Effect of chaotropes in Chagas disease and leishmaniasis cross-reacting serology assays for epidemiological surveys

Abstract INTRODUCTION: Serological cross-reactivity between leishmaniasis and Chagas disease, especially at low titers, leads to difficulties of the seroepidemiological interpretation. METHODS: We have studied the ability of urea as a chaotrope to select high-avidity antibodies in IgG ELISA, thus reducing low-avidity IgG cross-reactivity in serologically positive samples in both assays. RESULTS: Using 0.5M urea for diluting the sample efficiently defined leishmaniasis or double infections in high-avidity IgG ELISA and eliminated false-positive results. CONCLUSIONS: The use of a chaotropic diluting agent is useful for improving the specificity of Chagas disease and leishmaniasis immunoassays.

Influence of apoptosis on liver and spleen resistance in dogs with visceral leishmaniosis / Influência da apoptose na resistência hepática e esplênica de cães com leishmaniose visceral

Abstract The aim of this study was to evaluate apoptosis and parasite load in the liver and spleen of dogs with visceral leishmaniosis (VL), using immunohistochemistry. Liver and spleen samples from 71 dogs with VL were used. The parasite load in the spleen and liver showed significant difference between organs in infected group (P=0.0219). The density of the parasite load in the spleen (median=2.4) was higher than liver (median=0.8). Immunodetection of apoptotic cells was predominant in lymphocytes and differ between the infected and control group in spleen (P=0.0307) and liver (P=0.0346). There was a significant correlation between apoptosis and parasite load (P = 0.0084; r=0.3104) only in the spleen of the infected group, where it was observed that, when increasing the number of apoptotic cells increases the parasitic load. It was concluded that the liver and spleen of infected dogs presented greater numbers of cells undergoing apoptosis (lymphocytes) than the control group, thus suggesting that this process may be contributing towards the survival of Leishmania in these organs, because lymphocyte in apoptosis did not have the ability to present and recognize the antigen, allowing the survival of the parasite.

Resumo O objetivo deste estudo foi avaliar a apoptose e a carga parasitária no fígado e baço de cães com leishmaniose visceral (LV), pela técnica de imuno-histoquímica. Foram utilizadas amostras de fígado e baço de 71 cães com LV. A carga parasitária no baço e fígado mostrou diferença significativa entre os órgãos no grupo infectado (P=0,0219). A densidade da carga de parasita no baço (média=2,4) foi maior do que no fígado (média=0,8). A imunodetecção de células em apoptose foi predominante nos linfócitos, com diferenças entre o grupo infectado e controle no baço (P=0,0307) e fígado (P=0,0346). Houve uma correlação positiva fraca entre apoptose e carga parasitária (P=0,0084; r=0,3104) apenas no baço do grupo infectado, onde observou-se que quando aumentava o número de células em apoptose aumentava a carga parasitária. Concluiu-se que o fígado e o baço de cães infectados apresentam um maior número de células que sofrem apoptose (linfócitos) do que o grupo controle, sugerindo que este processo possa contribuir para a sobrevivência de Leishmania nestes órgãos, pois os linfócitos em apoptose não tiveram a capacidade de apresentar e reconhecer o antígeno, permitindo a sobrevivência do parasita.

Theranostic applications of phage display to control leishmaniasis: selection of biomarkers for serodiagnostics, vaccination, and immunotherapy

Phage display is a high-throughput subtractive proteomic technology used for the generation and screening of large peptide and antibody libraries. It is based on the selection of phage-fused surface-exposed peptides that recognize specific ligands and demonstrate desired functionality for diagnostic and therapeutic purposes. Phage display has provided unmatched tools for controlling viral, bacterial, fungal, and parasitic infections, and allowed identification of new therapeutic targets to treat cancer, metabolic diseases, and other chronic conditions. This review presents recent advancements in serodiagnostics and prevention of leishmaniasis -an important tropical parasitic disease- achieved using phage display for the identification of novel antigens with improved sensitivity and specificity. Our focus is on theranostics of visceral leishmaniasis with the aim to develop biomarker candidates exhibiting both diagnostic and therapeutic potential to fight this important, yet neglected, tropical disease.

