In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

PATHOGENICITY OF Blastocystis sp. TO THE GASTROINTESTINAL TRACT OF MICE: RELATIONSHIP BETWEEN INOCULUM SIZE AND PERIOD OF INFECTION / Histopatogenicidade de Blastocystis sp. para o trato gastrointestinal de camundongos: relação com inóculo e tempo de infecção

The pathogenic potential of Blastocystis sp. in experimental models requires further investigation. In this work, the pathogenicity of this parasite in the gastrointestinal tract of male Swiss mice was evaluated according to the inoculum size and period of infection. Animals were infected intragastrically, with 100, 500, 1,000, 5,000 and 10,000 Blastocystis sp. vacuolar forms obtained from a mixture of eight human isolates cultured axenically in Jones' medium. After seven, 14, 21, 28 and 60 days of infection, the animals were sacrificed and fragments of the small intestine (duodenum), large intestine, and cecum were subjected to histopathological analysis. Blastocystis sp. triggered an inflammatory response in the different tissues analyzed, with a predominance of mononuclear cells. The parasite was found in the muscular layer of the cecum, showing its invasive character. Larger inocula triggered inflammatory processes earlier (seven days) than smaller ones (from 21 days). We conclude that, in the proposed model, the pathogenicity of Blastocystis sp. isolates that were studied is related to inoculum size and period of infection.

Pouco é sabido sobre o potencial patogênico de Blastocystis sp. em modelos experimentais. Neste trabalho a patogenicidade desse parasito para o trato gastrointestinal de camundongos Swiss machos foi avaliada de acordo com o inóculo e tempo de infecção. Os animais foram infectados, via intragástrica, com 100, 500, 1.000, 5.000 e 10.000 formas vacuolares de Blastocystis sp. obtidos a partir de uma mistura de oito isolados humanos cultivados axenicamente em meio Jones. Após 7, 14, 21, 28 e 60 dias de infecção os animais foram sacrificados e fragmentos do intestino delgado (duodeno), grosso e ceco foram retirados para análise histopatológica. Blastocystis sp. desencadeou resposta inflamatória nos diferentes tecidos analisados, com predominância de infiltrado mononuclear. No ceco o parasito foi encontrado na túnica muscular mostrando seu caráter invasivo. Inóculos maiores desencadearam processos inflamatórios mais precocemente (7 dias) e inóculos menores mais tardiamente (a partir de 21 dias). Conclui-se que no modelo proposto a patogenicidade dos isolados de Blastocystis sp. estudados tem relação com o inóculo e tempo de infecção.

Single nucleotide polymorphisms in the interferon gamma gene are associated with distinct types of retinochoroidal scar lesions presumably caused by Toxoplasma gondii infection

The association of single nucleotide polymorphisms (SNPs) in the interferon (IFN)-γ gene ( IFNG ) with different types of retinal scar lesions presumably caused by toxoplasmosis were investigated in a cross-sectional population-based genetic study. Ten SNPs were investigated and after Bonferroni correction, only the associations between SNPs rs2069718 and rs3181035 with retinal/retinochoroidal scar lesions type A (most severe scar lesions) and C (least severe scar lesions), respectively, remained significant. The associations of two different IFNG SNPs with two different types of retinal lesions attributable to toxoplasmosis support the hypothesis that different inflammatory mechanisms underlie the development of these lesions. The in vitro analysis of IFN-γ secretion by peripheral blood mononuclear cells stimulated with Toxoplasma gondii antigens was also investigated. The association between SNP rs2069718 and type A scar lesions revealed that differential IFN-γ levels are correlated with distinct genotypes. However, no correlation was observed with IFN-γ secretion levels and the SNP rs3181035 , which was significantly associated with type C scar lesions. Our findings strongly suggest that immunogenetic studies of individuals with congenital or postnatally acquired infection are needed to better understand the role of IFN-γ and its polymorphisms in the pathogenesis of ocular toxoplasmosis.

Peripheral blood fibrocytes: new information to explain the dynamics of Leishmania infection

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease. .

