Field validation of a Leishmania (Leishmania) mexicana exo-antigens ELISA for diagnosing tegumentary leishmaniasis in regions of Leishmania (Viannia) predominance

Background: Several tests are performed to obtain better accuracy when diagnosing American tegumentary leishmaniasis (ATL). It is believed that antigens released via secretion, excretion and metabolism are more specific than are antigens released by the lysis of Leishmania parasites. Such antigens are known as exo-antigens (exo-Ag) and are formed from products released by cultured parasites in a way that is similar to that in which they cause infections in hosts. Objective: We attempted to validate a Leishmania mexicana ELISA exo-Ag for ATL diagnosis in Midwestern Brazil. Methods: A total of 281 patients were included in the study. We analysed pre-treatment blood from 98 ATL patients; out of those, 85.7% and 14.3% had cutaneous and mucosal forms, respectively. Results: The exo-Ag accuracy was 83.99% (95% CI = 79.24-87.81) with a sensitivity value of 90.82% (95% CI = 83.46-95.09) and an overall specificity value of 80.33% (95% CI = 73.97-85.44). The positive predictive value and negative predictive value were 71.20% (95% CI = 62.72-78.41) and 94.23% (95% CI = 89.40-96.94), respectively. Among healthy controls, exo-Ag had a specificity of 91.25% (95% CI = 83.02-95.70); additionally, the test had specificity rates of 66.67% (95% CI = 46.71-82.03) in Chagas disease patients, 60.61% (95% CI = 43.68-75.32) in patients with rheumatic diseases, 76.92% (95% CI = 49.74-91.82) in pemphigus foliaceus patients, 87.50% (95% CI = 52.91-97.76) in leprosy patients, 87.50% (95% CI = 63.98-96.50) in VRDL-positive patients, and 77.78 (95% CI = 45.26-93.68) in deep mycosis patients. Conclusion: Based on the indicators of validity, we conclude that the results obtained in this study enable the recommendation of the exo-Ag ELISA for ATL diagnosis once it presented a reasonable accuracy compared to classical methods. Cost evaluations are necessary to completely define the role of this technique in large scale. .

Consumo de cafeína durante a gestação e transtorno de déficit de atenção e hiperatividade (TDAH): uma revisão sistemática da literatura / Caffeine consumption during pregnancy and attention deficit hyperactivity disorder (ADHD): a systematic literature review / Consumo de cafeína durante el embarazo y trastorno por déficit de atención e hiperactividad (TDAH): una revisión sistemática de la literatura

O objetivo do presente estudo foi avaliar, mediante revisão sistemática da literatura, as evidências acerca da associação entre consumo materno de cafeína durante a gestação e transtorno de déficit de atenção e hiperatividade (TDAH) na infância. A busca na literatura ocorreu de forma sistemática, em múltiplas etapas, nas bases PubMed, LILACS, BIREME e PsycINFO, com limites para artigos publicados em português, inglês e espanhol, realizados em humanos. Foram encontradas 373 referências. Dessas, somente cinco foram mantidas, por atenderem ao objetivo deste estudo. Os cinco trabalhos foram realizados em países desenvolvidos; a maioria utilizou delineamento longitudinal e foi publicada nos últimos cinco anos. Apenas um estudo encontrou associação positiva. Estudos sobre o consumo de cafeína na gestação e TDAH são escassos, com resultados controversos e se deparam com várias dificuldades metodológicas, como falta de padronização na definição do desfecho.

This aim of this study was to conduct a systematic literature review on the association between maternal caffeine intake during pregnancy and attention deficit hyperactivity disorder (ADHD) in childhood. The systematic multiple-stage literature search in PubMed, LILACS, BIREME, and PsycINFO was limited to research in human subjects and published in Portuguese, English, and Spanish. A total of 373 references were retrieved. Of these, only five met the study's objectives and were kept in the review. Most of the studies employed a longitudinal design, were conducted in developed countries, and were published in the last five years. Only one study found a positive association. Studies on caffeine consumption during pregnancy and ADHD are scarce, with conflicting results and several methodological difficulties such as lack of standardized outcome measures.