Perfil da resposta imune celular em pacientes infectados pelo HIV com leishmaniose ou tuberculose / profile of the cellular immune response in HIV-infected patients with tuberculosis or leishmaniasis

A infecção pelo HIV promove a redução do número de linfócitos T CD4+ e, consequentemente, o surgimento de doenças oportunistas. A leishmaniose visceral e a tuberculose são comumente reconhecidas como doenças oportunistas importantes e associadas ao óbito de indivíduos infectados por HIV. Ambos os patógenos, Leishmania e Mycobacterium tuberculosis (Mtb) infectam cronicamente macrófagos. A imunidade protetora associada a estas infecções envolve linfócitos Th1 produtores de IFN-g. O prejuízo na resposta imune celular causado pelo HIV perturba a resposta imune contra estes patógenos. Não são bem determinadas quais alterações imunológicas causadas pelo HIV promovem o prejuízo na resposta imune específica contra a Leishmania spp. e Mtb, induzindo o desenvolvimento de formas atípicas e graves destas infecções. Deste modo, esta tese teve como objetivo descrever o perfil da resposta imune celular aos antígenos de Leishmania spp. ou Mtb em pacientes infectados com HIV. Para tal., foram recrutados pacientes infectados por HIV e com diagnóstico de leishmaniose (HIV/LV) e tuberculose (HIV/TB). Indivíduos não infectados por HIV e diagnóstico de leishmaniose (LV) ou tuberculose (TB) forma incluídos como controles. Foram avaliadas a linfoproliferação e a frequência das subpopulações de memória dos linfócitos T CD4+...

The HIV-infection promotes reduced number of CD4+ T-lymphocytes and manifestation of opportunistic diseases. Visceral leishmaniasis and tuberculosis are commonly known as main opportunistic infections and are associated with mortality in HIV-infected individuals. Both pathogens, Leishmania and Mycobacterium tuberculosis (Mtb), infect macrophages. The protect immune response involve T-lymphocytes help 1 (Th1) and producing of IFN-g. The impairment of cellular immune response caused by HIV disrupts the immune response against these pathogens. It is unclear which immunological alterations caused by HIV infection promote the damage in specific cellular immune response against Leishmania and Mtb and induces the development of atypical and severe forms. Thus, this thesis aimed to describe the profile of the cellular immune response to Leishmania antigens or Mtb in HIV infected patients. To this end, were recruited HIV infected patients with visceral leishmaniasis (HIV/VL) and HIV infected patients with active tuberculosis (HIV/TB). Moreover, HIV uninfected individuals with VL or TB were also included as controls. Lymphoproliferation and frequency of memory CD4+...

Perfil de transcrição gênica de células humanas estimuladas com a saliva de Lu. intermedia ou de Lu / Gene transcription profile of human cells stimulated with Lu saliva. Intermedia or Lu