Production of cytokine and chemokines by human mononuclear cells and whole blood cells after infection with Trypanosoma cruzi / Produção de citocinas e quimiocinas por células mononucleares e células do sangue total humano após infecção com Trypanosoma cruzi

INTRODUCTION: The innate immune response is the first mechanism of protection against Trypanosoma cruzi, and the interaction of inflammatory cells with parasite molecules may activate this response and modulate the adaptive immune system. This study aimed to analyze the levels of cytokines and chemokines synthesized by the whole blood cells (WBC) and peripheral blood mononuclear cells (PBMC) of individuals seronegative for Chagas disease after interaction with live T. cruzi trypomastigotes. METHODS: IL-12, IL-10, TNF-α, TGF-β, CCL-5, CCL-2, CCL-3, and CXCL-9 were measured by ELISA. Nitrite was determined by the Griess method. RESULTS: IL-10 was produced at high levels by WBC compared with PBMC, even after incubation with live trypomastigotes. Production of TNF-α by both PBMC and WBC was significantly higher after stimulation with trypomastigotes. Only PBMC produced significantly higher levels of IL-12 after parasite stimulation. Stimulation of cultures with trypomastigotes induced an increase of CXCL-9 levels produced by WBC. Nitrite levels produced by PBMC increased after the addition of parasites to the culture. CONCLUSIONS: Surface molecules of T. cruzi may induce the production of cytokines and chemokines by cells of the innate immune system through the activation of specific receptors not evaluated in this experiment. The ability to induce IL-12 and TNF-α contributes to shift the adaptive response towards a Th1 profile.

INTRODUÇÃO: A resposta imune inata é o primeiro mecanismo de proteção contra o Trypanosoma cruzi e a interação de células inflamatórias com moléculas do parasita pode ativar esta resposta e modular a resposta adaptativa. O objetivo deste trabalho foi analisar os níveis de citocinas e quimiocinas sintetizados por células do sangue total (WBC) e células mononucleares do sangue periférico (PBMC) de voluntários soronegativos para doença de Chagas depois da interação com Trypanosoma cruzi. MÉTODOS: IL-12, IL-10, TNF-α, TGF-β, CCL5, CCL2, CCL3, CXC-9 foram avaliados por ELISA. Níveis de nitrito foram determinados pelo método de Griess. RESULTADOS: Foram produzidos altos níveis de IL-10 por WBC quando comparado aos sintetizados por PBMC, inclusive após incubação com tripomastigotas. A produção de TNF-α foi significativamente maior nas culturas de PBMC e WBC após estímulo com o parasita. O aumento significativo dos níveis de IL-12 foi observado apenas em PBMC depois do estímulo com tripomastigotas. A adição de tripomastigotas nas culturas induziu aumento dos níveis de CXCL9 produzidos por WBC. Os níveis de nitrito produzidos pelos PBMCs de todos os voluntários após a adição de parasito nas culturas aumentaram. CONCLUSÕES: Moléculas de superfície do parasito podem induzir a produção de citocinas e quimiocinas pelas células da resposta imune inata através da ativação dos receptores específicos não avaliados neste experimento. A habilidade de induzir IL-12 e TNF-α contribui para direcionar uma resposta imune adaptativa de perfil Th1.

In vitro initial immune response against Leishmania amazonensis infection is characterized by an increased production of IL-10 and IL-13

The initial encounter of Leishmania with its host's immune system is important in the outcome of infection. Previous studies have shown that PBMCs from healthy volunteers (HV) exposed to Leishmania differ in IFN-γ production. We have expanded such observations evaluating the profile and kinetics of cytokines (IFN-γ, IL-12p70, IL-10, IL-13), chemokines (CCL5, CCL3, CCL4, CXCL10), and chemokine receptors (CCR1,CCR5, CXCR3, CCR4) in vitro L. amazonensis-stimulated of HV's PBMCs. HVs were divided in groups of high (HR) or low (LR) IFN-γ responders. In both groups, HR and LR, after L. amazonensis infection there was a predominance of IL-10 and IL-13 over IFN-γ production, while IL-12 was produced in similar amount. Regarding chemokines, a more striking difference was observed for CCL3 expression that was lower at 12 hours and 48 hours post infection in LR than in HR. Interestingly, a downregulation of CCR5 and a greater expression of CCR4 were found in low IFN-γ responders. These data suggest that early after L. amazonensis infection there is a cytokine milieu dominated by IL-13 and IL-10, and despite of this environment, IFN-γ is produced, supporting the complexity of the response. It is noteworthy that the pattern of immune response is mounted in first hours after Leishmania stimulation, with the definition of the differentiation of Th1 versus Th2 cells. It remains to be determined if such an in vitro difference has an in vivo counterpart in terms of susceptibility to infection.