El objetivo de este estudio fue evaluar, a través de una revisión sistemática de la literatura, evidencias sobre la asociación entre el consumo de cafeína durante el embarazo y el trastorno por déficit de atención e hiperactividad (TDAH) en la infancia. Se realizó una búsqueda sistemática en la literatura, por etapas múltiples, en PubMed, LILACS BIREME y PsycINFO, limitándose a artículos publicados en portugués, inglés y español, realizados en estudios sobre humanos. Fueron localizadas 373 referencias. De ellas, apenas se mantuvieron cinco, por cumplir el objetivo de este estudio. Los estudios se realizaron en países desarrollados; el diseño longitudinal fue el más utilizado y se trata de publicaciones de los últimos cinco años. Sólo un estudio encontró asociación positiva. Los estudios sobre el consumo de cafeína durante el embarazo y el TDAH son escasos, con resultados controvertidos, y enfrentan varias dificultades metodológicas, como la no estandarización de la evaluación del resultado.

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during metacyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

Immunological correlates of cure in the first American Cutaneous Leishmaniasis patient treated by immunotherapy in Argentina: A case report. / Correlatos inmunológicos de curación en el primer paciente con Leishmaniasis Cutánea Americana tratado con inmunoterapia en Argentina: Reporte de un caso

A patient with localized cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis infection was treated with an antigen containing heat-killed L. (L.) amazonensis promastigotes plus BCG. Expression of T-cell differentiation, memory and senescence receptors markers were analyzed on T cell subpopulations, in order to establish the correlation between the percentages of expression of these receptors and his clinical status, at different stages of his follow up. The following case reports on the achievement of a successful clinical outcome with complete resolution after receiving immunotherapy. A thorough clinical and immunological follow up supporting the healing process of this patient’s lesion is presented in detail.

Un paciente con leishmaniasis cutánea localizada producida por Leishmania (Leishmania) amazonensis fue tratado con un antígeno compuesto por promastigotes de L. (L.) amazonensis muertos por calor combinado con BCG. Se analizó la expresión de distintos receptores de diferenciación, de memoria y de senescencia en las subpoblaciones de células T, con el fin de establecer una relación entre los porcentajes de expresión de dichos receptores y la clínica del paciente en diferentes momentos del seguimiento. Se reporta en este caso un resultado exitoso, con resolución completa de la lesión después de recibir la inmunoterapia, y se presenta en detalle un seguimiento clínico e inmunológico completo durante el proceso de curación.

El rol de tres pruebas de ELISA con antígenos de promastigotes de Leishmania braziliensis, L. amazonensis y L. guyanensis en el diagnóstico de leishmaniasis tegumentaria / Role of three ELISA tests using promastigote homogenates of Leishmania braziliensis, L. amazonensis and L. guyanensis in the diagnosis of tegumentary leishmaniasis

Es importante conocer si la variabilidad de especies de Leishmania circulantes en una región afecta la performance de las pruebas de ELISA estandarizadas para el diagnostico de la leishmaniasis. El objetivo de este trabajo fue analizar la reactividad de la prueba de ELISA utilizando homogenados de promastigotes de Leishmania (V.) braziliensis (ELISAb), L (L) amazonensis (ELISAa) y L (V.) guyanensis (ELISAg) frente a distintos grupos de sueros. Se estudiaron muestras de personas con leishmaniasis cutánea (n = 37), leishmaniasis mucocutánea (n = 8), no infectados (n = 52), infectadas por Trypanosoma cruzi (n = 11) e infecciones mixtas (n = 14). Se calcularon las sensibilidades, especificidades, cut off, valores predictivos, y se compararon las tres pruebas usando ANOVA, índice de concordancia kappa, comparación de curvas ROC e intervalos de confianza construidos por el método de bootstrap. Se encontraron diferencias significativas al comparar los niveles de DO de los sueros de pacientes con leishmaniasis cutánea respecto a los controles negativos, pero no se encontraron diferencias entre pruebas. Las sensibilidades calculadas fueron de 84.6% para ELISAb y ELISAa y de 88.5 para ELISAg, mientras que el valor de especificidad para las tres pruebas fue de 96.2. El índice de concordancia kappa y la comparación de curvas ROC mostraron performances similares para las tres pruebas (p = 0.225). La elevada reactividad obtenida para estas ELISAs frente a sueros de pacientes con leishmaniasis mucocutánea indica un importante potencial de esta técnica como complemento en el diagnóstico de la enfermedad.

