Interleukin-7 potentiates MAPK10-elicited host-protective vaccine against Leishmania donovani.

Leishmania donovani causes the potentially fatal disease visceral leishmaniasis for which neither a vaccine nor an adjuvant for human use exists. Although interleukin-7 (IL-7) is implicated in CD4+ T-cell response stabilization, its anti-leishmanial function is uncertain. Therefore, we examined whether IL-7 would potentiate the efficacy of Leishmania major-expressed MAPK10 (LmjMAPK10; M10)-elicited anti-leishmanial host-protective response. We observed that aligning with IL-7R expression, IL-7 increased IFN-γ-secreting TH1 cell but reduced IL-4-producing TH2 cells and production of IL-10 and TGF-ß effectuating anti-leishmanial functions in susceptible BALB/c mouse-derived macrophages. Co-culturing IL-7-pre-treated L. donovani-infected macrophages with L. donovani-infected BALB/c-derived T cells induced IFN-γ-dominated TH1 type anti-leishmanial function. IL-7 treatment of L. donovani-infected BALB/c mice significantly reduced splenic and hepatic parasite loads. Co-culturing CD4+ T cells from IL to 7-treated mice with L. donovani-infected macrophages reduced amastigote numbers suggesting IL-7-elicited host-protective effector T cells. Priming BALB/c with M10 + IL-7 reduced the splenic parasite burden more effectively than that was observed in M10-primed mice. An enhanced protection against L. donovani infection was accompanied by enhanced IL-12 and IFN-γ, but suppressed IL-10 and IL-4, response and host-protective TH1 and memory T cells. These results indicate IL-7-induced leishmanial antigen-specific memory T cell response that protects a susceptible host against L. donovani infection.

Circadian Regulation of Leishmania Parasite Internalisation in Macrophages and Downstream Cellular Events.

Leishmania spp. parasites use macrophages as a host cell during infection. As a result, macrophages have a dual role: clearing the parasite as well as acting as host cells. Recently, studies have shown that macrophages harbour circadian clocks, which affect many of their functions such as phagocytosis, receptor expression and cytokine release. Interestingly, Leishmania major infection in hosts was also shown to be under circadian control. Therefore, we decided to investigate what underlies the rhythms of L. major infection within macrophages. Using a culture model of infection of bone marrow-derived macrophages with L. major promastigotes, we show that the parasites are internalised into macrophages with a 24-h variation dependent on a functional circadian clock in the cells. This was associated with a variation in the number of parasites per macrophage. The cell surface expression of parasite receptors was not controlled by the cells' circadian clock. In contrast, the expression of the components of the endocytic pathway, EEA1 and LC3b, varied according to the time of infection. This was paralleled by variations in parasite-induced ROS production as well as cytokine tumour necrosis factor α. In summary, we have uncovered a time-dependent regulation of the internalisation of L. major promastigotes in macrophages, controlled by the circadian clock in these cells, as well as subsequent cellular events in the endocytic pathway, intracellular signalling and cytokine production.

From classical approaches to new developments in genetic engineering of live attenuated vaccine against cutaneous leishmaniasis: potential and immunization.

Despite the development of a vaccine against cutaneous leishmaniasis in preclinical and clinical studies, we still do not have a safe and effective vaccine for human use. Given this situation, the search for a new prophylactic alternative to control leishmaniasis should be a global priority. A first-generation vaccine strategy-leishmanization, in which live Leishmania major parasites are inoculated into the skin to protect against reinfection, is taking advantage of this situation. Live attenuated Leishmania vaccine candidates are promising alternatives due to their robust protective immune responses. Importantly, they do not cause disease and could provide long-term protection following challenges with a virulent strain. In addition to physical and chemical methods, genetic tools, including the Cre-loxP system, have enabled the selection of safer null mutant live attenuated Leishmania parasites obtained by gene disruption. This was followed by the discovery and introduction of CRISPR/Cas-based gene editing tools, which can be easily and precisely used to modify genes. Here, we briefly review the immunopathology of L. major parasites and then present the classical methods and their limitations for the production of live attenuated vaccines. We then discuss the potential of current genetic engineering tools to generate live attenuated vaccine strains by targeting key genes involved in L. major pathogenesis and then discuss their discovery and implications for immune responses to control leishmaniasis.

Rapid and practical colorimetric biosensor for leishmaniasis diseases.

