigen are specific for Leishmanai spp. The 116kD antigen, on the other hand, exhibited poor specificity: about 30% of the individuals with a previous history of leishmaniasis did not respond to this antigen. A significant proportion of the population from an endemic area, but with no history of visceral leishmaniasis, responded to the 70kD antigen. This is an indicator of a prior exposure to Leishmania parasite and is consistent with previous studies which have shown that subclinical infections of visceral leishmaniasis do occur in such areas. Results from the present study suggest that the 70kD antigen is a strong candidate for vaccine development.

Cross-reactive studies in visceral leishmaniasis: comparison of antigens of some Leishmania species in Kenya.

Little is known about the common antigens of the various strains of Leishmania in Kenya, and their possible role in immunity to disease. Kenyan isolates of Leishmania donovani, L. Major and L. tropica were cultured, crude antigens prepared and electrophoresced in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots by pooled sera from cutaneous and visceral leishmaniasis patients revealed common antigens within the 110-116kD, 70kD and the 63kD ranges. Only the 112-116kD antigens from L. major strains were reacted to by both cutaneous and visceral leishmaniasis patient sera. Sera from normal individuals living in an endemic area for leishmaniasis also showed reactivity to the 116kD antigen. We suggest that these common antigens may be of value in future vaccine studies

Screening of metal ion chelators against Leishmania donovani-infected Syrian hamsters.

Leishmania donovani-infected Syrian hamsters were treated intraperitoneally with 0.23 mmoles/kg/day of EDTA, EGTA, HEEDTA and 100 mg/kg/day of Pentostam R. The control group received 0.1 ml of phosphate buffered saline. After 30 days of treatment, the animals were sacrificed. Of the Pentostam-treated animals, 5 out 6 had negative spleen cultures, while all the chelator and PBS-treated ones yielded parasites. While all the Pentostam-treated animals had negative bone marrow cultures, only 1 out of 6 HEEDTA-treated hamsters yielded parasites. Spleen, liver and bone marrow parasite- loads calculated from chelator-treated animals were consistently significantly higher than for Pentostam-treated animals. These results suggest that although metal ion chelators have some antileishmanial potential, their in vivo activity against L. donovani is low compared to Pentostam.

Comparison of Giemsa and acridine orange stains for the diagnosis of Leishmania donovani in biopsies of infected hamsters.

Identical impression smears of spleen, liver and bone marrow biopsy materials from Leishmania donovani-infected hamsters were stained using either acridine orange or Giemsa. Spleen parasite-loads calculated from the two stains for identical biopsy material were significantly different from each other. However, liver and bone marrow parasite- loads calculated from either Giemsa-stained or acridine orange-stained biopsies were not significantly different from each other. This study has shown that acridine orange, which is a quick and simple technique, has great potential in the diagnosis of kala-azar when liver and bone marrow biopsies are used.