It is important to know whether the variability of species of Leishmania parasites circulating in a region affects the performance of the ELISA test for the diagnosis of leishmaniasis. Therefore, the aim of this study was to analyze the reactivity of the ELISA using homogenates of promastigotes of Leishmania (V.) braziliensis (ELISAb), Leishmania (L) amazonensis (ELISAa) and Leishmania (V.) guyanensis (ELISAg) against different sera groups. Samples from individuals with cutaneous leishmaniasis (n = 37), mucocutaneous leishmaniasis (n = 8), healthy controls (n = 52), persons infected with Trypanosoma cruzi (n = 11) and mixed infections (n = 14) were included in the study. We calculated sensitivities, specificities, cut offs, and predictive values for the three tests and compared them using ANOVA, kappa index, ROC curves comparison, and confidence intervals calculated by the bootstrap method. Significant differences were found when comparing the OD levels of sera from patients with cutaneous leishmaniasis against healthy controls, but there were no differences when comparing the different ELISAs. The sensitivities calculated for ELISAb and ELISAa were 84.6 and of 88.5% for ELISAg, while the value of specificity for the three tests was 96.2. The kappa index (0.87) and comparison of ROC curves showed similar performance for the three ELISAs (p = 0.225). The high reactivity obtained for these ELISAs in sera of patients with mucocutaneous leishmaniasis indicates this test as an important complement in the diagnosis of the disease.

Determinación de la especificidad de IgA sérica producida en respuesta a antígenos de Leishmania (Leishmania) mexicana en leishmaniosis murina / Determination of the specificity of seric IgA produced in response to antigens of Leishmania (Leishmania) mexicana in murine leishmaniasis

En la leishmaniosis experimental, la función de los anticuerpos no está completamente clara, ya que algunos autores consideran que dichas proteínas no participan en la protección contra la infección; sin embargo, estudios histopatológicos en lesiones con leishmaniosis humana y experimental, muestran infiltrados de células plasmáticas positivas para IgA y secreción de IgM, IgG e IgA que podrían mediar la formación de complejos inmunológicos con antígenos parasitarios o propios, favoreciendo la necrosis lo que conlleva a la eliminación del parásito. En este trabajo se determinó si la IgA sérica en el modelo murino posee reactividad específica contra antígenos de Leishmania (Leishmania) mexicana de utilidad diagnóstica. Para ello, se utilizaron ratones susceptibles y resistentes a leishmaniosis cutánea, demostrándose mediante ELISA indirecta que la IgA sérica de ratones susceptibles es elevada en comparación con la producida por ratones resistentes. Aunque otros estudios en modelos murinos demuestran que la IgG sérica de ratones infectados con L. (L) mexicana presenta reactividad cruzada con antígenos parasitarios no relacionados obtenidos de Trypanosoma cruzi, al analizar la especificidad de IgA por antígenos de L. (L) mexicana y T. cruzi, mediante Western Blot, se demostró que la IgA sérica de ratones infectados con T. cruzi también reaccionan con antígenos de L. (L) mexicana, estos hallazgos sugieren que la IgA puede ser útil para el manejo clínico y pronóstico de la enfermedad.