In this article, a colorimetric biosensor for detection of Leishmania major surface protease (Gp63) antibody (anti-gp63) was developed by using gold nanoparticle (AuNP) as a color reagent. The dispersion or aggregation of AuNPs leads to a distinct and sensitive change in UV-vis spectra and solution color. For this purpose, kinetoplastid membrane protein-11 (KMP-11) was labeled with AuNPs surface directly. After that, Gp63 antibody was added in the KMP-11@AuNP solution and a color change from red/pink to purple/violet was observed. As a result, anti-gp63 solution diluted at a ratio of 1:640 can be detected with the developed colorimetric leishmania biosensor. The relative standard deviation value for 1:320 diluted anti-gp63 was calculated as 1.29 %. Furthermore, the linear range of the developed colorimetric biosensor was determined as 1:80 to 1:640. Moreover, developed Leishmania biosensor was applied for detection of leishmania parasite crude antigen and rabbit serum which were used as positive and negative samples respectively. As a result, the recovery values for the measurements of aforementioned samples were calculated as 95.3 % ± 0.02, 103.1 % ± 0.02, 96.2 % ± 0.01 and 95.5 % ± 0.03 for dilutions of 1:200, 1:160, 1:320 and 1:640 anti-gp63 solutions respectively.

Leishmania major MAPK4 intercepts and redirects CD40 signaling promoting infection.

The parasite Leishmania resides as amastigotes within the macrophage parasitophorous vacuoles inflicting the disease Leishmaniasis. Leishmania selectively modulates mitogen-activated protein kinase (MAPK) phosphorylation subverting CD40-triggered anti-leishmanial functions of macrophages. The mechanism of any pathogen-derived molecule induced host MAPK modulation remains poorly understood. Herein, we show that of the fifteen MAPKs, LmjMAPK4 expression is higher in virulent L. major. LmjMAPK4- detected in parasitophorous vacuoles and cytoplasm- binds MEK-1/2, but not MKK-3/6. Lentivirally-overexpressed LmjMAPK4 augments CD40-activated MEK-1/2-ERK-1/2-MKP-1, but inhibits MKK3/6-p38MAPK-MKP-3, phosphorylation. A rationally-identified LmjMAPK4 inhibitor reinstates CD40-activated host-protective anti-leishmanial functions in L. major-infected susceptible BALB/c mice. These results identify LmjMAPK4 as a MAPK modulator at the host-pathogen interface and establish a pathogen-intercepted host receptor signaling as a scientific rationale for identifying drug targets.

PLGA nanoparticle-delivered Leishmania antigen and TLR agonists as a therapeutic vaccine against cutaneous leishmaniasis in BALB/c mice.

Leishmaniasis, caused by Leishmania (L.) species, remains a neglected infection. Therapeutic vaccination presents a promising strategy for its treatment. In this study, we aimed to develop a therapeutic vaccine candidate using Leishmaniaantigens (SLA) and Toll-like receptor (TLR) 7/8 agonist (R848) encapsulated into the poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Moreover, TLR1/2 agonist (Pam3CSK4) was loaded onto the NPs. The therapeutic effects of these NPs were evaluated in L. major-infected BALB/c mice. Footpad swelling, parasite load, cellular and humoral immune responses, and nitric oxide (NO) production were analyzed. The results demonstrated that the PLGA NPs (SLA-R848-Pam3CSK4) therapeutic vaccine effectively stimulated Th1 cell responses, induced humoral responses, promoted NO production, and restricted parasite burden and lesion size.Our findings suggest that vaccination with SLA combined with TLR1/2 and TLR7/8 agonists in PLGA NPs as a therapeutic vaccine confers strong protection againstL. majorinfection. These results represent a novel particulate therapeutic vaccine against Old World cutaneous leishmaniasis.

Implication of the Annexin 1/FPR axis in leishmanial exosome-mediated Leishmania major skin hyperpathogenesis.

