The impact of cholesteryl ester transfer protein on the progression of cutaneous leishmaniasis.

Introduction: Pathogenesis of cutaneous leishmaniases involves parasite growth, persistent inflammation, and likely participation of lipoproteins (LP). The cholesteryl ester transfer protein (CETP), involved in LP remodeling, has been shown to participate in the inflammatory response and the evolution of infectious conditions. Methods: We evaluated the impact of the presence of CETP on infection by Leishmania (L.) amazonensis in an experimental model of cutaneous leishmaniasis using C57BL6/J mice transgenic for human CETP (CETP), having as control their littermates that do not express the protein, wild-type (WT) mice. The progression of the lesion after infection in the footpad was monitored for 12 weeks. Two groups of animals were formed to collect the plantar pad in the 4th and 12th week post-infection. Results: The lesion increased from the 3rd week onwards, in both groups, with a gradual decrease from the 10th week onwards in the CETP group compared to the WT group, showing a reduction in parasitism and an improvement in the healing process, a reduction in CD68+ cells, and an increase in CD163+ and CD206, characterizing a population of M2 macrophages. A reduction in ARG1+ cells and an increase in INOS+ cells were observed. During infection, the LP profile showed an increase in triglycerides in the VLDL fraction in the CETP group at 12 weeks. Gene expression revealed a decrease in the CD36 receptor in the CETP group at 12 weeks, correlating with healing and parasite reduction. In vitro, macrophages derived from bone marrow cells from CETP mice showed lower parasite load at 48 h and, a reduction in arginase activity at 4 h accompanied by increased NO production at 4 and 24 h compared to WT macrophages, corroborating the in vivo findings. Discussion: The data indicate that the presence of CETP plays an important role in resolving Leishmania (L.) amazonensis infection, reducing parasitism, and modulating the inflammatory response in controlling infection and tissue repair.

ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Unusual manifestation of genital cutaneous leishmaniasis in an immunocompetent patient from São Paulo, Brazil: A case report

Abstract A 31-year-old male patient developed an ulcer on the glans penis that evolved for three months without healing. We diagnosed it as leishmaniasis using polymerase chain reaction. No immunosuppression or associated diseases were observed. The patient was treated with meglumine antimoniate that cured the lesion in a month post-treatment. Here, we report this case of cutaneous leishmaniasis lesion at the unusual location of glans penis in an immunocompetent individual. The lesion likely developed due to the bite of a vector, highlighting the need for considering cutaneous leishmaniasis among differential diagnosis of sexually transmitted diseases in areas endemic for leishmaniasis.

In vivo assessment of antiretroviral therapy-associated side effects

Antiretroviral therapy has been associated with side effects, either from the drug itself or in conjunction with the effects of human immunodeficiency virus infection. Here, we evaluated the side effects of the protease inhibitor (PI) indinavir in hamsters consuming a normal or high-fat diet. Indinavir treatment increased the hamster death rate and resulted in an increase in triglyceride, cholesterol and glucose serum levels and a reduction in anti-oxLDL auto-antibodies. The treatment led to histopathological alterations of the kidney and the heart. These results suggest that hamsters are an interesting model for the study of the side effects of antiretroviral drugs, such as PIs.

Infecção por leptospiras em ovinos e caracterização da resposta inflamatória renal / Leptospirosis infection in sheep and characterization of the renal inflammatory response

As Leptospiroses são zoonoses graves de distribuição mundial que afetam o homem e outros animais. A infecção em animais, geralmente, é inaparente, ou os sintomas quando presentes são similares a outras infecções. Neste estudo foram colhidos soros de 119 ovinos e seus respectivos rins durante abate em feiras livres no município de Teresina-Piauí. Pela técnica de soroaglutinação microscópica (SAM) obtiveram-se 34 amostras sorológicas positivas para um ou mais sorovares de Leptospira spp., com taxa de ocorrência de 28,6 por cento de anticorpos anti-leptospiras, sendo 23 casos de infecção para um único sorovar e 11 com coaglutinações para dois ou mais sorovares. Dentre os sorovares patogênicos, o de maior ocorrência foi o Autumnalis (29,4 por cento). A análise histopatológica de 36 fragmentos de rins revelou alterações túbulo intersticiais em 33 (91,7 por cento) animais soro-reagentes. Lesões tubulares foram observadas em 20 (55,5 por cento) animais soro-reagentes. A presença de leptospiras, pela técnica de Warthin Starry, foi observada em 8 (22,20 por cento) amostras positivas. Pela técnica de imunoperixidase, de 20 casos analisados, foi verificada a presença de leptospira em 12 (60 por cento) de 20 amostras positivas. Nos animais soro-reagentes, o infiltrado inflamatório foi significantemente mais evidente na região córtico-medular e cortical do que na região medular (p=0,000), mas não houve diferença entre animais soro-reagentes e soro não-reagentes. Cilindros hialinos nos túbulos proximais estavam presentes em quantidade significantemente maior nos animais soro-reagentes comparados aos não-reagentes (p=0,0001). Em glomérulos, foi observada lesão discreta. Os resultados deste estudo mostram que ovinos soro-reagentes para leptospiras apresentam lesões renais túbulo intersticiais, com presença de leptospiras nos túbulos, o que confere a esses animais a condição de disseminadores da infecção.