In experimental leishmaniasis, the role of antibodies is not entirely clear, as some authors consider that these proteins are not involved in protection against infection. However, histopathological studies in human and experimental leishmaniasis lesions, show plasma cell infiltrates positive for IgA and secretion of IgM, IgG and IgA could mediate the formation of immune complexes with parasite antigens or self components, favoring necrosis leading to the elimination of the parasite. In this study, we determined if the serum IgA in the murine model has specific reactivity against antigens of Leishmania (Leishmania) mexicana of diagnostic utility. To do this, we used mice either susceptible or resistant to cutaneous leishmaniasis, and demonstrated by indirect ELISA that serum IgA is elevated in susceptible mice compared with that produced by resistant mice. Although other studies in murine models show that the serum IgG from mice infected with L. (L) mexicana present cross reactivity with unrelated parasite antigens derived from Trypanosoma cruzi, the analysis of the specificity of IgA by antigens of L. (L) mexicana and T. cruzi, by Western Blot, showed that the IgA serum of mice infected with T. cruzi reacts too with antigens of L. (L) mexicana. These findings suggest that IgA may be useful for the clinical management and prognosis of the disease.

Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human leishmaniasis in CBA mice

Leishmania amazonensis causes different diseases depending on the host and parasitic virulence factors. In this study, CBA mice were infected with L. amazonensis isolates from patients with localized (Ba125), diffuse cutaneous (Ba276) or visceral leishmaniasis (Ba109). Mice infected with Ba125 and Ba276 progressed rapidly and lesions displayed an infiltrate rich in parasitized macrophages and were necrotic and ulcerated. Ba109 induced smaller lesions and a mixed inflammatory infiltrate without necrosis or ulceration. Ba109 induced an insidious disease with lower parasite load in CBA mice, similar to human disease. Levels of IFN-γ, IL-4 and IL-10 did not differ among the groups. Because all groups were unable to control the infection, expression of IL-4 associated with low production of IFN-γ in the early phase of infection may account for susceptibility, but others factors may contribute to the differences observed in inflammatory responses and infection progression. Evaluation of some parasitic virulence factors revealed that Ba276 exhibits higher ecto-ADPase and 5'-nucleotidase activities compared to the Ba109 and Ba125 strains. Both Ba276 and Ba125 had higher arginase activity in comparison to Ba109. Finally, these data suggest that the differences in enzyme activities among parasites can account for differences in host inflammatory responses and infection progression.

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.

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.

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasiinfection. These immunogenic preparations were composed of Leishmania amazonensisor Leishmania braziliensisantigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasiby intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensisantigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasiantigen. No change was detected in the production of IFN-γ. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.

Reacción de antígenos de Leishmania (Leishmania) mexicana con sueros de pacientes con leishmaniosis cutánea de Sinaloa, México / Reaction of Leishmania (Leishmania) mexicana antigens by sera of patients with cutaneous leishmaniasis from Sinaloa, Mexico

OBJETIVO: Detectar los antígenos de Leishmania (Leishmania) mexicana que reaccionan con sueros de pacientes con leishmaniosis cutánea (LC) de Sinaloa, México. MATERIAL Y MÉTODOS: Un extracto crudo de L. (L.) mexicana fue usado como antígeno para Western blots 2-D empleando sueros de cinco pacientes con LC y controles originarios de Sinaloa, México, durante el 2008. RESULTADOS: Cinco antígenos fueron detectados sólo por los sueros de los cinco pacientes estudiados; estos son: 26 kDa (pI 7.8), 27 kDa (pI 8.1), 28 kDa (pI 8.6), 29 kDa (pI 8.5) y 31 kDa (pI 9.0). CONCLUSIONES: Se detectaron nuevos antígenos de L. (L.) mexicana potencialmente inmunodominantes, lo que sugiere a este parásito como el agente causal de la LC en Sinaloa.

