Eliana T. Nascimento

Oxidative Responses of Human and Murine Macrophages During Phagocytosis of Leishmania chagasi

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2− during phagocytosis of opsonized promastigotes. Addition of the O2− scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2− enhances macrophage leishmanicidal activity. The importance of NO· produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO· in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor NG-monomethyl-l-arginine inhibited IFN-γ-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-γ. These data suggest that O2− and NO· each contribute to intracellular killing of L. chagasi in human and murine macrophages.

Activation of TGF-β by Leishmania chagasi: Importance for Parasite Survival in Macrophages

TGF-β is a potent regulatory cytokine that suppresses expression of inducible NO synthase and IFN-γ, and suppresses Th1 and Th2 cell development. We examined whether functionally active TGF-β is present in the local environment surrounding the invading protozoan Leishmania chagasi. Our prior data showed that TGF-β levels are significantly increased in L. chagasi-infected mice. In the current study, we found TGF-β was also abundant in bone marrows of humans with acute visceral leishmaniasis but not in those of uninfected controls. Furthermore, L. chagasi infection caused an increase in biologically active TGF-β in human macrophage cultures without changing the total TGF-β. Therefore, we investigated the means through which leishmania could augment activated but not total TGF-β. Incubation of latent TGF-β with Leishmania sp. promastigotes caused active TGF-β to be released from the latent complex. In contrast, the nonpathogenic protozoan Crithidia fasciculata could not activate TGF-β. TGF-β activation by leishmania was prevented by inhibitors of cysteine proteases and by the specific cathepsin B inhibitor CA074. Physiologic concentrations of TGF-β inhibited killing of intracellular L. chagasi in macrophages, although the phagocytosis-induced respiratory burst remained intact. In contrast, supraphysiologic concentrations of TGF-β had no effect on parasite survival. We hypothesize that the combined effect of abundant TGF-β stores at extracellular sites during infection, and the ability of the parasite to activate TGF-β in its local environment, leads to high levels of active TGF-β in the vicinity of the infected macrophage. Locally activated TGF-β could, in turn, enhance parasite survival through its effects on innate and adaptive immune responses.

An urban outbreak of visceral leishmaniasis in Natal, Brazil

The epidemiological pattern of visceral leishmaniasis in north-eastern Brazil is changing. The disease was typically seen in rural, endemic areas, but is now occurring as an epidemic in the city of Natal where 316 cases have been reported since 1989; 49% were in children less than 5 years of age. The principle clinical and laboratory findings were weight loss, fever, hepato-splenomegaly, anaemia, leucopenia and hypergammaglobulinaemia. Elevated transaminases and hyperbilirubinaemia were also observed. The diagnosis was confirmed in 87% of cases by identifying amastigotes in aspirates from bone marrow or spleen. Five isolates were identified as Leishmania (L.) chagasi by isoenzyme analysis. The mortality rate was 9%; all deaths occurred during the first week in hospital. One person had concurrent human immunodeficiency virus infection. Among 210 household contacts and neighbours of patients from the endemic area examined for evidence of L. (L.) chagasi infection, 6 additional cases of visceral leishmaniasis were diagnosed. Thirty-eight percent of house-mates and neighbours gave a positive Montenegro skin test reaction, indicating prior subclinical infection.

An Emerging Peri-Urban Pattern of Infection with Leishmania chagasi, the Protozoan Causing Visceral Leishmaniasis in Northeast Brazil

Peri-urban visceral leishmaniasis (VL) caused by Leishmania chagasi is emerging in a new epidemiologic pattern in Brazilian cities. We studied peri-urban VL in endemic neighborhoods surrounding Natal, Brazil, identified through hospitalized individuals with VL. Clinical and environmental information obtained for 1106 members of 216 families living in endemic neighborhoods enabled us to identify 4 groups: VL: individuals with current or prior symptomatic visceral leishmaniasis (n=135); DTH+: individuals with positive delayed-type hypersensitivity response with no history of VL (n=390); Ab+: individuals with negative DTH response and seropositive (n=21); DTH−: individuals with negative DTH and seronegative (n=560). The mean±SD age of VL was 9.3±12.3 y. The gender distribution was nearly equal below age 5, but skewed toward males at higher ages. Acutely infected VL subjects had significantly lower hematocrits, neutrophils, and eosinophils than other categories. AB+ subjects also had lower eosinophil counts than others, a possible immune marker of early infection. VL was not associated with ownership of dogs or other animals, raising the question whether the reservoir differs in peri-urban settings. This new pattern of L. chagasi infection enables us to identify epidemiological and host factors underlying this emerging infectious disease.