Host immune response to Toxoplasma gondii and Ascaris lumbricoides in a highly endemic area: evidence of parasite co-immunomodulation properties influencing the outcome of both infections

Toxoplasmosis and ascaridiasis evoke polar Th-1 and Th-2 host immune responses, respectively. A study to investigate the specific cytokine profile production by in vitro cultures of peripheral blood mononuclear cells from individuals living under precarious sanitary conditions in a highly endemic area for the parasites Toxoplasma gondii and Ascaris lumbricoides was conducted. High levels of both IFN-³ (Th-1) and IL-13 (Th-2) were observed in groups of co-infected individuals presenting toxoplasmic ocular lesions. Significantly lower IL-10 and TGF-² levels were produced by co-infected individuals in comparison with groups of individuals not infected with A. lumbricoides and either positive or negative for T. gondii living under good sanitary conditions (control groups). The possible influence of co-parasitism on the clinical presentation of ocular toxoplasmosis is discussed.

Estabelecimento de ensaios in vitro para avaliação da resposta imune de cães a Leishmania chagasi / Establishment of in vitro tests to evaluate the immune response of dogs to Leishmania chagasi

A leishmaniose visceral (LV) é apontada como doença re-emergente e sua ascensão se relaciona com a ineficácia dos métodos de controle. Há evidências de que o cão é o principal reservatório da forma zoonótica da LV e que L. infantum/L. chagasi seja mantida em comunidades urbanas e peri-urbanas através de um ciclo cão-inseto-cão. Cães com leishmaniose visceral canina (LVC) exibem doença progressiva e geralmente fatal (cão susceptível ou sintomático) ou uma aparente resistência (cão assintomático). Animais doentes apresentam depressão das funções mediadas pelas células T. Em contraste, cães infectados e assintomáticos apresentam resistência ao parasito associada com uma resposta imune celular efetiva. A elucidação dos mecanismos que vão mediar a resposta imune na LVC pode auxiliar o desenvolvimento de vacinas ou estratégias de imunoterapia. O objetivo desta tese foi estabelecer dois ensaios in vitro. O primeiro ensaio foi estabelecido para avaliar se a resposta protetora à infecção por L. infantum/L. chagasi de cães imunizados e assintomáticos se reproduz num sistema de re-estimulação in vitro de PBMC. O segundo ensaio teve como objetivo estabelecer um sistema de estimulação primária in vitro (PIV), que consistiu em utilizar co-culturas de PBMC de cães sadios, previamente estimulados com promastigotas de L. infantum/L. chagasi, e macrófagos autólogos infectados. O primeiro ensaio mostrou que sobrenadantes de PBMC de cães assintomáticos são capazes de estimular macrófagos de cães normais a reduzir a infecção por L. chagasi. A detecção de IFN-y no sobrenadante de PBMC desses cães imunizados e re-estimulados in vitro com antígeno de L. infantum/L. chagasi foi associada à produção de NO pelos macrófagos caninos estimulados com sobrenadantes desses PBMC. O segundo ensaio estabelecido, um sistema de PIV, mostrou que a expressão de IFN-y e IL-4 pelos PBMC estimulados primariamente in vitro com L. infantum/L. chagasi apresenta uma correlação negativa com a infecção. O aumento da expressão dessas citocinas pelos PBMC estimulados primariamente com L. infantum/L. chagasi durante seis dias e co-cultivadas com macrófagos está correlacionado a uma diminuição do percentual de macrófagos infectados. A expressão de IL-1O desses PBMC foi variável e não pôde ser correlacionada à infecção. Esse é o primeiro relato de um sistema de PIV com PBMC e macrófagos caninos infectados por L. infantum/1. chagasi. Esse ensaio, utilizando cães não expostos ao parasito, permitiu discriminar in vitro a resposta de macrófagos a L. infantum/L. chagasi. Os dois ensaios estabelecidos poderão ser utilizados como testes para predizer a resposta do cão à infecção ou na avaliação de candidatos vacinais contra a LVC.