A saliva dos flebotomíneos transmissores do parasita Leishmania possui uma variedade de agentes farmacológicos, como anticoagulantes, vasodilatadores além de moléculas imunomoduladoras e anti-inflamatórias. A saliva de Lu. intermedia e de Lu. longipalpis provocam o aumentam da infecção por diferentes espécies de Leishmania, em modelos experimentais.Entretanto a pré-exposição à saliva de Lu. longipalpis confere proteção a infecção, enquanto a pré-exposição à saliva de Lu. intermedia causa exacerbação da doença. Neste trabalho estimulamos as células do sangue de voluntários sadios com a saliva de Lu. intermedia ou de Lu. longipalpis e, posteriormente, o RNA foi extraído e utilizado no sequenciamento em larga escala (RNAseq). O estudo demonstrou que a saliva de Lu. intermedia e de Lu.longipalpismodula a expressão de uma série de genes e os processos biológicos mais requentes são semelhantes após a estimulação com as duas diferentes salivas. Identificamos seis processos biológicos comuns às salivas dos dois lebotomineos:Taxis,Chemotaxis,Locomotory behavior, Positive regulation of immune system process, Regulation of cytokine production e Regulation of cell activation. Dentre os genes que caracterizam esses seis processos,detectamosgenes que codificam quimiocinas, citocinas além de moléculas de superfície tais como CCL19, CCL2, CCL3, CCL4, CD80, CD83, IL10, IL1B, IL4, IL6.Definimos um conjunto de genes encontrados exclusivamente na amostra estimulada com a saliva de Lu. intermedia (ADA, CCL23, CCL3, CCL3L1, CXCL11, PDGFB, PDPN, PLAU e TNFSF15). Da mesma forma, encontramos um outro conjunto de genes expresso exclusivamenteem células estimuladas com a saliva de Lu. longipalpis(ENPP2, EREG, IDO1,IL1A e VEGFA). Essas diferenças podem fornecer pistas para o desenvolvimento da leishmaniose em indivíduos continuamente expostos à saliva de Lu. intermedia.

The saliva of sand flies, vectors of leishmania parasite, has a variety of pharmacological agents, such as anticoagulants, vasodilators as well as immunomodulatory and antiinflammatory molecules. Lu. intermedia and Lu. longipalpis increase the infection by different species of leishmania in experimental models. However, the pre-exposure to Lu. longipalpis saliva provides protection to infection, while the pre-exposure Lu. intermedia saliva causes exacerbation of the disease. In this work, we stimulated peripheral blood cells from healthy volunteers with salivary glands from Lu. intermedia or Lu. longipalpis and later RNA was extracted and used in large-scale sequencing (RNAseq). This study demonstrated that Lu. intermedia and Lu. longipalpis saliva modulate the expression of a number of genes and the most frequent biological processes are similar in cells stimulated with both saliva. We identified six biological processes commonly upregulated following stimulation with saliva of the two sand flies: taxis, chemotaxis, locomotory behavior, positive regulation of immune system process, regulation of cytokine production and regulation of cell activation. Among the genes that characterize these six biological processes, we detected genes encoding chemokines, cytokines plus surface molecules such as:CCL19, CCL2, CCL3, CCL4, CD80, CD83, IL10, IL1B, IL4 and IL6. We found a set of genes upregulated exclusively in cells stimulated with Lu. intermedia saliva (ADA, CCL23, CCL3, CCL3L1, CXCL11, PDGFB, PDPN, PLAU and TNFSF15). Similarly, another set of genes was expressed only in cells stimulated Lu. longipalpis saliva (ENPP2, EREG, IDO1,IL1A and VEGFA). These differences may provide clues for the development of leishmaniasis in individuals continuously exposed to Lu. intermedia saliva.

Leishmania donovani secretory serine protease alters macrophage inflammatory response via COX-2 mediated PGE-2 production.

Leishmania parasites determine the outcome of the infection by inducing inflammatory response that suppresses macrophage’s activation. Defense against Leishmania is dependent on Th1 inflammatory response by turning off macrophages’ microbicidal property by upregulation of COX-2, as well as immunosuppressive PGE-2 production. To understand the role of L. donovani secretory serine protease (pSP) in these phenomena, pSP was inhibited by its antibody and serine protease inhibitor, aprotinin. Western blot and TAME assay demonstrated that pSP antibody and aprotinin significantly inhibited protease activity in the live Leishmania cells and reduced infection index of L. donovani-infected macrophages. Additionally, ELISA and RT-PCR analysis showed that treatment with pSP antibody or aprotinin hold back COX-2-mediated immunosuppressive PGE-2 secretion with enhancement of Th1 cytokine like IL-12 expression. This was also supported in Griess test and NBT assay, where inhibition of pSP with its inhibitors elevated ROS and NO production. Overall, our study implies the pSP is involved in down-regulation of macrophage microbicidal activity by inducing host inflammatory responses in terms of COX-2-mediated PGE-2 release with diminished reactive oxygen species generation and thus suggests its importance as a novel drug target of visceral leishmaniasis.