Genetic predisposition to self-curing infection with the protozoan Leishmania chagasi : A genomewide scan

The protozoan Leishmania chagasi can cause disseminated, fatal visceral leishmaniasis (VL) or asymptomatic infection in humans. We hypothesized that host genetic factors contribute to this variable response to infection. A family study was performed in neighborhoods of endemicity for L. chagasi near Natal in northeastern Brazil. Study subjects were assessed for the presence of VL or asymptomatic infection, which was defined by a positive delayed-type hypersensitivity (DTH) skin test response to Leishmania antigen without disease symptoms. A genomewide panel of 385 autosomal microsatellite markers in 1254 subjects from 191 families was analyzed to identify regions of linkage. Regions with potential linkage to the DTH response on chromosomes 15 and 19, as well as a novel region on chromosome 9 with potential linkage to VL, were identified. Understanding the genetic factors that determine whether an individual will develop symptomatic or asymptomatic infection with L. chagasi may identify proteins essential for immune protection against this parasitic disease and reveal strategies for immunotherapy or prevention.

Genes at human chromosome 5q31.1 regulate delayed-type hypersensitivity responses associated with Leishmania chagasi infection.

Visceral leishmaniasis (VL) caused by Leishmania chagasi is endemic to northeast Brazil. A positive delayed-type hypersensitivity skin test response (DTH+) is a marker for acquired resistance to disease, clusters in families and may be genetically controlled. Twenty-three single nucleotide polymorphisms (SNPs) were genotyped in the cytokine 5q23.3–q31.1 region IRF1-IL5-IL13-IL4-IL9-LECT2-TGFBI in 102 families (323 DTH+; 190 DTH−; 123 VL individuals) from a VL endemic region in northeast Brazil. Data from 20 SNPs were analyzed for association with DTH+/− status and VL using family-based, stepwise conditional logistic regression analysis. Independent associations were observed between the DTH+ phenotype and markers in separate linkage disequilibrium blocks in LECT2 (OR 2.25; P=0.005; 95% CI=1.28–3.97) and TGFBI (OR 1.94; P=0.003; 95% CI=1.24–3.03). VL child/parent trios gave no evidence of association, but the DTH− phenotype was associated with SNP rs2070874 at IL4 (OR 3.14; P=0.006; 95% CI=1.38–7.14), and SNP rs30740 between LECT2 and TGFBI (OR 3.00; P=0.042; 95% CI=1.04–8.65). These results indicate several genes in the immune response gene cluster at 5q23.3–q31.1 influence outcomes of L. chagasi infection in this region of Brazil.

The emergence of concurrent HIV-1/AIDS and visceral leishmaniasis in Northeast Brazil

HIV has become increasingly prevalent in the Northeast region of Brazil where Leishmania infantum chagasi is endemic, and concurrent AIDS and visceral leishmaniasis (VL) has emerged. In this study, persons with HIV/AIDS and VL (n=17) had a mean age of 37.3 years (range 29-53 years) compared with 12.5 years (1-80 years) for persons with VL alone (n=2836). Males accounted for 88% of cases with concurrent VL and AIDS and 65% of those with VL alone. The mean CD4 count and antileishmanial antibody titre were lower and recurrence of VL and death were more likely with co-infection. Considering the prevalences of L.i. chagasi and HIV in the region, this may herald the emergence of an important public health problem.

Forum: geographic spread and urbanization of visceral leishmaniasis in Brazil. Postscript: new challenges in the epidemiology of Leishmania chagasi infection

as an example of this process in Brazil, was similar to that observed in the Northeast, with subsequent spread to other cities in the South-east and Central-West. It is worrisome that these endemic areas have a high population density, thus placing the population at increased risk of infection.Dogs have traditionally been considered the principal reservoirs of

Genetic Admixture in Brazilians Exposed to Infection with Leishmania chagasi

Visceral leishmaniasis (VL) in northeast Brazil is a disease caused by infection with the protozoan Leishmania chagasi. Infection leads to variable clinical outcomes ranging from asymptomatic infection to potentially fatal disease. Prior studies suggest the genetic background of the host contributes to the development of different outcomes after infection, although it is not known if ancestral background itself influences outcomes. VL is endemic in peri‐urban areas around the city of Natal in northeast Brazil. The population of northeast Brazil is a mixture of distinct racial and ethnic groups. We hypothesized that some sub‐populations may be more susceptible than others to develop different clinical outcomes after L. chagasi infection. Using microsatellite markers, we examined whether admixture of the population as a whole, or markers likely inherited from a distinct ethnic background, differed between individuals with VL, individuals with an asymptomatic infection, or individuals with no infection. There was no apparent significant difference in overall population admixture proportions among the three clinical phenotype groups. However, one marker on Chr. 22 displayed evidence of excess ancestry from putative ancestral populations among different clinical phenotypes, suggesting this region may contain genes determining the course of L. chagasi infection.