Davy K. Koech

A Low Interleukin-10 Tumor Necrosis Factor-α Ratio Is Associated with Malaria Anemia in Children Residing in a Holoendemic Malaria Region in Western Kenya

The balance between Th1 cytokines (tumor necrosis factor [TNF]-alpha, interferon [IFN]-gamma) and Th2 cytokines (interleukin [IL]-10, -4) may be critical in the development of severe falciparum malaria. Therefore, plasma concentrations of these cytokines were determined in children with various manifestations of malaria. Plasma levels of IFN-gamma and IL-4 were undetectable in most children. However, TNF-alpha and IL-10 were significantly elevated in children with high-density parasitemia and malaria anemia compared with children in control groups. In children with mild malaria, IL-10, but not TNF-alpha, was significantly elevated. While the highest concentrations of TNF-alpha were found in children with malaria anemia, IL-10 levels were highest in children with high-density uncomplicated malaria. The mean ratio of IL-10 to TNF-alpha was significantly higher in children with mild and high-density parasitemia (4.64, P<.005) than in children with malaria anemia (1.77). Thus, higher levels of IL-10 over TNF-alpha may prevent development of malaria anemia by controlling the excessive inflammatory activities of TNF-alpha.

In utero exposure to helminth and mycobacterial antigens generates cytokine responses similar to that observed in adults.

Neonates exposed to parasite antigens (Ags) in utero may develop altered fetal immunity that could affect subsequent responses to infection. We hypothesized that cord blood lymphocytes (CBL) from offspring of mothers residing in an area highly endemic for schistosomiasis, filariasis, and tuberculosis in Kenya would either fail to respond or generate a predominantly Th2-associated cytokine response to helminth and mycobacterial antigens (PPD) in vitro compared to maternal PBMC. Kenyan CBL generated helminth Ag-specific IL-5 (range 29-194 pg/ml), IL-10 (121-2,115 pg/ml), and/or IFN-gamma (78 pg/ml-10.6 ng/ml) in 26, 46, and 57% of neonates, respectively (n = 40). PPD induced IFN-gamma in 30% of Kenyan CBL (range 79-1,896 pg/ml), but little or no IL-4 or IL-5. No Ag-specific IL-4, IL-5, or IFN-gamma release was detected by CBL obtained in the United States (n = 11). Ag-driven cytokine production was primarily CD4-dependent. Cytokine responses to helminth and mycobacterial Ags by maternal PBMC mirrored that observed in neonates. CBL from helminth infected and/or PPD-sensitized mothers produced more Ag-specific cytokines compared to CBL from uninfected mothers (P < 0.05). These data demonstrate that the human fetus develops similar patterns of cytokine production observed in adults and indicates that prenatal exposure may not lead to tolerance or altered fetal immunity. .

HLA-DR-Promiscuous T Cell Epitopes from Plasmodium falciparum Pre-Erythrocytic-Stage Antigens Restricted by Multiple HLA Class II Alleles

Previously, we identified and established the antigenicity of 17 CD8+ T cell epitopes from five P. falciparum Ags that are restricted by multiple common HLA class I alleles. Here, we report the identification of 11 peptides from the same Ags, cicumsporozoite protein, sporozoite surface protein 2, exported protein-1, and liver-stage Ag-1, that bind between at least five and up to 11 different HLA-DR molecules representative of the most common HLA-DR Ags worldwide. These peptides recall lymphoproliferative and cytokine responses in immune individuals experimentally immunized with radiation-attenuated Plasmodium falciparum sporozoites (irradiated sporozoites) or semi-immune individuals naturally exposed to malaria in Irian Jaya or Kenya. We establish that all peptides are recognized by individuals of each of the three populations, and that the frequency and magnitude of helper T lymphocyte responses to each peptide is influenced by the intensity of exposure to P. falciparum sporozoites. Mean frequencies of lymphoproliferative responses are 53.2% (irradiated sporozoites) vs 22.4% (Kenyan) vs 5.8% (Javanese), and mean frequencies of IFN-γ responses are 66.3% (irradiated sporozoites) vs 27.3% (Kenyan) vs 8.7% (Javanese). The identification of HLA class II degenerate T cell epitopes from P. falciparum validates our predictive strategy in a biologically relevant system and supports the potential for developing a broadly efficacious epitope-based vaccine against malaria focused on a limited number of peptide specificities.

EFFECTIVENESS OF AMODIAQUINE AS TREATMENT FOR CHLOROQUINE-RESISTANT PLASMODIUM FALCIPARUM INFECTIONS IN KENYA

Studies were conducted in Malindi, Kenya, to assess the response of Plasmodium falciparum to chloroquine and amodiaquine in vivo (by an extended 14-day test) and in vitro (with the Rieckmann micro test). In-vivo resistance was demonstrated in 19 of 69 (28%) infections treated with chloroquine, but in only 2 of 60 (3.3%) of those treated with amodiaquine (p less than 0.001). In-vitro resistance to chloroquine was demonstrated in 15 of 23 (65%) tests. In contrast, 22 of the same 23 isolates were sensitive to amodiaquine in vitro. Effective concentrations by probit analysis for 50% and 99% (EC50 and EC99) inhibition, respectively, were 180.7 and 4319.6 nmol/l for chloroquine and 12.2 and 147.0 nmol/l for amodiaquine. The results suggest that amodiaquine is effective for the treatment of chloroquine-resistant falciparum malaria in Kenya.

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-119) were detected in 24 of 92 (26%) newborns (4–192 IFN-γ and 3–88 IL-4-secreting cells per 106/cord blood lymphocytes). Peptide epitopes in the N-terminal block 3 region of MSP-1 also drove IFN-γ 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-119 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-119 in vitro. Neonatal B cell IgG responses were restricted to the Q-KNG allele of MSP-119, the major variant in this endemic area, whereas T cells responded to all four MSP-119 alleles evaluated. In utero sensitization to MSP-1 correlated with the presence of malaria parasites in cord blood (χ2 = 20, p < 0.0001). These data indicate that prenatal sensitization to blood-stage Ags occurs in infants born in malaria endemic areas.