OBJECTIVE: To detect Leishmania mexicana antigens reacting with sera of patients with cutaneous leishmaniasis (CL). MATERIAL AND METHODS: A crude extract of L. mexicana was used as antigen for 2-D Western blot using sera from 5 patients with CL and controls from Sinaloa, Mexico during 2008. RESULTS: Five antigens were detected in the five infected patients analyzed; their molecular weights and isoelectric points were: 26 kDa (pI 7.8), 27 kDa (pI 8.1), 28 kDa (pI 8.6), 29 kDa (pI 8.5) and 31 kDa (pI 9.0). CONCLUSION: New potentially immunodominant L. mexicana antigens were detected, suggesting that this parasite could be the species responsible for human infection in Sinaloa.

Comparison of serological and parasitological methods for cutaneous leishmaniasis diagnosis in the state of Paraná, Brazil

We evaluated the effectiveness of serological and parasitological methods for cutaneous leishmaniasis (CL) diagnosis in patients from the central region of Paraná state, southern Brazil. Five groups were compared: clinical diagnosis, parasitological diagnosis, communicants, inhabitants of a non-endemic area and carriers of other etiologies. Two antigens were prepared from promastigotes of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis for indirect immunofluorescence assay, ELISA and immunoblotting. The parasitological approaches detected 79.3 percent of the patients with a clinical diagnosis; the parasites were identified by PCR as L. (V.) braziliensis. Serological methods showed 95 percent sensitivity for homologous antigens. Immunoblotting revealed specific proteins for diagnosis of CL and detected 96.6 percent of the patients when L. (V.) braziliensis was used as an antigen, and 83.3 percent with L. (L.) amazonensis. This study demonstrated the importance of differential diagnosis for leishmaniasis; the association of two or more indirect methods increased diagnosis sensitivity.

Aureobasidium-Derived Soluble Branched (1,3-1,6) beta-Glucan (Sophy beta-glucan) Enhances Natural Killer Activity in Leishmania amazonensis-Infected Mice

The beta-glucans derived from yeast cell walls have been reported for having many immunomodulatory activities in vivo and in vitro. In this study, Aureobasidium-derived soluble branched (1,3-1,6) beta-glucan (Sophy beta-glucan) was checked for natural killer (NK) activity and for the production of IFN-gamma and IL-4 in Leishmania amazonensis infection. The main experiment was performed with a group of female C57BL/6 and BALB/c mice, orally supplemented with 5% of Sophy beta-glucan and infected with promastogotes of L. amazonensis (1 x 10(7)) into the footpad. Increase in the footpad thickness with time was observed in BALB/c mice in spite of the oral Sophy beta-glucan supplement, but it was less in C57BL/6 mice. The difference in overall mean footpad thickness between 'infection only' versus 'infection + glucan' groups was statistically significant (P < 0.001). High NK activity in C57BL/6 than BALB/c mice was observed in 'glucan only' group compared to the control group and also in 'infection + glucan' group compared to 'infection only' group. The difference in the NK activity among these groups was significant (P < 0.05). The IFN-gamma level increased at weeks 7 and 8 post-infection in C57BL/6 mice and was significantly high in 'infection + glucan' group compared to the 'infection only' group (P < 0.05). IL-4 levels did not increase up to detectable levels throughout the study. The results led a conclusion that Sophy beta-glucan enhances NK activity and cellular immunity in L. amazonensis-infected mice.