TLR4-mediated activation of MyD88 signaling induces protective immune response and IL-10 down-regulation in Leishmania donovani infection.

In visceral leishmaniasis, a fragmentary IL-12 driven type 1 immune response along with the expansion of IL-10 producing T-cells correlates with parasite burden and pathogenesis. Successful immunotherapy involves both suppression of IL-10 production and enhancement of IL-12 and nitric oxide (NO) production. As custodians of the innate immunity, the toll-like receptors (TLRs) constitute the first line of defense against invading pathogens. The TLR-signaling cascade initiated following innate recognition of microbes shapes the adaptive immune response. Whereas numerous studies have correlated parasite control to the adaptive response in Leishmania infection, growing body of evidence suggests that the activation of the innate immune response also plays a pivotal role in disease pathogenicity. In this study, using a TLR4 agonist, a Leishmania donovani (LD) derived 29 kDa β 1,4 galactose terminal glycoprotein (GP29), we demonstrated that the TLR adaptor myeloid differentiation primary response protein-88 (MyD88) was essential for optimal immunity following LD infection. Treatment of LD-infected cells with GP29 stimulated the production of IL-12 and NO while suppressing IL-10 production. Treatment of LD-infected cells with GP29 also induced the degradation of IKB and the nuclear translocation of NF-kB, as well as rapid phosphorylation of p38 MAPK and p54/56 JNK. Knockdown of TLR4 or MYD88 using siRNA showed reduced inflammatory response to GP29 in LD-infected cells. Biochemical inhibition of p38 MAPK, JNK or NF-kB, but not p42/44 ERK, reduced GP29-induced IL-12 and NO production in LD-infected cells. These results suggested a potential role for the TLR4-MyD88–IL-12 pathway to induce adaptive immune responses to LD infection that culminated in an effective control of intracellular parasite replication.

Animal models frr the study of leishmaniasis immunolggy / Modelos animales para el estudio de la inmunologia de la leishmaniosis

Leishmaniasis remains a major public health problem worldwide and is classified as Category I by the TDR/WHO, mainly due to the absence of control. Many experimental models like rodents, dogs and monkeys have been developed, each with specific features, in order to characterize the immune response to Leishmania species, but none reproduces the pathology observed in human disease. Conflicting data may arise in part because different parasite strains or species are being examined, different tissue targets (mice footpad, ear, or base of tail) are being infected, and different numbers (“low” 1×102 and “high” 1×106) of metacyclic promastigotes have been inoculated. Recently, new approaches have been proposed to provide more meaningful data regarding the host response and pathogenesis that parallels human disease. The use of sand fly saliva and low numbers of parasites in experimental infections has led to mimic natural transmission and find new molecules and immune mechanisms which should be considered when designing vaccines and control strategies. Moreover, the use of wild rodents as experimental models has been proposed as a good alternative for studying the host-pathogen relationships and for testing candidate vaccines. To date, using natural reservoirs to study Leishmania infection has been challenging because immunologic reagents for use in wild rodents are lacking. This review discusses the principal immunological findings against Leishmania infection in different animal models highlighting the importance of using experimental conditions similar to natural transmission and reservoir species as experimental models to study the immunopathology of the disease.