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.

Comparison of different chemotherapy strategies against Schistosoma mansoni in Machakos District, Kenya : effects on human infection and morbidity

A comparison was made of the long-term impact of different methods of administration of chemotherapy (oxamniquine, 30 mg/kg in divided doses; or praziquantel, 40 mg/kg) on prevalence and intensity of Schistosoma mansoni infection in four areas in Kangundo Location, Machakos District, Kenya. In Area A, treatment was offered in October 1983 and again in April 1985 to all infected individuals. In Area H, treatment was offered in April 1985 to individuals excreting greater than or equal to 100 eggs per gram (epg) of faeces. In Area S, treatment was offered in April 1985 to all infected school children, within the framework of the primary schools. In the witness area, Area W, treatment was given in April 1985, for ethical reasons, to a small number of individuals excreting greater than or equal to 800 epg. Prevalence and intensities of infection were subsequently monitored at yearly intervals for three complete post-treatment years. In the Area S schools, clinical examination was also carried out at yearly intervals. Treatment of all infected individuals on two occasions (Area A) was the most effective and long-lasting way of reducing prevalence and intensity of infection. In this area, however, some earlier interventions had been carried out and pre-treatment intensities were lower than in the other areas. Treatment only of infected schoolchildren (Area S) also had a marked and prolonged effect, comparable to or better than treatment of individuals with heavy infections (Area H). Treatment of infected schoolchildren also caused a persistent reduction in the prevalence of hepatomegaly, and there was suggestive evidence from intensities of infection in community stool surveys (but not from incidence rates) of an effect on transmission. In all study areas, reinfection was most rapid and most intense among children. These findings are discussed in the light of theoretical considerations and of results from other studies, both on schistosomiasis and on intestinal helminths. We conclude that, in areas of low morbidity such as Kangundo, chemotherapy of schoolchildren only, at intervals of up to 3 years, is a satisfactory way of producing a long-term reduction in both intensity of infection and morbidity.

Seroepidemiology and serodiagnosis of schistosomiasis in Kenya using crude and purified egg antigens of Schistosoma mansoni in ELISA

The performance of antibody detection for the diagnosis of schistosomiasis has been evaluated in Kenya. Approximately 1500 blood samples from 3 areas with endemic schistosomiasis (Schistosoma mansoni only, S. haematobium only, and a mixed infection area), and from a non-endemic control area, were tested for their antibody reactivity in an enzyme-linked immunosorbent assay (ELISA). The results were compared with infection status determined by parasitological examination. Two test antigens were used: unfractionated S. mansoni egg homogenate (SEA), and CEF6, a previously described, partially purified fraction of SEA containing 2 cationic antigens. The antigens prepared from eggs of Kenya and Puerto Rico S. mansoni isolates gave very similar results. Bloods from patients with S. haematobium infection cross-reacted significantly with the two S. mansoni antigen preparations, but reactivity against CEF6 appeared more specifically indicative of S. mansoni infection. Of 254 blood samples from schoolchildren in the non-endemic area, 100% gave ELISA optical density readings at 492 nm (OD492) < 0.20 against SEA, and 98% were < 0.20 against CEF6. With 887 blood samples from subjects of all ages in the area endemic for S. mansoni alone, using an ELISA OD492 cut-off point of 0.20, SEA and CEF6 had sensitivities of 94% and 97% respectively, and specificities of 64% and 59% respectively. Increasing the OD492 cut-off value reduced the sensitivity and increased the specificity of both test antigens. Specificity of both antigens was poor with samples from 234 children in an area endemic for both S. mansoni and S. haematobium (< 20% for both antigens at an OD492 cut-off value of 0.20).(ABSTRACT TRUNCATED AT 250 WORDS)

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 (Tregs) in utero, cord blood mononuclear cells (CBMC) were obtained from 44 Kenyan newborns of women with and without malaria at delivery. CD4+CD25lo T cells and CD4+CD25hi FOXP3+ cells (Tregs) were enriched from CBMC. Treg frequency and HLA-DR expression on Tregs 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 CD25hi 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 Tregs to CD4+CD25lo 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, Tregs only partially suppressed malaria-induced Th2 cytokines. IL-10 or TGF-β did not mediate this suppression. Thus, prenatal exposure to malaria blood-stage Ags induces Tregs that primarily suppress Th1-type recall responses to P. falciparum blood-stage Ags. Persistence of these Tregs postnatally could modify a child’s susceptibility to malaria infection and disease.

Quantitation of malaria sporozoites transmitted in vitro during salivation by wild Afrotropical Anopheles

Abstract. The malaria transmission potential of wild, infective Anopheles from western Kenya was evaluated by determining the number of sporozoites transmitted in vitro by salivation when their mouthparts were inserted into capillary tubes containing either sucrose or blood. With sucrose, 86.6% of 102 infective Anopheles transmitted a geometric mean (GM) of 3.84 sporozoites (range 1–34). With blood, 23.1% of 104 infective Anopheles, tested on the day of collection, transmitted a GM of 2.30 sporozoites (range 1–117). For Anopheles held 5 days postcapture before testing with blood, 53.6% of 56 transmitted a GM of 6.04 sporozoites (range 1–420). Transmitting Anopheles contained significantly more salivary gland sporozoites than non‐transmitters. No significant differences were detected between Anopheles gambiae Giles sensu lato and Anopheles funestus Giles in sporozoite transmission by individuals with sporozoites in their salivary glands.