Serological reactivity of different antigenic preparations of Leishmania (Leishmania) amazonensis and the Leishmania braziliensis complex / Reatividade sorológica frente a diferentes preparações antigênicas de Leishmania (Leishmania) amazonensis e do complexo Leishmania braziliensis

Total antigen from Leishmania (Leishmania) amazonensis and isolates from the Leishmania braziliensis complex, along with their respective antigenic fractions obtained by affinity chromatography on concanavalin-A-Sepharose and jacalin-agarose columns evaluated using immunoenzymatic ELISA assay. For this, serum samples from 229 patients were used, grouped as American tegmental leishmaniasis (nº=58), visceral leishmaniasis (nº=28), Chagas disease (nº=49), malaria (nº=32), tuberculosis (nº=13) and healthy volunteers (nº=49). Samples from American tegmentary leishmaniasis showed higher reactivity with antigens isolated from the Leishmania braziliensis complex than with antigens from Leishmania amazonensis (p<0.001). ELISA assays showed a sensitivity range from 60 percent to 95 percent with antigens isolated from the Leishmania braziliensis complex. There was marked nonspecific reactivity among serum samples with the use of antigenic fractions binding with concanavalin-A and jacalin from both Leishmania complexes, in comparison with other antigens (p<0.001). The results presented in this study suggest that the use of homologous antigens increases the efficiency of anti-Leishmania immunoglobulin detection, which may be very valuable for diagnostic purposes.

Antígeno total de Leishmania (Leishmania) amazonensis e isolado do complexo Leishmania brazilienis, assim como suas respectivas frações antigênicas obtidas por cromatografia de afinidade em coluna de concanavalina-A ligada a sepharose e Jacalina ligada a agarose foram avaliadas por ensaio imunoenzimático ELISA. Para tanto, foram utilizadas amostras de soros de 229 pacientes agrupadas em leishmaniose tegumentar americana (nº=58), leishmaniose visceral (nº=28), doença de Chagas (nº=49), malaria (nº=32), tuberculose (nº=13) e voluntários saudáveis (nº=49). Houve maior reatividade das amostras de leishmaniose tegumentar americana com a utilização dos antígenos obtidos do isolado do complexo Leishmania braziliensis quando comparado com antígenos de Leishmania amazonensis (p<0,001). Observou-se ainda que a sensibilidade do teste ELISA variou de 60 a 95 por cento entre os antígenos obtidos do isolado do complexo Leishmania braziliensis. Houve acentuada reatividade inespecífica das amostras de soros com a utilização das frações antigênicas ligantes de Concanavalina-A e Jacalina de ambos os complexos Leishmania em comparação aos demais antígenos (p<0,001). Os resultados apresentados no presente trabalho sugerem que a utilização de antígenos homólogos aumentam a eficiência de detecção de imunoglobulina anti-Leishmania o que pode ser de grande valia para o propósito de diagnóstico.

Role of Leishmania (Leishmania) amazonensis amastigote glycosphingolipids in macrophage infectivity

The role of glycosphingolipids (GSLs) present in amastigote forms of Leishmania (Leishmania) amazonensis during infection of macrophages was analyzed, with particular emphasis on GSLs presenting the terminal Galpß1-3Galpa disaccharide. Macrophage invasion by L. (L.) amazonensis amastigotes was reduced by 37 percent when the disaccharide Galpß1-3Galp (1 mM) was added to the culture medium. The putative macrophage receptor/lectin for ß-Gal-globotriaosylceramide (Galpß1-3Galpa1-4Galpß1-4Glc pß1-1Cer) and other structurally related GSLs from L. (L.) amazonensis amastigotes were analyzed by micelles and parasite binding assay to peritoneal macrophage proteins fractionated by SDS-PAGE under nonreducing conditions. Micelles containing purified amastigote GSLs or a suspention of L. (L.) amazonensis amastigotes fixed with 2 percent formaldehyde were incubated with nitrocellulose membrane containing the macrophage proteins transferred by Western blotting. Binding of micelles containing purified GSLs from amastigote forms or fixed L. (L.) amazonensis amastigotes to nitrocellulose membrane was probed using monoclonal antibody ST-3, which recognizes the glycoepitope Galpß1-3Galpa1-R present either in the micelle preparation or on the amastigote surface. Macrophage protein with molecular mass ~30 kDa bound the amastigote GSL and appeared to be a doublet on electrophoresis. The specificity of this interaction was confirmed using fixed L. (L.) chagasi amastigotes, which do not express GSLs such as ß-Galp-globotriaosylceramides, and which do not bind to 30-kDa protein.