Introduction: Exosomes produced by the protozoan parasite Leishmania (LeishEXO) are well-established drivers of virulence, though mechanisms underlying their exacerbation of experimental leishmaniasis remain elusive. Expression of Annexin A1 (ANXA1), a protein implicated in exosome-mediated pathologies and viral internalization, has been shown to correlate with cutaneous leishmaniasis severity. Given ANXA1's regulation of myeloid cells - the canonical hosts for Leishmania - we studied the potential role of ANXA1 and its receptors FPR1/2 in exerting LeishEXO's effects. Methods: Murine and in vitro ANXA1-/- models were used to study the generation of protective TH1 responses during experimental L. major infection with and without LeishEXO. Recruitment of inflammatory cells was assessed using a peritoneal cell recruitment assay and immunophenotyping, and production of inflammatory mediators was measured using a cytokine and chemokine array. Treatment of experimental models with FPR2 antagonist WRW4 and FPR1/2 agonist WKYMVm was used to delineate the role of the FPR/ANXA1 axis in LeishEXO-mediated hyperpathogenesis. Results: We established that ANXA1 deficiency prohibits LeishEXO-mediated pathogenesis and myeloid cell infection, with minimal alterations to adaptive and innate immune phenotypes. FPR2 blockade with WRW4 similarly inhibited leishmanial hyperpathogenesis, while direct activation of FPRs with WKYMVm enhanced infection and recapitulated the LeishEXO-mediated phenotype. This research describes LeishEXO's utilization of the ANXA1/FPR axis to facilitate parasitic internalization and pathogenesis, which may be leveraged in the development of therapeutics for leishmaniasis.

The Route of Leishmania tropica Infection Determines Disease Outcome and Protection against Leishmania major in BALB/c Mice

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

Low and high-dose intradermal infection with Leishmania majorand Leishmania amazonensis in C57BL/6 mice

A model of skin infection with Leishmania amazonensiswith low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 10³ or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 10³ parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensisdisplayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensisin the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.

Subcutaneous immunization against Leishmania major - infection in mice: efficacy of formalin-killed promastigotes combined with adjuvants / Imunização subcutânea na infecção do camundongo pela Leishmania major: eficácia dos promastigotos mortos pela formalina combinada com adjuvantes

Formalin-killed promastigotes (FKP) of Leishmania major, in combination with Montanide ISA 720 (MISA), BCG or alum were used in vaccination of an inbred murine model against cutaneous leishmaniasis (CL). Significant and specific increases in anti-FKP IgG responses were detected for both alum-FKP and BCG-FKP compared to MISA-FKP (p < 0.001). Significant increases in splenic lymphocyte recall proliferation was obtained in the MISA-FKP vaccinated mice compared to alum-FKP or BCG-FKP vaccinated groups (p < 0.01). The highest interferon-ã responses were observed in the BCG-FKP group followed by the MISA-FKP while the alum-FKP gave the least responses. Significantly reduced lesion sizes were obtained in the MISA-FKP group compared to the BCG/alum adjuvants-FKP vaccinated groups. Although the BCG-FKP group showed the highest IFN-ã responses, it failed to control cutaneous lesions. Significant reductions in parasite numbers were observed in the MISA-FKP and BCG-FKP vaccinated groups (p < 0.001). There was a good correlation between parasite burden and IFN-ã level indicating IFN-ã response as a sensitive parameter of the immune status. In conclusion, MISA-FKP is the most efficacious vaccine formulation against murine cutaneous leishmaniasis.

Promastigotos mortos pela formalina (FKP) de Leishmania major combinados com Montanide ISA 720 (MISA), BCG ou alumen foram usados na vacinação de modelo murino cutâneo de leishmaniose (CL). Aumento significante e específico de resposta IgG anti FKP foram detectados tanto no FKP com alumen como naquele com BCG comparados ao MISA-FKP (p < 0,001). Aumento significante da proliferação esplênica de linfócitos de memória foi obtida nos camundongos vacinados com MISA-FKP quando comparados aos grupos vacinados com alumen-FKP ou BCG-FKP (p < 0,01). As maiores respostas por interferon-gama foram observadas no grupo BCG-FKP seguido pelo MISA-FKP enquanto que o alumen-FKP deu a menor resposta. No grupo MISA-FKP foram obtidas reduções significantes do tamanho das lesões quando comparado aos grupos vacinados com BCG/adjuvante de alumen-FKP. Embora o grupo BCG-FKP tenha mostrado a maior resposta por interferon-gama, não houve controle das lesões cutâneas. Redução significante no número de parasitas foi observada tanto no grupo vacinado com MISA-FKP como no BCG-FKP (p < 0,001). Houve boa correlação entre a carga parasitária e o nível de interferon-gama indicando que a resposta do interferon-gama é parâmetro sensível do estado imunológico. Em conclusão, MISA-FKP é a forma mais eficaz de vacina contra a leishmaniose cutânea murina.