Las leishmaniosis siguen siendo un importante problema de salud pública a nivel mundial y se clasifican como categoría I por el programa TDR/WHO, debido principalmente a la ausencia de control. Muchos modelos experimentales tales como roedores, perros y monos han sido desarrollados, cada uno con características específicas, para caracterizar la respuesta inmune a las diferentes especies de Leishmania, sin embargo ninguno reproduce la patología observada en la enfermedad humana. La diversidad en los resultados obtenidos podría deberse en parte a que diferentes cepas de parásitos o especies están siendo examinadas, diferentes tejidos (cojinete plantar, oreja o base de la cola) han sido infectados y diferente número (“bajo” 1×102 y “alto” 1×106) de promastigotes metacíclicos han sido inoculados. Recientemente, nuevos enfoques han sido propuestos con el fin de obtener datos más significativos en cuanto a la respuesta inmune del huésped y a la patogénesis, de tal forma que reproduzcan lo que ocurre en la enfermedad humana. El uso de la saliva del insecto y de un número de parásitos menor en las infecciones experimentales ha permitido reproducir la transmisión natural, identificar nuevas moléculas, así como mecanismos inmunes que deberían ser considerados en el diseño de vacunas y estrategias de control. Adicionalmente, se ha propuesto como una buena alternativa el uso de roedores silvestres como modelos experimentales tanto para el estudio de las relaciones huésped-patógeno como para probar nuevas vacunas. A la fecha, el uso de reservorios naturales para estudiar la infección por Leishmania ha sido un reto, debido a la carencia de reactivos inmunológicos para uso en roedores silvestres. Esta revisión describe los principales hallazgos inmunológicos ante la infección por Leishmania, en los diferentes modelos animales, destacando la importancia del uso de condiciones experimentales similares a la transmisión natural y de reservorios como modelos experimentales para el estudio de la inmunopatología de la enfermedad.

Aspectos clínico-imunológicos da Leishmania recidiva cútis (LRC) causada por Leishmania Viannia Braziliensis / Clinical and immunological aspects of Leishmania relapse skin (LRC) caused by Leishmania Viannia Braziliensis

Introdução: As leishmanioses representam um grande problema de saúde pública sendo consideradas como doença emergente e sem controle - doença de categoria 1 - e que o foco das pesquisas deve ser a aquisição de novos conhecimentos e medidas eficazes de controle. Sob o ponto de vista clínico, a leishmaniose tegumentar (LT) nas Américas apresenta as seguintes modalidades: infecção inaparente, ganglionar, cutânea localizada (LCL), cutânea disseminada (LD), cutânea difusa (LCD), recidiva cútis (LRC) e mucosa. A primeira descrição sobre LRC ocorreu na leishmaniose no Oriente com o nome “chronic lúpus-like”, definidas como lesões de aparecimento tardio e refratariedade aos tratamentos ou como uma reação peculiar do sistema imune do hospedeiro ao parasito. A LRC é uma variante da LT convencional, uma forma de cicatrização, sendo importante entender aspectos relacionados à resposta imune do hospedeiro. Objetivo: Avaliar aspectos clínico-imunológicos e terapêuticos da LRC em pacientes da região do Vale de Jiquiriçá, Bahia, Brasil. Metodologia: O estudo foi realizado no Município de Jiquiriçá, pertencente à região Sudoeste da Bahia. Os pacientes foram atendidos no ambulatório de Doenças infecciosas e Parasitárias do Centro de Referência em Doenças Endêmicas do Vale do Jiquiriçá (CERDEJJC)...

Introduction: Leishmaniasis represent a major public health problem being considered as emerging and uncontrolled disease - disease of category 1 - and the focus of research should be new knowledge and effective control measures. In relation clinical modalities, cutaneous leishmaniasis (CL) in the Americas presents the following forms: inapparent infection, lymphnode and localized cutaneous leishmaniasis (LCL), disseminated cutaneous leishmaniasis (DL), diffuse cutaneous leishmaniasis (DCL), cutis recidivans leishmaniasis (CRL) and mucosal form. The first description of CRL occurred in Orient leishmaniasis called "chronic lupus-like lesions” defined as a late compromise and refractory to treatment or as a peculiar reaction of the immune response of the host to the parasite. The CRL is a variant of the conventional TL, a form of wound healing. Objective: To evaluate clinical, immunological and therapeutic aspects of the CRL in patients of the Jiquiriçá Valley region, Bahia, Brazil. Methodology: The study was conducted in Jiquiriçá municipality, belonging to southwest Bahia state. The patients were enrolled at the outpatient Infectious and Parasitic Diseases of the Reference Center for Endemic Diseases Jiquiriçá Valley (CERDEJJC)...

