Alloys S. S. Orago

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.

Vervet monkeys vaccinated with killed Leishmania major parasites and interleukin-12 develop a type 1 immune response but are not protected against challenge infection

ABSTRACT Leishmania major is a protozoan parasite that causes chronic cutaneous lesions that often leave disfiguring scars. Infections in mice have demonstrated that leishmanial vaccines that include interleukin-12 (IL-12) as an adjuvant are able to induce protective immunity. In this study, we assessed the safety, immunopotency, and adjuvant potential of two doses of IL-12 when used with a killed L. major vaccine in vervet monkeys. The induction of cell-mediated immunity following vaccination was determined by measuring delayed-type hypersensitivity, in vitro lymphocyte proliferation, and gamma interferon (IFN-γ) production. Protection was assessed by challenging the animals with L. major parasites and monitoring the course of infection. At low doses of IL-12 (10 μg), a small increase in the parameters of cell-mediated immunity was observed, relative to those in animals that received antigen without IL-12. However, none of these animals were protected against a challenge infection. At higher doses of IL-12 (30 μg), a substantial increase in Leishmania-specific immune responses was observed, and monkeys immunized with antigen and IL-12 exhibited an IFN-γ response that was as great as that in animals that had resolved a primary infection and were immune. Nevertheless, despite the presence of correlates of protection, the disease course was only slightly altered, and protection was low compared to that in self-cured monkeys. These data suggest that protection against leishmaniasis may require more than the activation of Leishmania-specific IFN-γ-producing T cells, which has important implications for designing a vaccine against leishmaniasis.

Combining Evidence of Natural Selection with Association Analysis Increases Power to Detect Malaria-Resistance Variants

Statistical power to detect disease variants can be increased by weighting candidates by their evidence of natural selection. To demonstrate that this theoretical idea works in practice, we performed an association study of 10 putative resistance variants in 471 severe malaria cases and 474 controls from the Luo in Kenya. We replicated associations at HBB (P=.0008) and CD36 (P=.03) but also showed that the same variants are unusually differentiated in frequency between the Luo and Yoruba (who historically have been exposed to malaria) and the Masai and Kikuyu (who have not been exposed). This empirically demonstrates that combining association analysis with evidence of natural selection can increase power to detect risk variants by orders of magnitude--up to P=.000018 for HBB and P=.00043 for CD36.

Cellular Immune Responses of Schistosomiasis Patients Are Altered by Human Immunodeficiency Virus Type 1 Coinfection

In vitro studies suggest that CD4(+) cells with a T helper 2 (Th2) phenotype better support human immunodeficiency virus type 1 (HIV-1) replication than do cells of the Th1 phenotype. As a result, Th2-type immune responses may be substantially affected by HIV-1 coinfection. To test this hypothesis, a comparison was done of proliferation and cytokine production by peripheral blood mononuclear cells from patients with schistosomiasis who were positive or negative for HIV-1. Patients with schistosomiasis with HIV-1 coinfections had significantly lower interleukin (IL)-4 and IL-10 production than did HIV-1-negative individuals. In contrast, interferon-gamma production levels were similar between the 2 groups. Furthermore, in patients with HIV-1, a decrease in CD4(+) T cells was correlated with an increased Th1:Th2 cytokine production ratio. The effect of praziquantel treatment on proliferation and cytokine responses also differed between HIV-1 infection groups. Thus, HIV-1 infection affects immune response patterns of patients with schistosomiasis.

Measurement of Antibody Levels against Region II of the Erythrocyte-Binding Antigen 175 of Plasmodium falciparum in an Area of Malaria Holoendemicity in Western Kenya