Análise TG-ROC de testes de imunofluorescência no diagnóstico de leishmaniose visceral canina / TG-ROC analysis of immunofluorescence assays in canine visceral leishmaniasis diagnosis

OBJETIVO: Analisar a acurácia do diagnóstico de dois protocolos de imunofluorescência indireta para leishmaniose visceral canina. MÉTODOS: Cães provenientes de inquérito soroepidemiológico realizado em área endêmica nos municípios de Araçatuba e de Andradina, na região noroeste do estado de São Paulo, em 2003, e área não endêmica da região metropolitana de São Paulo, foram utilizados para avaliar comparativamente dois protocolos da reação de imunofluorescência indireta (RIFI) para leishmaniose: um utilizando antígeno heterólogo Leishmania major (RIFI-BM) e outro utilizando antígeno homólogo Leishmania chagasi (RIFI-CH). Para estimar acurácia utilizou-se a análise two-graph receiver operating characteristic (TG-ROC). A análise TG-ROC comparou as leituras da diluição 1:20 do antígeno homólogo (RIFI-CH), consideradas como teste referência, com as diluições da RIFI-BM (antígeno heterólogo). RESULTADOS: A diluição 1:20 do teste RIFI-CH apresentou o melhor coeficiente de contingência (0,755) e a maior força de associação entre as duas variáveis estudadas (qui-quadrado=124,3), sendo considerada a diluição-referência do teste nas comparações com as diferentes diluições do teste RIFI-BM. Os melhores resultados do RIFI-BM foram obtidos na diluição 1:40, com melhor coeficiente de contingência (0,680) e maior força de associação (qui-quadrado=80,8). Com a mudança do ponto de corte sugerido nesta análise para a diluição 1:40 da RIFI-BM, o valor do parâmetro especificidade aumentou de 57,5 por cento para 97,7 por cento, embora a diluição 1:80 tivesse apresentado a melhor estimativa para sensibilidade (80,2 por cento) com o novo ponto de corte. CONCLUSÕES: A análise TG-ROC pode fornecer importantes informações sobre os testes de diagnósticos, além de apresentar sugestões sobre pontos de cortes que podem melhorar as estimativas de sensibilidade e especificidade do teste, e avaliá-los a luz do melhor custo-benefício.

OBJECTIVE: To analyze the accuracy of the diagnosis of two protocols of indirect immunofluorescence assays for canine visceral leishmaniasis. METHODS: Dogs from the seroepidemiological survey conducted in an endemic area of the cities of Araçatuba and Andradina, in Northwestern São Paulo state, in 2003, and in a non-endemic area of the metropolitan region of São Paulo, were used to assess two protocols of indirect immunofluorescence assay (IFA) for leishmaniasis: one using a Leishmania major heterologous antigen (IFA-BM) and another using a Leishmania chagasi homologous antigen (IFA-CH). Two-graph receiver operating characteristic (TG-ROC) analysis was used to estimate accuracy. TG-ROC analysis compared 1:20 dilution readings of the homologous antigen (IFA-CH), considered as reference test, with IFA-BM dilutions (heterologous antigen). RESULTS: The 1:20 dilution used in the IFA-CH test showed the best contingency coefficient (0.755) and the highest strength of association between the two variables studied (chi-square=124.3). Thus, it was considered the test reference dilution in comparisons with different IFA-BM test dilutions. The best IFA-BM results were obtained from 1:40 dilutions with the best contingency coefficient (0.680) and highest strength of association (chi-square=80.8). With the change in the cut-off point, recommended for the IFA-BM 1:40 dilution in this analysis, the specificity parameter value rose from 57.5 percent to 97.7 percent, even though the 1:80 dilution showed the best sensitivity estimate (80.2 percent), with the new cut-off point. CONCLUSIONS: TG-ROC analysis can provide important information about diagnostic tests, in addition to offering suggestions on cut-off points that can improve test sensitivity and specificity estimates and assessing these tests in terms of the best cost-benefit ratio.

Inhibition of caspase-8 activity reduces IFN-gamma expression by T cells from Leishmania major infection

Following infection with Leishmania major, T cell activation and apoptosis can be detected in draining lymph nodes of C57BL/6-infected mice. We investigated the mechanisms involved in apoptosis and cytokine expression following Tcellactivation. After two weeks of infection, apoptotic T cells were not detected in draining lymph nodes but activation with anti-CD3 induced apoptosis in both CD4 and CD8 T cells. Treatment with anti-FasLigand, caspase-8 or caspase- 9 inhibitors did not block activation-induced T-cell death. We also investigated whether the blockade of caspase-8 activity would affect the expression of type-1 or type-2 cytokines. At early stages of infection, both CD4 and CD8 T cells expressed IFN-gamma upon activation. Treatment with the caspase-8 inhibitor zIETD-fmk (benzyl-oxycarbonyl-Ile- Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone) reduced the proportion of CD8 T cells and IFN-gamma expression in both CD4 and CD8T cells. We conclude that a non apoptotic role of caspase-8 activity may be required for T cell-mediated type-1 responses during L. major infection.