Histopathological and immunohistochemical aspects of American cutaneous leishmaniasis before and after different treatments* / Aspectos histopatológicos e imuno-histoquímicos da leishmaniose tegumentar americana antes e depois de diferentes tratamentos

The histopathology and immune responses of the healing process of leishmaniasis are still poorly studied. This study aimed to examine the histopathological and immunological aspects of lesions of patients with cutaneous leishmaniasis before and after different therapeutic methods. We studied 23 individuals grouped according to the treatments: Glucantime, Glucantime + Leishvacin and Glucantime + Leishvacin associated with Bacillus Calmette-Guerin. For analysis of the histopathological changes present in the dermis and epidermis, histological sections were stained with hematoxylin and eosin. The samples were immunostained before and after treatment to analyze the expression of interferon (IFN)-γ, interleukin (IL) 12, IL-10 and IL-4. Before treatment the presence of intense infiltrates of mononuclear cells was noticed and after treatment, even with a diagnosis of clinical cure, the subjects still showed a moderate inflammatory process. In the immunohistochemical analyses, we noticed a difference between the cytokines, with increased expression of cytokines IFN-γ and IL-12 compared to IL 10 and IL-4, both before and after treatment and, comparatively, the difference in this expression was more intense before treatment. However, the cytokine expression analyzed by treatment group showed no statistically significant difference. We conclude that a clinical cure does not always coincide with the histopathological ...

A histopatologia e as respostas imunológicas do processo de cura da leishmaniose são ainda pouco estudadas. Este estudo teve como objetivo avaliar os aspectos histopatológicos e imunológicos das lesões de pacientes com leishmaniose tegumentar, antes e após diferentes métodos terapêuticos. Foram estudados 23 indivíduos agrupados de acordo com os tratamentos: Glucantime, Glucantime + Leishvacin e Glucantime + Leishvacin associado com Bacilo Calmette-Guerin. Para a análise das alterações histopatológicas presentes na derme e epiderme, cortes histológicos foram corados com hematoxilina e eosina. Para avaliar a expressão de interferon (IFN)-γ, interleucina (IL) 12, IL-10 e IL-4 foi utilizada a técnica de imuno-histoquímica antes e após o tratamento. Antes do tratamento houve um intenso infiltrado de células mononucleares, após o tratamento, mesmo com um diagnóstico de cura clínica, apresentou-se ainda um moderado processo inflamatório. Na análise imuno-histoquímica, notamos uma diferença entre as citocinas, com expressão aumentada de citocinas IFN-γ e IL-12 em comparação com IL-10 e IL-4 tanto antes quanto depois do tratamento, e comparativamente, a diferença nesta expressão mostrou-se mais intensa antes do tratamento. No entanto, a expressão das citocinas analisadas por grupo de tratamento não mostraram diferenças ...

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.

Leishmaniasis / Leishmaniasis

La leishmaniasis se considera una enferemedad zoonótica que puede afectar al hombre al entrar en contacto con el ciclo de transmisión del parásito, convirtiéndose en una antropozoonosis. En la actualidad, es una de las seis endemias consideradas prioridad en el mundo. Las zonas de mayor prevalencia de leishmaniasis en paraguay, corresponden a los nuevos asentamientos poblacionales, especialmente de los departamentos de canindeyú y San Pedro, en ñareas rurales boscosas de tierra poco explotadas producto del avance de la frontera agrícola. El objetivo de este trabajo es brindar información actualizada y completa de la epidemiología, etiología, ciclo biológico, reservorios, histopatología, diagnostico, clínica y tratamiento. Con el propósito de que sirva al profesional de la salud como material de lectura acerca estas zoonosis tan freceuntes en nuestro país, de manera que se logre diagnosticar precozmente al paciente, y así sentar base al tratamiento adecuado.