Lepitospirosis is a serious worldwide distribution disease which affects man and other animals. The infection is generally asymptomatic in animals. In cases whose symptoms are present, symptoms are similar to other infections. In the present study serum samples from 119 sheep and their kidneys were collected during their slaughter in outdoor markets in the city of Teresina, Piauí, Brazil. The Microscopic Agglutination Test (MAT) obtained 34 positive serological samples for one or more Leptospira spp. serovar with occurrence rate of 28.6 percent of leptospiral antibodies. There were 23 cases of infection for a single serovar, and 11 cases with coagglutination for two or more serovars. Autumnalis had the highest occurrence (29.4 percent) among the pathogenic serovars. The histopathological analysis of 36 kidney fragments revealed tubulo-interstitial alterations in 33 (91.7 percent) positive animals. Tubular lesions were observed in 20 (55.5 percent) positive animals. The Warthin Starry staning revealed the presence of Leptospira in 8 (22.20 percent) of the 36 positive samples. The immunoperoxidase staining revealed the presence of Leptospira in 12 (60 percent) of 20 positive samples. The inflammatory infiltrate in the positive animals was significantly more evident in the cortical-medullar and cortical regions than in the medullar region (p=0.000), however, there was no difference between positive and negative animals. The presence of hyaline casts in the proximal tubules was significantly higher in positive animals compared to the negative ones (p=0.0001). Discrete lesion was observed in glomeruli. In conclusion, the results from this study showed that sheep which are positive for Leptospira present tubulo-intersticial renal lesions with the presence of Leptospira in the tubules, conferring to these animals the condition of asymptomatic carriers.

Vigilância da leishmaniose visceral em localidades epidemiologicamente distintas em Juruti, um município minerário do Estado do Pará, Brasil / Surveillance of visceral leishmaniasis in epidemiologically distinct locations in Juruti, a mining municipality in Pará State, Brazil

Realizaram-se ações de vigilância para leishmaniose visceral humana (LVH) em Juruti, município minerário do Estado do Pará. Foram selecionadas as localidades de Santa Maria (SM), periurbana, e Capiranga (CA), rural, com e sem LVH, respectivamente, para quatro inquéritos sorológicos semestrais (ELISA-Lisado) nas populações caninas (SM = 94; CA =45) e três levantamentos entomológicos (armadilhas luminosas CDC, 18-6hx4). Posteriormente, investigaram-se clínico e infecção por em 53 cães (SM = 28; CA = 25) com diagnóstico parasitológico (medula/linfa, Giemsa), molecular (leucócitos do sangue periférico, kDNA-PCR) e sorológico (ELISA), avaliando-se diferentes antígenos (Lisado, k39, Hsp83 - , curva ROC). Soroprevalências variaram em SM (45; 40; 15; 15 por cento) e CA (22; 30; 8,5; 0 por cento), com médias crescentes de IgG em SM(320; 378; 951; 1866; p<0,05), apesar da eutanásia de cães após segundo inquérito, e estáveis em CA (100; 159, 141; 0), onde não houve eutanásia. A frequência de spp diferiu em SM (279/296) e CA (4/6). Os resultados clínicos e laboratoriais assemelharam-se para cães de SM e CA, respectivamente, quanto à infecção (parasitológico: 86 e 84 por cento; kDNA-PCR: 100 por cento), clínico (assintomáticos: 43 e 56 por cneto; sintomáticos: 57 e 44 por cento) e especificidade no ELISA (100 por cento), mas variaram sensibilidades(Lisado: 44 e 18 por cento; Hsp83: 48 e 27 por cento; k39: 48 e 41 por cento) e níveis de IgG ( 6.400; 200). O perfil da infecção canina nas localidades com e sem transmissão de LVH diferiu apenas em níveis/evolução de IgG, o que torna necessária a temporalidade dos inquéritos, sobretudo em áreas silenciosas, isoladas com baixa densidade do vetor, onde seria dispensável a eutanásia de cães. O melhor teste sorológico foi ELISA-k39.