A ativação e a morte por apoptose de linfócitos T foram observadas em linfonodos drenantes de camundongos C57BL/6 infectados com Leishmania major. Investigamos os mecanismos envolvidos na apoptose e na expressão de citocinas após a ativação de linfócitos T. Após duas semanas de infecção, embora as células apoptóticas ainda não sejam detectadas em linfonodos drenantes, células T CD4 e CD8 sofrem apoptose após ativação com anti-CD3. O tratamento com anticorpo antagonista anti-Ligante de Fas, ou com inibidores das caspases-8 e 9, não bloqueou a morte induzida por ativação das células T. Investigamos também se a inibição da atividade da caspase-8 poderia afetar a expressão de citocinas tipo-1 ou tipo-2. Nos estágios iniciais da infecção, células T CD4 e CD8 de animais infectados com L. major expressaram IFN-gama após ativação. O tratamento com o inibidor de caspase-8 zIETD (benzoil-oxicarbonil-Ile-Glu(OMe)-Thr-Asp(OMe)-fluorometilcetona) durante a estimulação de células T reduziu a proporção de células T CD8 e a expressão de IFN-gama por células T CD4 e CD8. Concluimos que a atividade não apoptótica de caspase-8 pode ser necessária para o estabelecimento da imunidade mediada por células T durante a infecção por L. major.

Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice

Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response.

Identification of novel Leishmania major antigens that elicit IgG2a response in resistant and susceptible mice

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania Major in BALB/c mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.

Nitric oxide mediates the microbicidal activity of eosinophils

There are several experimental evidences that nitric oxide (NO) is involved in the microbicidal activity of macrophages against a number of intracellular pathogens including Leishmania major, Trypanosoma cruzi, Toxoplasma gondii. It is also well known that eosinophils (EO) have microbicidal activity against many parasites such as Schistosoma mansoni, Trichenella spiralis, T. cruzi and L. amazonensis. The purpose of this study was to investigate if NO is involved in the microbicidal activity of EO against L. major. Eosinophils harvested from peritoneal cavity of rats released spontaneously after 24 and 48 hr a small amount of nitrite. This release was enhanced by the treatment of cells with IFN-gamma (200 IU/ml). This release was blocked by addition of the NO synthase inhibitor, L-NIO (100µM) into the culture. To determine the leishmanicidal activity of eosinophils the parasites were incubated with activated eosinophils with IFN-gamma and the abiblity of surviving parasites to incorporate [3H] thymidine was evaluated. IFN-gamma-activated eosinophils were able to kill L. major and to release high levels of nitrite. The ability to destroy L. major and the release of NO were completely blocked by L-NIO. These results indicate that activated eosinophils release NO which is involved in the microbicidal activity of these cells against L. major.

Leishmania (Leishmania) major-infected rhesus macaques (Macaca mulatta) develop varying levels of resistance against homologous re-infections

Seven rhesus macaques were infected intradermally with 10(7) promastigotes of Leishmania (Leishmania) major. All monkeys developed a localized, ulcerative, self-healing nodular skin lesion at the site of inoculation of the parasite. Non-specific chronic inflammation and/or tuberculoid-type granulomatous reaction were the main histopathological manifestations of the disease. Serum Leishmania-specific antibodies (IgG and IgG1) were detected by ELISA in all infected animals; immunoblot analyses indicated that numerous antigens were recognized. A very high degree of variability was observed in the parasite-specific cell-mediated immune responses [as detected by measuring delayed-type hypersensitivity (DTH) reaction, in vitro lymphocyte proliferation, and gamma interferon (IFN-gamma) production] for individuals over time post challenge. From all the recovered monkeys (which showed resolution of the lesions after 11 weeks of infection), 57.2 percent (4/7) and 28.6 percent (2/7) animals remained susceptible to secondary and tertiary infections, respectively, but the disease severity was altered (i.e. lesion size was smaller and healed faster than in the primary infection). The remaining monkeys exhibited complete resistance (i.e. no lesion) to each rechallenge. Despite the inability to consistently detect correlates of cell-mediated immunity to Leishmania or correlation between resistance to challenge and DTH, lymphocyte transformation or IFN-gamma production, partial or complete acquired resistance was conferred by experimental infection. This primate model should be useful for measuring vaccine effectiveness against the human disease