Fagócitos mononucleares inflamatórios potencialmente envolvidos no transporte de leishmania em hospedeiros vertebrados / Fagócitos mononuclear potentially inflammatory involved in the transport of leishmaniasis in vertebrate hosts

A infecção por protozoários do gênero Leishmania modula a função de integrinas em fagócitos inflamatórios. Essa alteração pode interferir na migração celular e apresentação de antígenos ao sistema imune. O objetivo deste estudo é identificar populações de fagócitos mononuc1eares inflamatórios potencialmente envolvidos no transporte de Leishmania em hospedeiros vertebrados. Inicialmente, definimos um modelo que permite o estudo do processo de migração em um grande número de fagócitos mononucleares inflamatórios. Para isso, foi induzida peritonite em camundongos da linhagem BALB / c com a injeção de Tioglicolato e foram examinadas a cinética de migração celular para o linfonodo regional e as populações de fagócitos mononuc1eares que compõem o exsudato e sua susceptibilidade à infecção com Leishmania. O influxo celular para o peritônio foi crescente, atingindo o pico de peritonite por volta do quarto dia. A partir de então, o número de células diminuiu, atingindo um platô por volta dos quarenta dias, permanecendo estável até o centésimo dia. Inicialmente, entre 4 horas e o primeiro dia, houve aumento de polimorfonuc1eares (9+ - 4 por cento, - 12+ - 8 por cento das células), seguindo um predomínio de fagócitos mononuc1eares (primeiro ao quarto dia) e posterior aparecimento de linfócitos (quadragésimo ao centésimo dia). A migração de fagócitos mononuc1eares para os linfonodos regionais foi observada 8 horas após o estímulo inflamatório, tornando-se máxima a partir do quarto dia. A migração das células foi confirmada em ensaios de injeção e rastreamento de células marcadas, observando-se a substancial migração dessas células para o linfonodo em um período de doze-vinte e quatro horas...

Evaluación de la efectividad de la vía de inmunización transcutánea contra la leishmanisis / Evaluation of the effectivity of the patway of transcutaneous immunization again the Leismaniasis

La leishmaniasis comprende un grupo de enfermedades causadas por protozoarios parásitos del género Leishmania. Dado que la piel representa la vía de entrada natural de estos parásitos al organismo y es el sitio de desarrollo de la infección por las especies responsables de las formas cutáneas, en este trabajo se quiso evaluar la efectividad de la vía de inmunización transcutánea (IT) contra la leishmaniasis en un modelo múrido susceptible a la infección. La IT consiste en la aplicación de antígenos y adyuvantes sobre la piel hidratada, y persigue la inducción de respuestas inmunitarias específicas mediante la estimulación local de las células de Langerhans. Para ello, se aplicó un lisado de promastigotes de Leishmania (L.) mexicana (Ag), en presencia de la toxina colérica (Ag+TC), un oligonucleótido contentivo del motivo CpG (Ag+CpG), o una combinación de ambos (Ag+TC+CpG) como adyuvante, a ratones BALB/c. Se pudo demostrar que la inmunización transcutánea con Ag+TC, a través de la piel la de la oreja, induce el desarrollo de una potente respuesta celular contra el parásito, en ausencia de anticuerpos específicos, incapaz de conferir protección frente a la infección. La introducción de CpG, conocido estimulador de respuestas Th1, como segundo adyuvante, si bien mostró un efecto modulador sobre la proliferación inducida por la toxina, no logró mejorar su efecto protector frente a la infección. De manera interesante, la IT con la combinación Ag+CpG confirió cierta protección, aunque moderada y transitoria, contra un reto con el parásito. Este efecto podría estar correlacionado con un aumento en la proporción de los linfocitos T CD8+, mas no parece tener relación con la estimulación de una respuesta linfoproliferativa específica, con la producción de anticuerpos dirigidos contra el parásito, o con alteraciones en el perfil de citocinas en los órganos linfoides, de los animales protegidos con respecto a los no protegidos.