Surveillance actions for human visceral leishmaniasis (HVL) were carried out in Juruti, a mining municipality in Pará State. A peri-urban (Santa Maria-SM) and a rural (Capiranga-CA) location were selected with or without HVL, respectively, for the execution of four biannual serologic inquiries (lysate ELISA) in canine populations (SM = 94, CA = 45) and three entomological surveys (CDC light traps, 18-6 h x4). Subsequently, the clinical status, as well as the infection by leishmania , was investigated in 53 dogs (SM = 28; CA = 25) with parasitological (bone marrow/lymph, Giemsa), molecular(peripheral blood leukocytes, kDNA-PCR) and serological (ELISA) diagnoses assessing different antigens (lysate, k39, Hsp83 - screen test, ROC curve). Seroprevalence varied in SM (45; 40; 15; 15 percent) and in CA (22; 30; 8.5; 0 percent), presenting increasing average IgG rates in SM (320; 378; 951; 1866; p <0.05) despite the euthanasia of dogs after the second survey, and stable average IgG rates in CA (100; 159; 141; 0), where euthanasia was not conducted. The frequency rates of lutzomyia longpalpis/Lutzomia spp. differed in SM (279/296) and CA (4/6). Clinical and laboratory results were similar for dogs from SM and CA, respectively: infection (parasitological examination: 86 and 84 percent; kDNA-PCR: 100 percent), clinical status (asymptomatic: 43 and 56 percent; symptomatic: 57 and 44 percent), and specificity by ELISA (100 percent). On the other hand, sensitivity (lysate: 44 and 18 percent; Hsp83: 48 and 27 percent; k39: 48 and 41 percent) and IgG levels ( 6,400; 200) varied, respectively. The profile of canine infection in localities with or without HVL transmission differed only in terms of the level/evolution of IgG, which makes the temporality of investigations necessary, especially in quiet and isolated areas that present a low vector density and where the euthanasia of dogs would become unnecessary. The best serological test was ELISA-k39.

Immunoactivation and immunopathogeny during active visceral leishmaniasis / Imunoativação e imunopatogenia durante leishmaniose visceral ativa

Visceral leishmaniasis is caused by protozoan parasites of the Leishmania donovani complex. During active disease in humans, high levels of IFN-γ and TNF-α detected in blood serum, and high expression of IFN-γ mRNA in samples of the lymphoid organs suggest that the immune system is highly activated. However, studies using peripheral blood mononuclear cells have found immunosuppression specific to Leishmania antigens; this poor immune response probably results from Leishmania antigen-engaged lymphocytes being trapped in the lymphoid organs. To allow the parasites to multiply, deactivating cytokines IL-10 and TGF-β may be acting on macrophages as well as anti-Leishmania antibodies that opsonize amastigotes and induce IL-10 production in macrophages. These high activation and deactivation processes are likely to occur mainly in the spleen and liver and can be confirmed through the examination of organ samples. However, an analysis of sequential data from studies of visceral leishmaniasis in hamsters suggests that factors outside of the immune system are responsible for the early inactivation of inducible nitric oxide synthase, which occurs before the expression of deactivating cytokines. In active visceral leishmaniasis, the immune system actively participates in non-lymphoid organ lesioning. While current views only consider immunocomplex deposition, macrophages, T cells, cytokines, and immunoglobulins by diverse mechanism also play important roles in the pathogenesis.

