Induction of haem oxygenase-1 increases infection of dog macrophages by L. infantum.

We aimed to induce and inhibit HO-1, ascertaining its effect on infection rate, parasite load and the levels of superoxide, reactive oxygen species (ROS), nitric oxide (NO), TNF-alpha and IL-10 in cultured macrophages from healthy dogs infected by Leishmania infantum. Macrophages obtained from 15 healthy dogs were cultured alone or infected with L. infantum, with or without association of HO-1 inducer and inhibitor. The infection rate and the parasite load were determined by the number of infected macrophages and number of promastigotes per macrophage, respectively. HO-1 levels and gene expression, as well as IL-10 and TNF-alpha levels were also measured in these cultures. Superoxide, ROS and NO levels in macrophages were measured through flow cytometry. Induction of HO-1 increased the infection rate and parasite load, while its inhibition decreased the infection rate and IL-10 production. There was a positive correlation between HO-1 and infection rate or parasite load. Increased infection rate was associated with decreased superoxide, ROS and NO levels. Induction of HO-1 metabolism in dogs infected by L. infantum is possibly one of the mechanisms responsible for increasing the infection of macrophages, mainly through reduction in the oxidative and nitrosative metabolisms of these cells.

M1 polarization and the effect of PGE2 on TNF-α production by lymph node cells from dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is caused by the intracellular parasite Leishmania infantum. Increased levels of arginase, nitric oxide (NO2 ) and prostaglandin E2 (PGE2 ) can play a regulatory role regarding the immune response in CVL cases. This study aimed to evaluate the arginase activity in adherent macrophages cultured from the lymph nodes of healthy and naturally infected dogs and to examine the NO2 and PGE2 levels in the supernatant of these cultures. In addition, the regulatory effect of PGE2 on the production of tumour necrosis factor (TNF-α) and interleukin-10 (IL-10) in supernatants from the total lymph node was observed in leucocyte cultures. The arginase activity was lower in the adherent macrophages cultured from the lymph nodes of naturally infected dogs and there were higher concentrations of NO2 and PGE2 in the supernatants of these cultures. Higher TNF-α and IL-10 concentrations were observed in supernatants from total lymph node leucocytes cultures, from infected dogs, and the presence of indomethacin only decreased TNF-α in the supernatant of these cultures. We conclude that the low arginase activity in macrophages suggested that M1 polarization and PGE2 were participating in the immune response and were increasing TNF-α in CVL.

Effects of P-MAPA immunomodulator on Toll-like receptor 2, ROS, nitric oxide, MAPKp38 and IKK in PBMC and macrophages from dogs with visceral leishmaniasis.

Leishmania (L.) chagasi is the etiologic agent of visceral leishmaniasis (VL) that can be transmitted to humans and dogs. VL in Brazil represents a serious public health problem; therefore, it is important to study new alternatives to treat infected dogs. In dogs, the therapeutic arsenal against canine VL is limited. The immunomodulator protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) improves immunocompetence when the immune system is impaired, but its dependence on Toll-like receptors (TLRs) and the mechanisms involved in immune response remain unclear. The in vitro action of P-MAPA on the expression of TLR2 and TLR4, reactive oxygen species (ROS), nitric oxide (NO) and p38 mitogen-activated protein kinase (p38 MAPK) and IKK phosphorylation was studied in mononuclear cells from peripheral blood and macrophages from healthy and Leishmania-infected dogs. The PBMC or macrophages were isolated and cultured with different concentrations of P-MAPA (20,100 and 200 µg/ml) in a humid environment at 37°C with 5% CO(2). Observation revealed that Leishmania-infected dogs showed a decrease in TLR2 in macrophages compared with healthy dogs and in induction with P-MAPA. ROS were increased in PBMCs from Leishmania spp.-infected dogs compared with healthy dogs and P-MAPA improved ROS production. NO production was increased in culture supernatant from macrophages stimulated by P-MAPA in both healthy and Leishmania spp. infected dogs. Treatment of macrophages from healthy dogs with immunomodulatory P-MAPA induced p38 MAPK and IKK phosphorylation, suggesting signal transduction by this pathway. These findings suggest that P-MAPA has potential as a therapeutic drug in the treatment of canine visceral leishmaniasis.