ABSTRACT Region II of the 175-kDa erythrocyte-binding antigen (EBA-175RII) of Plasmodium falciparum is functionally important in sialic acid-dependent erythrocyte invasion and is considered a prime target for an invasion-blocking vaccine. The objectives of this study were to (i) determine the prevalence of anti-EBA-175RII antibodies in a naturally exposed population, (ii) determine whether naturally acquired antibodies have a functional role by inhibiting binding of EBA-175RII to erythrocytes, and (iii) determine whether antibodies against EBA-175RII correlate with immunity to clinical malaria. We treated 301 lifelong residents of an area of malaria holoendemicity in western Kenya for malaria, monitored them during a high-transmission season, and identified 33 individuals who were asymptomatic despite parasitemia (clinically immune). We also identified 50 clinically susceptible individuals to serve as controls. These 83 individuals were treated and monitored again during the subsequent low-transmission season. Anti-EBA-175RII antibodies were present in 98.7% of the individuals studied. The antibody levels were relatively stable between the beginning and end of the high-transmission season and correlated with the plasma EBA-175RII erythrocyte-binding-inhibitory activity. There was no difference in anti-EBA-175RII levels or plasma EBA-175RII erythrocyte-binding-inhibitory activity between clinically immune and clinically susceptible groups. However, these parameters were higher in nonparasitemic than in parasitemic individuals at enrollment. These results suggest that although antibodies against EBA-175RII may be effective in suppressing some of the wild parasite strains, EBA-175RII is unlikely to be effective as a monovalent vaccine against malaria, perhaps due to allelic heterogeneity and/or presence of sialic acid-independent strains.

Naturally acquired hemozoin by monocytes promotes suppression of RANTES in children with malarial anemia through an IL-10-dependent mechanism.

Regulated upon activation, normal T-cell expressed, and secreted (RANTES, CCL-5) is an important immunoregulatory mediator that is suppressed in children with malarial anemia (MA). Although pro-inflammatory (e.g., TNF-alpha, IL-1beta and IFN-gamma) and anti-inflammatory (e.g., IL-4, IL-10 and IL-13) cytokines regulate RANTES production, their effect on RANTES in children with MA has not been determined. Since intraleukocytic malarial pigment, hemozoin (Hz), causes dysregulation in chemokine and cytokine production, the impact of naturally acquired Hz (pfHz) on RANTES and RANTES-regulatory cytokines (TNF-alpha, IFN-gamma, IL-1beta, IL-4, IL-10, and IL-13) was examined. Circulating RANTES levels progressively declined with increasing levels of pigment-containing monocytes (PCM) (P=0.035). Additional experiments in cultured peripheral blood mononuclear cells (PBMC) showed that monocytic acquisition of pfHz (in vivo) was associated with suppression of RANTES under baseline (P=0.001) and stimulated conditions (P=0.072). Although high PCM levels were associated with decreased circulating IFN-gamma (P=0.003) and IL-10 (P=0.010), multivariate modeling revealed that only PCM (P=0.048, beta=-0.171) and IL-10 (P<0.0001, beta=-0.476) were independently associated with RANTES production. Subsequent in vitro experiments revealed that blockade of endogenous IL-10 significantly increased RANTES production (P=0.028) in PBMC from children with naturally acquired Hz. Results here demonstrate that monocytic acquisition of Hz suppresses RANTES production in children with MA through an IL-10-dependent mechanism.

Distinct pattern of class and subclass antibodies in immune complexes of children with cerebral malaria and severe malarial anaemia.

Plasmodium falciparum infection can lead to deadly complications such as severe malaria‐associated anaemia (SMA) and cerebral malaria (CM). Children with severe malaria have elevated levels of circulating immune complexes (ICs). To further investigate the quantitative differences in antibody class/subclass components of ICs in SMA and CM, we enrolled 75 children with SMA and 32 children with CM from hospitals in western Kenya and matched them to 74 and 52 control children, respectively, with uncomplicated symptomatic malaria. Total IgG IC levels were always elevated in children with malaria upon enrolment, but children with CM had the highest levels of any group. Conditional logistic regression showed a borderline association between IgG4‐containing IC levels and increased risk of SMA (OR = 3·11, 95% CI 1·01–9·56, P = 0·05). Total IgG ICs (OR = 2·84, 95% CI 1·08–7·46, P = 0·03) and IgE‐containing ICs (OR = 6·82, OR 1·88–24·73, P ≤ 0·01) were associated with increased risk of CM. These results point to differences in the contribution of the different antibody class and subclass components of ICs to the pathogenesis of SMA and CM and give insight into potential mechanisms of disease.