A leishmaniose visceral é causada por protozoários do gênero do complexo Leishmania donovani. Durante a doença ativa no homem são detectados altos níveis de IFN-γ e de TNF-α no soro, e elevada expressão de mRNA de IFN-γ em amostras de órgãos linfóides sugerindo um estado intensamente ativado do sistema imunológico. A visão atual, no entanto, refere-se à imunossupressão específica aos antígenos de Leishmania com base em estudos utilizando células mononucleares do sangue periférico; a explicação para sua resposta deficiente seria provavelmente porque os linfócitos compometidos com antígeno de Leishmania são sequestrados nos órgãos linfóides. Para permitir a proliferação do parasito, citocinas desativadoras IL-10 e TGF-β atuariam nos macrófagos, bem como os anticorpos anti-Leishmania opsonizando amastigotas e induzindo a produção IL-10 pelos macrófagos. Estes processos de intensa ativação e desativação provavelmente ocorreriam no baço e fígado, principalmente, e confirmados com amostras de órgãos. No entanto, analisando dados seqüenciais obtidos na leishmaniose visceral no hamster, sugere-se provável presença de fatores fora do sistema imunológico como responsável pela inativação inicial de sintase induzível do óxido nítrico que ocorre antes da expressão de citocinas desativadoras. Na leishmaniose visceral ativa o sistema imunológico participa ativamente na lesão de órgãos não linfóides. Contrária à visão existente que considera somente mecanismos de deposição de imunocomplexos, observa-se na patogenia a participação de macrófagos, células T, citocinas e imunoglobulinas por mecanismo alternativo.

Cytokine profile in Montenegro skin test of patients with localized cutaneous and mucocutaneous leishmaniasis / Perfil de citocinas na intradermorreação de Montenegro em doentes de leishmaniose cutânea localizada e cutâneo-mucosa

American tegumentary leishmaniasis presents as two major clinical forms: localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). The immune response in leishmaniasis is efficiently evaluated by the response to Leishmania antigen through the Montenegro skin test (MST). Both LCL and MCL present positive response to MST, indicating that the patients present cell-mediated immunity against the parasite - Leishmania. In spite of the presence of immunity in MCL, this is not sufficient to stop disease progression and prevent resistance to treatment. In this study we demonstrated interleukin (IL) 2, 4, 5 and interferon (IFN) gamma expression in biopsies of MST of ten patients with American tegumentary leishmaniasis. The obtained results were compared between LCL (n = 5) and MCL (n = 5) patients. The MST of MCL patients displayed a higher expression of IL-2, IL-4 and IL-5, in comparison to LCL. There was no significant difference in IFN-gamma expression between groups. The obtained results suggest the role of IL-4 and IL-5 in the maintenance of the immunopathogenic mechanism of the destructive lesions that characterize MCL.

A leishmaniose tegumentar americana apresenta duas formas clínicas mais comuns: a leishmaniose cutânea localizada e a leishmaniose cutâneo-mucosa. A imunidade da leishmaniose é avaliada pela resposta ao antígeno Leishmania através da Intradermorreação de Montenegro. Estas duas formas apresentam resposta positiva, indicando que o paciente apresenta imunidade celular contra o parasita Leishmania. Apesar da presença da imunidade celular na leishmaniose cutâneo-mucosa, esta não é suficiente para barrar a progressão da doença e a resistência ao tratamento. Neste estudo, detectamos quatro citocinas por imunohistoquímica, IL-2, IL-4, IL-5 e IFN-gama nas biópsias da intradermorreação de Montenegro de pacientes com leishmaniose tegumentar americana (n = 10), cinco com leishmaniose cutânea e cinco com cutâneo-mucosa. Os resultados mostraram uma alta expressão significativa de IL-2, IL-4, IL-5 na leishmaniose cutâneo-mucosa comparada com a leishmaniose cutânea localizada, mas sem diferença significante na expressão do IFN-γ entre os grupos. Estes resultados sugerem a importância da participação da citocina IL-4 e IL-5 na manutenção do mecanismo imunopatogênico das lesões destrutivas da forma cutâneo-mucosa.

Efeitos de diferentes emulsões lipídicas sobre a expressão de moléculas de superfície envolvidas no processo de apresentação de antígenos em células mononucleares humanas in vitro / Effects of different lipid emulsions over the expression of surface molecules involved in the antigen presenting process of in vitro human mononuclear cells

Emulsões lipídicas parenterais (EL) podem modular funções leucocitárias, influenciando a resposta imune. O processo de apresentação de antígenos por leucócitos ocorre pela expressão de moléculas de superfície específicas e constitui o ponto-chave para o desencadeamento de resposta imune eficiente / Parenteral lipid emulsion (LE) can modulate leukocytes function and modify immune response. The antigen presenting process by leucocytes occurs through the expression of specific surface molecules and constitutes the key point for the start of an efficient immune response...