Desempenho de tilápias-do-nilo alimentadas com farelo da casca de pequi / Performance of Nile tilapia fed with bran made of pequi peel

Avaliou-se o desempenho de tilápias-do-nilo alimentadas com farelo da casca de pequi (Caryocar brasiliense). Foram utilizados 200 alevinos, com idade de 37 dias e peso corporal médio de 0,63±0,25g, distribuídos em delineamento inteiramente ao acaso, com quatro tratamentos ­ zero, 20, 40 e 60% de substituição de ração comercial por farelo da casca de pequi ­ e cinco repetições representadas por caixas de cloreto de polivinila com capacidade para 130L, contendo 10 peixes cada, totalizando 20 unidades experimentais. As características de desempenho avaliadas foram consumo de ração, peso corporal, ganho de peso, conversão alimentar, comprimento total e viabilidade criatória. A conversão alimentar ­ 1,96µ; 2,21µ; 2,63µ; 3,12µ - piorou linearmente com a inclusão do farelo de casca da pequi, enquanto as demais variáveis de desempenho não foram influenciadas pelos tratamentos. Conclui-se que a inclusão do farelo da casca de pequi na ração piora a conversão alimentar, sem alterar as demais variáveis de desempenho.

The performance of Nile tilapia fed with bran made of pequi peel was evaluated. Two hundred fingerlings, at 37 days of age and with mean body weight of 0.63±0.25 g, were distributed in a completely randomized design with four treatments ­0, 20, 40 and 60% of replacement of commercial diet with bran made of pequi peel ­with five repetitions represented by boxes of polyvinyl chloride (PVC) with capacity for 130 L, with 10 fish each, totalizing 20 experimental units. The performance characteristics evaluated were feed intake, body weight, weight gain, feed conversion, total length, and live viability. Feed conversion ­1.96µ; 2.21µ; 2.63µ; 3.12µ­ increased linearly with the inclusion of bran made of pequi peel, while the other performance variables were not influenced by treatments. The conclusion is that the inclusion of bran peel in the pequi diet worsened feed conversion, without changing other performance variables.

Distribution of human immunodeficiency virus type 1 subtypes in the state of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible "homogenous" subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country.

Fungos aeróbios no intestino grosso de borregos e de ovelhas criados em pastagens tropicais / Aerobic fungi in the large intestine of lambs and ewes raised on tropical pastures

Fungi populations were evaluated in large intestine from sheep raised in the North of Minas Gerais, Brazil. The samples were from 39 Santa Inês crossbred ewes and 30 lambs raised on Tanzania grass (Panicum maximum cv. Tanzania), both supplemented with mineral mixtures. The clinical specimens were directly collected from rectal ampoule with sterile swabs. The development of fungal mycelium was observed in all lamb samples and positive cultures were observed in 34 ewe specimens (87.2 percent). After microculture of 40 colonies from lambs, 34 isolates of the genus Aspergillus, three of Paecilomyces spp., one Acremonium sp., and one Trichoderma sp. were identified. Out of the 39 isolates from ewes, 15 Paecilomyces spp., 11 Aspergillus spp., 11 Malbranchea spp., and one Onychocola sp. were identified. The results showed the predominance of the genus Aspergillus in the lambs, while the genus Paecilomyces, Malbranchea, and Aspergillus were predominant, in ewes.

Apoptotic effect of fludarabine is independent of expression of IAPs in B-cell chronic lymphocytic leukemia.

Despite the efficiency of fludarabine in the induction of clinical responses in B-cell chronic lymphocytic leukemia (B-CLL) patients, resistance to this drug has been documented. The present study tested whether resistance to fludarabine is related to the expression of inhibitor of apoptosis proteins (IAPs) family members. We analyzed the expression of c-IAP1, c-IAP2 and XIAP, by immunocytochemistry, in 30 blood samples from B-CLL patients and correlated protein expression to fludarabine-induced apoptosis estimated by an annexin-V assay. Expression of c-IAP1, c-IAP2 and XIAP were found predominantly in the cytoplasm, and a wide range of staining intensities was observed among distinct samples. No correlation was found between the levels of IAPs expression and prognostic factors such as age, gender, lymphocyte doubling time, white blood cell count or previous treatment. The expression of IAPs also failed to predict the sensitivity to fludarabine-induced apoptosis. Alternative pathways of cell death may explain the independence of fludarabine-induced apoptosis from the high expression of IAPs.

Characterization and distribution of extracellular matrix components and receptors in GH3B6 prolactin cells.

GH3B6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50-70% of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH3B6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: alpha5beta1 (FN receptor), alpha6beta1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75%) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH3B6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.

Neuropathy of metachromatic leucodystrophy.

We describe two patients with metachromatic leucodystrophy who presented with peripheral neuropathy and had no evidence of central nervous system involvement at the time of presentation. Such an occurrence is not well recognized and the diagnosis is usually missed until the other features of the disease manifest themselves. Early recognition of this disease may become of practical importance from the point of view of therapy, judging from recent developments, and is also important from the point of view of genetic counselling. We suggest that cases of peripheral neuropathy in infancy and childhood, should be subjected to peripheral nerve biopsy when the aetiology is not obvious.