Schistosoma mansoni antigen-driven interleukin-10 production in infected asthmatic individuals

Asthmatics infected with Schistosoma mansoni have a less severe course of asthma and an inhibition of the Th2 inflammatory response that seems to be mediated by interleukin (IL-10). The objective of this study was to evaluate the capacity of some S. mansoni antigens to stimulate IL-10 production in vitro by cells of asthmatic infected individuals. Peripheral bloods mononuclear cells were stimulated with the S. mansoni recombinant antigens Sm22.6, Sm14, P24, and PIII antigen. IL-10 was measured in the supernatants of cultures. As the recombinant antigens were cloned in Escherichia coli, we blocked contaminant endotoxin with polymyxin B added to the cultures. We demonstrated that all antigens used drove high production of IL-10 in S. mansoni infected individuals (n = 13, 408 ± 514 and 401 ± 383 pg/ml, 484 ± 245 pg/ml, 579 ± 468 pg/ml, respectively). In asthmatics infected with S. mansoni (n = 21) rP24 induced higher levels of IL-10 (565 ± 377 pg/ml) when compared to PIII, rSm14 and rSm22.6 (184 ± 209 pg/ml; 292 ± 243 pg/ml; 156 ± 247 pg/ml, respectively). Conclusion: the S. mansoni antigens evaluated in this study stimulated IL-10 production by cells from infected individuals and therefore they have the potential to be used as a modulator of the inflammatory response in asthma.

Dynamics and kinetics of natural killer cell cytotoxicity in human malaria as evaluated by a novel stepwise cytotoxicity assay

Malaria causes important functional alterations of the immune system, but several of them are poorly defined. To evaluate thoroughly the natural killer cell cytotoxicity in patients with malaria, we developed a technique capable to assess both the dynamics and the kinetics of the process. For the kinetics assay, human peripheral blood mononuclear cells were previously incubated with K562 cells and kept in agarose medium, while for the dynamics assay both cells were maintained in suspension. NK activity from patients with vivax malaria presented a kinetics profile faster than those with falciparum malaria. NK cytotoxicity positively correlated with parasitemia in falciparum malaria. The dynamics of NK cytotoxicity of healthy individuals was elevated at the beginning of the process and then significantly decreased. In contrast, malaria patients presented successive peaks of NK activity. Our results confirmed the occurrence of alteration in NK cell function during malaria, and added new data about the NK cytotoxicity process.

A malária causa importantes alterações do sistema imunitário, muitas ainda mal definidas. Para permitir uma avaliação abrangente da atividade citotóxica das células natural killer em pacientes com malária, desenvolvemos um teste capaz de avaliar concomitantemente a dinâmica e a cinética do processo. Para a avaliação da cinética, células mononucleares do sangue periférico interagiram com células K562 e foram mantidas em agarose, enquanto para avaliar a dinâmica as células eram mantidas em suspensão. A cinética da atividade citotóxica das células NK foi mais rápida em pacientes com Plasmodium vivax, do que naqueles infectados com P. falciparum. Nestes, houve correlação positiva entre a atividade citotóxica das células NK e a parasitemia. O padrão da dinâmica da atividade citotóxica nos pacientes com malária foi bem diferente daquele apresentado pelos indivíduos sadios. Enquanto nestes, a atividade estava muito aumentada no início da incubação das células, sofrendo posteriormente uma redução, nos indivíduos infectados foram detectados sucessivos picos de atividade citotóxica. Nossos resultados confirmam a ocorrência de alteração funcional das células NK na malária humana e acrescentam novos dados sobre a dinâmica e a cinética da atividade citotóxica.

Immune response to Leishmania infection.

Antileishmanial immune response is shown to be host genotype dependent so that some inbred strains of mouse are susceptible while others are resistant. The resistance is conferred by T-helper type-1 (Th1) cells while the susceptibility is conferred by Th2 cells. Th1 cells secrete IL-2 and IFN-gamma but Th2 cells secrete IL-4, IL-5 and IL-10. It has been shown that IFN-gamma activates macrophages to express iNOS2, the enzyme catalyzing the formation of nitric oxide. Nitric oxide kills the intracellular amastigotes. In contrast, Th2 immune response limits the action of Th1 functions via IL-10 and IL-4, which deactivate macrophages helping intracellular parasite growth and disease progression. Being a parasite, Leishmania ensures its own survival by modulating host immune system either by inducing immunosuppression or by promoting pro-parasitic host functions. A detailed knowledge of this host-parasite interaction would help in designing prophylactic and therapeutic strategies against this infection.