Imunidade inata e imunopatogenia nas leishmanioses experimentais / Innate immunity and immunopathogenesis in experimental leishmaniasis

Pesquisas na imunidade inata e na imunopatogenia das leishmanioses experimentais são apresentadas. Avaliamos a importância dos elementos inespecíficos, complemento, células “natural killer” e polimorfonucleares neutrófilos e fator de crescimento insulina-símile I na proteção e na suscetibilidade à infecção. Estudamos ainda na leishmaniose visceral, imunossupressão e a implicação da apoptose de diferentes células na evolução da infecção: a de macrófagos no desenvolvimento da infecção e a de amastigotas no controle do parasitismo. Na imunopatogenia das lesões na leishmaniose visceral, principalmente de alterações glomerulares, definimos a participação de linfócitos T, moléculas de adesão e imunoglobulinas, estas por um mecanismo diferente de deposição de imunocomplexos / Research data on innate immunity and immunopathogenesis of experimental leishmaniasis are presented. Role of non-specific elements, complement, natural killer cells, polymorphonuclear neutrophils and insulin-like growth factor-I are shown in the protection and susceptibility of the host. We also present data on immunosuppression and contribution of apoptosis of different cell populations in the evolution of visceral leishmaniasis: macrophages in the development of the infection, and of amastigotes in the control of parasitism. In immunopathogenesis of lesions in visceral leishmaniasis, mainly glomerular lesions, the involvement of CD4+ T cells, adhesion molecules and immunoglobulins, these by a mechanism not related to immune complex deposition, are revealed...

Leishmania (viannia) panamensis: induced cutaneous leishmaniasis in susceptible and resistant mouse strains

Estudamos a susceptibilidade a Leishmania (Viannia) panamensis em linhagens de camundongos BALB/c, DBA/2J, CBA/HJ e C57BL/6. Os camundongos da linhagem C57BL/6 eram resistentes, apresentando lesoes autolimitantes na pata. Os das linhagens BALB/c e DBA/2J eram suscetiveis, apresentando edema na pata, evidente aos 20 dias pos-infeccao que progrediu para uma lesao tipo tumoral nas fases tardias. Os animais da linhagem CBA/HJ apresentaram resistencia intermediaria. Em contraste a outros modelos de leishmaniose cutanea murina, a lesao nos camundongos infectados pela L. (V.) panamensis mostrou ser restrita ao local de inoculacao na pele. Estudamos tambem o desenvolvimento de resposta celular e anticorpos anti-Leishmania nas linhagens BALB/c e C57BL/6. A resposta proliferativa de celulas do linfonodo a antigenos de L. (V.) panamensis foi bifasica em ambas as linhagens...

Malária experimental: contaminaçäo de cepas e animais de biotério por Eperythrozoon Coccoides / Experimental malaria: contamination of strains and experimental animals by Eperythrozoon coccoides

Neste artigo descrevemos a contaminaçäo acidental de uma cepa de malária de roedor (Plasmodium berghei) por um hemoparasita (Eperythrozoon coccoides), levando a alteraçöes importantes no comportamento da malária experimental. A demonstraçäo do parasita foi feita por microscopia óptica e eletrônica e a fonte de contaminaçäo foi detectada em roedores normalmente utilizados na manutençäo da cepa, obtidos do mesmo biotério. As medidas disponíveis para o controle deste tipo de infecçäo säo discutidas propondo-se a utilizaçäo de tetraciclina em matrizes e posterior utilizaçäo de animais F1 näo tratados. Comenta-se a importância deste tipo de contaminaçäo experimental

Modulaçåo da resposta imune induzida por ciclofosfamida em Leishmaniose expeimental de Hamster / Effect of Cyclophosphamide in the modulation of immune response in the experimental Leishmaniose of the Hamster

Estudou-se a resposta imune da leishmaniose visceral em hamsteres inoculados com (10) x (7) amastigotas da Leishmania sp. cepa 70 por via intraperitonial. As alteraçöes imunológicas encontradas foram: produçåo de anticorpos anti-leishmânia, aumento da densidade linfocitária na zona B-dependente e reaçåo de hipersensibilidade tardia específica negativa. A modulaçåo da resposta imune com o uso da ciclofosfamida mostrou uma diminuiçåo no nível de anticorpos anti-leishmânia, depleçåo linfocitária da zona B-dependente, aparecimento da reaçåo de leishmania e sugeriu a existência de célula reguladora da reaçåo de hipersensibilidade tardia, sensível à ciclofosfamida, em hamster