The Levels of CD16/Fcγ Receptor IIIA on CD14+ CD16+ Monocytes Are Higher in Children with Severe Plasmodium falciparum Anemia than in Children with Cerebral or Uncomplicated Malaria

ABSTRACT Fc gamma receptor IIIA (CD16/FcγRIIIA) on monocytes/macrophages may play an important role in the pathogenesis of severe malarial anemia (SMA) by promoting phagocytosis of IgG-coated uninfected red cells and by allowing the production of tumor necrosis factor alpha (TNF-α) upon cross-linking by immune complexes (ICs). However, not much is known about the differential expression of this receptor on monocytes of children with severe malaria and uncomplicated malaria. Therefore, we investigated the expression of CD16/FcγRIIIA on monocytes of children with SMA, cerebral malaria (CM), and their age-matched uncomplicated malaria controls by flow cytometry. Since CD14low (CD14+) monocytes are considered more mature and macrophage-like than CD14high (CD14++) monocytes, we also compared the level of expression of CD16/FcγRIIIA according to the CD14 level and studied the relationship between CD16/FcγRIIIA expression and intracellular TNF-α production upon stimulation by ICs. CD16/FcγRIIIA expression was the highest overall on CD14+ CD16+ monocytes of children with SMA at enrollment. At convalescence, SMA children were the only ones to show a significant decline in the same parameter. In contrast, there were no significant differences among groups in the expression of CD16/FcγRIIIA on CD14++ CD16+ monocytes. A greater percentage of CD14+ CD16+ monocytes produced TNF-α upon stimulation than any other monocyte subset, and the amount of intracellular TNF-α correlated positively with CD16/FcγRIIIA expression. Furthermore, there was an inverse correlation between hemoglobin levels and CD16/FcγRIIIA expression in children with SMA and their controls. These data suggest that monocytes of children with SMA respond differently to Plasmodium falciparum infection by overexpressing CD16/FcγRIIIA as they mature, which could enhance erythrophagocytosis and TNF-α production.

The levels of CD16/Fc gamma receptor III A on CD14+CD16+ monocytes are higher in children with severe Plasmodium falciparum anemia than in children with cerebral or uncomplicated malaria

Fc gamma receptor IIIA (CD16/FcRIIIA) on monocytes/macrophages may play an important role in the pathogenesis of severe malarial anemia (SMA) by promoting phagocytosis of IgG-coated uninfected red cells and by allowing the production of tumor necrosis factor alpha (TNF-) upon cross-linking by immune complexes (ICs). However, not much is known about the differential expression of this receptor on monocytes of children with severe malaria and uncomplicated malaria. Therefore, we investigated the expression of CD16/FcRIIIA on monocytes of children with SMA, cerebral malaria (CM), and their age-matched uncomplicated malaria controls by flow cytometry. Since CD14low (CD14) monocytes are considered more mature and macrophage-like than CD14high (CD14) monocytes, we also compared the level of expression of CD16/FcRIIIA according to the CD14 level and studied the relationship between CD16/FcRIIIA expression and intracellular TNF- production upon stimulation by ICs. CD16/FcRIIIA expression was the highest overall on CD14 CD16 monocytes of children with SMA at enrollment. At convalescence, SMA children were the only ones to show a significant decline in the same parameter. In contrast, there were no significant differences among groups in the expression of CD16/FcRIIIA on CD14 CD16 monocytes. A greater percentage of CD14 CD16 monocytes produced TNF- upon stimulation than any other monocyte subset, and the amount of intracellular TNF- correlated positively with CD16/FcRIIIA expression. Furthermore, there was an inverse correlation between hemoglobin levels and CD16/FcRIIIA expression in children with SMA and their controls. These data suggest that monocytes of children with SMA respond differently to Plasmodium falciparum infection by overexpressing CD16/FcRIIIA as they mature, which could enhance erythrophagocytosis and TNF- production. Severe malarial anemia (SMA) and cerebral malaria (CM) are two of the most serious manifestations of Plasmodium falciparum malaria and are important causes of childhood mortality and morbidity in sub-Saharan Africa. However, the pathogenesis of these complications remains unclear. A better understanding of the factors involved in the pathogenesis of severe malaria is essential for the identification of at-risk populations and the development of effective prophylactic and therapeutic measures. There is abundant evidence to suggest that destruction of uninfected red cells plays an important role in the development of SMA. Uninfected red cells of humans and mice infected with P. falciparum or rodent malaria have a reduced life span (26, 27, 48), and the life span is more reduced in patients with splenomegaly (26), a common finding in children with SMA. Uninfected erythrocytes can continue to be removed from circulation even following malaria treatment, suggesting that this phenomenon is not due to their continued invasion and rupture by parasites (27). A mathematical model suggested that