A dhfr-ts- Leishmania major Knockout Mutant Cross-protects against Leishmania amazonensis

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65 percent smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75 percent in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57 percent by SC and 49 percent by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization

Evaluation of Leishmania antigens preparation and storage for use in enzyme immunoassays

The influence of time and temperature on the storage of an alkaline antigen of L. major-like and L.(V.) braziliensis promastigotes added or not of a proteases inhibitor (PMSF) was evaluated by means of an IgG-ELISA. Antibodies in assays using L. major-like antigen stored at -20 degrees C for 6 months had a statistically lower geometric mean titer (GMT) and different 95% confidence interval limits (CL) than antigens stored otherwise, as assessed by the [quot ]t[quot ] statistic. The PMSFL. major-like antigen after storage for 6 months at a temperature of 4 degrees C had the same GMT and 95% CL displayed at time zero as well as when storage for 4 and 6 months at -20 degrees C. Significant differences were not found when L.(V.) braziliensis antigens were stored at times and temperatures mentioned; the PMSF antigen stored for 2 months at -70 degrees C resulted in a lower serum GMT and 95% CL than any other, as assessed by the [quot ]t[quot ] statistic. Antigen performance did not show any statistical difference associated to the addition of PMSF within the same species; the largest difference between antigens was that between PMSF-L. (V.) braziliensis and L. major-like without PMSF.

A influência do tempo e temperatura de estocagem de antígenos alcalinos de promastigotas de L. major-like e L. (V.) braziliensis adicionados ou não de um inibidor de proteases foi avaliada por meio de reações de IgG-ELISA. A reação que empregava o antígeno de L. major-like estocado por 6 meses a -20oC mostrou que médias geométricas dos títulos (MGT)e intervalos de confiança 95% (IC 95%) eram estatisticamente inferiores àquelas obtidas com antígenos estocados em outros intervalos de tempo, medido pela estatística "t". O antígeno PMSF-L. major-like depois de 6 meses de estocagem à temperatura de 4oC tinha a mesma MGT e IC 95% do tempo zero assim como quando ele foi estocado a -20oC por 4 e 6 meses. Não foram observadas diferenças estatisticamente diferentes com os antígenos de L. (V.) braziliensis estocados nas mesmas condições de tempo e temperatura exceto o antígeno PMSF estocado por 2 meses a -70oC que apresentou MGT e IC 95% inferiores a quaisquer outras como aferido pela estatísitca "t". Quando comparados os desempenhos dos antígenos não houve direrenças estatisticamente significantes entre a adição ou não de PMSF para qualquer dos parasitas. A análise do cruzamento entre antígenos mostrou que a maior diferença netre eles foi a do contraste entre L. (V.) braziliensis adicionado de PMSF e L. major-like sem adição de PMSF.

Reproducibility of alkaline antigens of Leishmania major-like and Leishmania (V.) braziliensis evaluated by IgG-ELISA: comparison of antigens added of a protein inhibitor (PMSF) or not

Neste trabalho estudou-se o comportamento de 10 partidas de antigenos alcalinos de L. major-like e L. (V.) braziliensis adicionados ou nao de um inibidor de protease (PMSF) avaliados em tres dias consecutivos por meio de ELISA-IgG empregando soros padrao positivo de pacientes com diagnostico de leishmaniose mucocutanea previamente testados para a presenca de anticorpos anti-leishmania por ELISA. A analise estatistica mostrou que para o antigeno de L. (V.) braziliensis adicionado de PMSF nao houve diferenca significante entre as partidas ou dias de teste. Para o antigeno sem PMSF houve diferenca entre as partidas mas nao entre os dias de teste. Uma ANOVA bi-caudal mostrou diferencas entre os antigenos com e sem PMSF. Os antigenos de L. major-like preparados com e sem adicao de PMSF mostraram diferencas significativas entre as partidas; os tres dias de teste foram significativamente diferentes para o antigeno preparado com PMSF mas somente os dias 1 e 2 o foram com o antigeno sem adicao de inibidor...