Inmunofluorescencia con Crithidia luciliae para la deteccion de anticuerpos anti-adn: imagenes atipicas y su relacion con enfermedad de chagas y leishmaniasis: imagenes atipicas y su relacion con enfermedad de chagas y leishmaniasis / Immunofluorescence assay with Crithidia luciliae for the detection of anti-DNA antibodies: atypical images and their relationship with Chagas' disease and leishmaniasis

Los anticuerpos anti-ADN nativo pueden detectarse por inmunofluorescencia indirecta con Crithidia luciliae, observándose tinción fluorescente anular del kinetoplasto que contiene ADN de doble cadena. En algunos casos pueden observarse imágenes fluorescentes en flagelo, membrana y corpúsculo basal, consideradas atípicas. Como C. luciliae pertenece a la familia Trypanosomatidae, que incluye patógenos para el hombre como Trypanosoma cruzi y Leishmania spp., se consideró que las imágenes atípicas pudieran deberse a reacciones cruzadas. Se realizaron estudios serológicos para Chagas a 105 muestras provenientes de zona endémica (Corrientes) y no endémica (Buenos Aires) para T. cruzi que presentaban imágenes atípicas con C. luciliae. La serología para Chagas resultó positiva en el 64.7% de las muestras de Buenos Aires y en el 78.3% de las de Corrientes que presentaban frente a C. luciliae imagen conjunta de membrana y flagelo. No presentaron la imagen conjunta ninguna de las muestras de dadores de sangre normales, ni de pacientes con enfermedades del tejido conectivo, excepto dos con lupus que también eran chagásicos. Todas las muestras de pacientes chagásicos analizadas frente a C. luciliae presentaron la imagen conjunta. Se estudiaron también 46 muestras de pacientes con leishmaniasis, 28 de ellos coinfectados con T. cruzi. La imagen conjunta se observó en el 88.0% de las muestras de leishmaniásicos y en el 78.5% de las de coinfectados. Los resultados sugieren que C. luciliae podría ser un sustrato alternativo, económico y de bajo riesgo para el diagnóstico serológico de enfermedad de Chagas, aunque no discrimina la infección por Leishmania. El hallazgo de la imagen conjunta en la detección de anti-ADN nativo señala la conveniencia de realizar en esos pacientes, estudios clínicos y de laboratorio para enfermedad de Chagas y leishmaniasis.

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.

Leishmaniasis: Biological understanding and beyond

An attempt has been made to briefly review the literature regarding current molecular understanding of host parasite relationship in Leishmaniasis. Sequencing of genomes for both human and important microbes including Leishmania major are in progress. The genetic information from both human and parasite have led towards molecular understanding of the interaction between parasite virulence factors and the host response factors. Gene codes for natural resistance associated macrophage protein 1 [NRAMP1], which controls the susceptibility to Leishmania donovani, Salmonella typhimurium and Mycobacterium bovis has been cloned in both mice and human. Research into the host genetics of Leishmaniasis has revealed the fundamental immunological mechanisms determining outcome of infection. It has been shown very conclusively that Tumour Necrosis Factor Alpha [TNF a] MHC and T helper cells [Th] all are associated to determine the susceptibility or resistance to leishmanial infection. Biological insights regarding parasite virulence genes identified as part of the leishmania genome project may be specifically targeted for vaccines or designing drugs for new treatments