Interferon-gamma is required for the late but not early control of Leishmania amazonensis infection in C57Bl/6 mice

The critical role of interferon-gamma (IFN-g) in the resistance of C57Bl/6 mice to Leishmania major is widely established but its role in the relative resistance of these animals to L. amazonensis infection is still not clear. In this work we use C57Bl/6 mice congenitally deficient in the IFN-g gene (IFN-g KO) to address this issue. We found that IFN-g KO mice were as resistant as their wild-type (WT) counterparts at least during the first two months of infection. Afterwards, whereas WT mice maintained lesion growth under control, IFN-g KO mice developed devastating lesions. At day 97 of infection, their lesions were 9-fold larger than WT controls, concomitant with an increased parasite burden. At this stage, lesion-draining cells from IFN-g KO mice had impaired capacity to produce interleukin-12 (IL-12) and tumour necrosis factor-a in response to parasite antigens whereas IL-4 was slightly increased in comparison to infected WT mice. Together, these results show that IFN-g is not critical for the initial control of L. amazonensis infection in C57Bl/6 mice, but is essencial for the developmente of a protective Th1 type immune response in the later stages.

Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin®)

Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin®), produced by Biobrás (Biochemistry of Brazil ), Montes Claros, State of Minas Gerais, Brazil, were identified and purified by polyacrylamide electrophoresis gel and electroelution. C57BL/10 mice were vaccinated with proteins with estimated molecular weights of 42, 46, 63, 66, 73, 87, 97, and 160kDa in three doses of 30æg of each protein at 15-day intervals combined with 250æg of Corynebacterium parvum followed by a challenge infection with 10(5) infective promastigotes from Leishmania (Leishmania) amazonensis. The ability of these proteins to induce immune response and protection was analyzed. No statistical difference was observed in the level of IFN-g induced by proteins in vaccinated groups in comparison with control groups. Six months after challenge infection, protection levels of 28.57; 42.86; 57.14; 42.86; 42.86, 57.14; 42.86 and 57.14 percent were demonstrated for each purified protein

Indivíduos não expostos à Leishmania diferem na resposta imune inicial ao parasito, in vitro e in vivo / Non exposed individuals difer in their initial immune responde, in vivo and in vivo

(...) Neste trabalho, foram avaliados os eventos iniciais da resposta imune humana à Leishmania amazonensis, através da sensibilização in vitro de CMSP com promastigotas vivas de indivíduos sadios, sem exposição prévia à Leishmania. Os mesmos voluntários foram submetidos à vacinação contra Leishmania amazonensis com uma vacina monocepa produzida pela BIOBRAS, CMSP de 32 voluntários de voluntários sadios foram sensibilizados in vitro com promastigotas vivas, e, em diferentes momentos, foram determinadas as concentrações de citocinas nos sobrenadantes das culturas e o fenótipo das células. (...) Houve uma correlação positiva significativa para todas as citocinas da resposta in vitro, com a da resposta pós vacina inicial, com 40 dias, sugerindo que o sistema de sensibilização in vitro, utilizado neste estudo, tem a capacidade de prever a resposta de sensibilização pós vacinação. Isto indica que este sistema de sensibilização in vitro sisgnifica um importante instrumento no estudo da imuno-regulação da leishmaniose e na triagem de antígenos para vacina.

Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-gamma by lymphocyte from subjects vaccinated with Leishvacin. The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mu/g of each protein at 15 days interval. One hundred mu/g of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86 percent of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14 percent of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43 percent protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60) and 10.77UI/ml of IFN-gamma production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14 percent of protection was found after the first challenge and 28.57 percent after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis