Differential regulation of E-NTPdases during Leishmania amazonensis lifecycle and effect of their overexpression on parasite infectivity and virulence.

Infections caused by Leishmania amazonensis are characterized by a persistent parasitemia due to the ability of the parasite to modulate the immune response of macrophages. It has been proposed that ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases) could be able to suppress the host immune defense by reducing the ATP and ADP levels. The AMP generated from E-NTPDase activity can be subsequently hydrolyzed by ecto-nucleotidases, increasing the levels of adenosine, which can reduce the inflammatory response. In the present work, we provide new information about the role of E-NTPDases on infectivity and virulence of L. amazonensis. Our data demonstrate that not only the E-NTPDase activity is differentially regulated during the parasite development but also the expression of the genes ntpd1 and ntpd2. E-NTPDase activity increases significantly in axenic amastigotes and metacyclic promastigotes, both infective forms in mammalian host. A similar profile was found for mRNA levels of the ntpd1 and ntpd2 genes. Using parasites overexpressing the genes ntpd1 and ntpd2, we could demonstrate that L. amazonensis promastigotes overexpressing ntpd2 gene show a remarkable increase in their ability to interact with macrophages compared to controls. In addition, both ntpd1 and ntpd2-overexpressing parasites were more infective to macrophages than controls. The kinetics of lesion formation by transfected parasites were similar to controls until the second week. However, twenty days post-infection, mice infected with ntpd1 and ntpd2-overexpressing parasites presented significantly reduced lesions compared to controls. Interestingly, parasite load reached similar levels among the different experimental groups. Thus, our data show a non-linear relationship between higher E-NTPDase activity and lesion formation. Previous studies have correlated increased ecto-NTPDase activity with virulence and infectivity of Leishmania parasites. Based in our results, we are suggesting that the induced overexpression of E-NTPDases in L. amazonensis could increase extracellular adenosine levels, interfering with the balance of the immune response to promote the pathogen clearance and maintain the host protection.

Geographic clustering of leishmaniasis in northeastern Brazil.

To determine whether disease outcomes and clades of Leishmania braziliensis genotypes are associated, we studied geographic clustering of clades and most severe disease outcomes for leishmaniasis during 1999-2003 in Corte de Pedra in northeastern Brazil. Highly significant differences were observed in distribution of mucosal leishmaniasis versus disseminated leishmaniasis (DL) (p<0.0001). Concordance was observed between distribution of these disease forms and clades of L. braziliensis genotypes shown to be associated with these disease forms. We also detected spread of DL over this region and an inverse correlation between frequency of recent DL diagnoses and distance to a previous DL case. These findings indicate that leishmaniasis outcomes are distributed differently within transmission foci and show that DL is rapidly spreading in northeastern Brazil.

American tegumentary leishmaniasis: a quantitative analysis of Langerhans cells presents important differences between L. (L.) amazonensis and Viannia subgenus.

A quantitative study was conducted on the density of Langerhans cells (LCs) CD1a+ in specimens obtained from patients with American tegumentary leishmaniasis (ATL) lesions without previous treatment, as well as from control healthy individuals. LC density was significantly higher among infected patients when compared to controls and also higher in longer term ones. Regarding parasite quantities, these were proportionally inverse and diminished in chronic patients. Localized cutaneous leishmaniasis (LCL) showed an increase in cell population when compared to diffuse cutaneous leishmaniasis (DCL). A tendency towards density increase was observed in LC Leishmania (Leishmania) amazonensis patients when compared to Leishmania (Viannia) sp. Regarding the delayed hypersensitivity test (DTH, Montenegro skin test), L. (L.) amazonensis demonstrated a peculiar behavior because it is a poor cell immune inducer, presenting--among LCL patients--higher density in negative Montenegro patients than in positive ones. Negative DTH responses are usually poor in LC, although this was not evidenced in this study, possibly due to cell reposition, in order to stimulate immune response. Such results confirm the important role of LC in ATL, while suggesting that L. (L.) amazonensis may be a good model for LC studies as APC in ATL, due to its spectral immunological and clinical behavior.

Alteraçoes ósseas na leishmaniose cutânea difusa (LCD) no Estado do Maranhão: aspectos clínicos, de imagens e histopatológico / Bone changes in cutaneous diffuse leishmaniasis (CDL) in the Maranhão state: clinical, imaging and histopathological aspects

Estudo das alterações ósseas na leishmaniose cutânea difusa (LCD) e associação com Leishmania (Leishmania) amazonensis em oito pacientes do Estado do Maranhão. Foram realizados exames de imagens, histopatológico do tecido ósseo e estudo experimental utilizando Leishmania e camundongos BALB/c. O estudo mostrou deformidades nas extremidades distais dos membros superiores e inferiores, limitação funcional, associação com lesões cutâneas e processos inflamatórios múltiplos. A histopatologia mostrou osteomielite crônica por Leishmania. O modelo experimental confirmou a infecção óssea crônica. Propõe-se a inclusão do protozoário como agente etiológico de osteomielite crônica.../Study of bone changes in Diffuse Cutaneous Leishmaniasis (DCL) and association with Leishmania (Leishmania) amazonensis in eight patients from Maranhão state. Imaging, histopathology exams of the bone tissue and experimental study using Leishmania and BALB/c mice were done. The studies showed deformity of extremities of upper and lower extremities, functional limitation, association with cutaneous lesions and multiple inflammations. The histopathology showed chronic osteomyelitis by Leishmania. The experimental model confirmed the bone chronic inflammation. We propose the inclusion of the protozoan as cause of chronic osteomyelitis

Intermediate or chronic cutaneous leishmaniasis: leukocyte immunophenotypes and cytokine characterisation of the lesion.

The American cutaneous forms of leishmaniasis include immune-responder individuals with localised cutaneous leishmaniasis (LCL) and non-responder individuals with diffuse cutaneous leishmaniasis (DCL). Patients with intermediate or chronic cutaneous leishmaniasis (ICL) have increased morbidity due to the length of their illness, atypical forms and areas of compromise. In the present study, we evaluated the expression of the leukocyte antigens (CD4, CD8, CLA: cutaneous lymphocyte antigen, CD69, CD83 and CD1a) and cytokines (IFN-gamma, IL-4, IL-10 and TGF-beta 1) in the lesions of patients with ICL (n = 18) using an immunocytochemical procedure. ICL results were compared with the information for LCL (n = 19) and DCL (n = 4). The numbers of CD4+ and CD8+ T cells in ICL were similar to those of LCL lesions, but significantly different (P < or = 0.05) from DCL lesions. LCL lesions have about half the numbers of early activated CD69+ cells as ICL, but most are CLA+ skin homing memory T cells, whereas ICL lesions have the highest number of CD69+ T cells, but about one-third of these cells expressed CLA. This suggests that the granuloma of ICL patients contains many activated T cells that are unprimed to cutaneous-launched antigens, thus contributing to an aberrant immune response. In contrast, DCL granulomas presented the lowest numbers of activated CD69+ and CLA+ cells, associated with the characteristic tolerogenic state of these patients. The immunolocalisation of cytokines showed a mixed cytokine pattern in ICL lesions with many positive cells for IL-10, TGF-beta 1, IL-4 and IFN-gamma, with a preponderance of the first two, and different from the prevalent Th1 and Th2 responses associated with LCL and DCL lesions, respectively. CD1a+ Langerhans cells were decreased (P < or = 0.05) in both ICL (271 +/- 15 cells/mm2) and DCL (245 +/- 19 cells/mm2) as compared to LCL (527 +/- 54 cells/mm2) epidermis. The percentage of IL-10+ epidermal Langerhans cells in ICL (33.69), from the total CD1a+ population, was higher than in LCL (17.45). In addition, fewer CD83+ primed Langerhans cells were present in ICL epidermis. The diminished participation of epidermal Langerhans cells, causing a defective signalling by the epidermis, in ICL lesions may account for the tissue-damaging state observed in these patients.

Leishmaniose tegumental americana com extenso acometimento de mucosas: relato de caso e revisäo da literatura / American diffuse leishmaniosis with extensive attacking of mucous membranes: case report and literature review

A leishmaniose tegumentar americana é uma doença de evolução crônica que acomete, isoladamente ou em associação, a pele e as mucosas do nariz, boca, faringe e laringe (4). É causada por protozoários do gênero Leishmania e transmitida por insetos do gênero flebotomíneos. Os autores fazem uma revisão bibliográfica e relatam um caso de leishmaniose mucocutânea em um paciente de 26 anos, que apresentava lesão no palato duro e toda orofaringe, evoluindo com emagrecimento importante. A lesão foi diagnosticada através de biópsia de mucosa, a qual evidenciou o protozoário. O interesse do relato se dá pela ascensão do número de casos de leishmaniose em nosso meio, sendo importante o diagnóstico diferencial

Leishmaniose tegumentar americana (LTA) difusa em paciente infectado pelo vírus da imunodeficiência humana (HIV): relato de caso / American cutaneous leishmaniasis (LTA) in an hiv-positive patient: a case report

Paciente acompanhado pelo Setor de Doenças Infecciosas e Parasitárias do Hospital Universitário Regional do Norte do Paraná (HURNP), desde janeiro de 1991, com o diagnóstico de infecçäo pelo vírus da imunodeficiência humana (HIV), feito na ocasiäo por teste imunoenzimático (Elisa) e Western Blot. Manteve-se assintomático até 1994, quando passou a apresentar lesäo papuloeritematosa ulcerada no punho direito, cuja biópsia permitiu estabelecer o diagnóstico de leishmaniose tegumentar americana. Foi, entäo, submetido a tratamento com N-metilglucamina, sem resposta clínica. A terapêutica com anfotericina B, iniciada em seguida, teve bom resultado, mas foi interrompida pelo paciente, que näo mais compareceu ao ambulatório. Em março de 1995, retornou ao Serviço, com quadro de monilíase oral, diarréia e lesöes maculares, melanodérmicas e polimórficas generalizadas, predominantemente localizadas nos membros superiores e inferiores. A biópsia dessas lesöes confirmou, mais uma vez, o diagnóstico de LTA, tendo sido positiva (1:640) a pesquisa no soro, por imunofluorescência indireta, de IgG anti-Leishmania braziliensis. Foi, entäo reintroduzido o tratamento com anfotericina B

Infection of BALB/c, C57B1/6 mice and F1 hybrid CB6F1 mice with strains of Leishmania mexicana isolated from Mexican patients with localized or diffuse cutaneous leishmaniasis.

Mice from the syngeneic strains BALB/c, C57B1/6 and (BALB/cxC57B1/6)F1 hybrids (CB6F1) were infected in the footpad with six different strains of Leishmania mexicana mexicana isolated from Mexican patients. Three Leishmania strains were isolated from patients with localized cutaneous leishmaniasis (LCL, the benign form of the disease) and three from patients with diffuse cutaneous leishmaniasis (DCL, the malignant form of the disease). In BALB/c mice, four Leishmania strains showed a sustained fast growth from 4 to 5 weeks postinfection until the end of the experiment (15 weeks), and the other two grew slowly up to 10 or 12 weeks after infection and then started to grow faster. In C57B1/6 mice four Leishmania strains showed a limited to moderate growth up to 6 to 11 weeks postinfection and then started to decrease. One strain showed a moderate growth during the entire experiment and one strain grew as fast as in BALB/c mice up to 11 weeks postinfection and then started to decrease. The CB6F1 hybrid behaved like the C57B1/6 parent strain with five Leishmania strains but was much more resistant to one Leishmania strain than the C57B1/6 mice. Sex of the mouse did not influence the outcome of infection. One important purpose of this work was to see if the Leishmania strains that cause DCL are intrinsically more virulent than those that cause the benign form (LCL). Although important variations in virulence among the Leishmania strains were observed, especially in BALB/c mice, they were not correlated with the type of disease caused in humans.