Pothomorphe umbellata: antifungal activity against strains of Trichophyton rubrum.

Trichophyton rubrum is a dermatophyte, which can cause infections in human skin, hair and nail. Pothomorphe umbellata (L.) Miq. (Piperaceae) is a native Brazilian plant, in which phytochemical studies have demonstrated the presence of steroids, 4-nerolidylcatechol, sesquiterpenes and essential oils. The objective of this study was to analyze the in vitro activity of extracts and fractions of P. umbellata on resistant strains of T. rubrum. The microdilution plate method was utilized to test Tr1, H6 and ΔTruMDR2 strains of T. rubrum; ΔTruMDR2 strain was obtained from H6 by TruMDR2 gene rupture, which is involved in multiple drugs resistance. The highest antifungal activity to all strains was observed for dichloromethane and hexane fractions of the 70% ethanolic extract which showed minimal inhibitory concentration (MIC) and minimal fungicide concentration (MFC) of 78.13 µg/mL. This antifungal activity was also obtained by 70% ethanolic extract, which presented MIC and MFC of 78.13 µg/mL to ΔTruMDR2, whereas the MIC values for Tr1 and H6 were 78.13 and 156.25 µg/mL, respectively. Our results suggest the potential for future development of new antifungal drugs from P. umbellata, especially to strains presenting multiple resistance.

Assessment of genetic relationship between Klebsiella pneumoniae and Klebsiella oxytoca samples isolated from a dental office

The present study aimed to analyze the genetic similarity between genomic profiles of thirteen Klebsiella oxytoca and seven Klebsiella pneumoniae samples isolated from two different collections carried out in different places of dental offices. Random amplified polymorphic DNA (RAPD) technique and similarity coefficients (calculated by Sorensen-Dice and simple matching) were applied to determine their genetic profile of randomic DNA sequences. The majority of the isolates of K. pneumoniae and K. oxytoca presented similar coefficient values (¡Ý 0.80). Thus, it was possible to identify that strain dissemination occurred mainly via the hands of the surgeon-dentists and, finally, to determine the genetic similarity of the strains from dental office environments.(AU)

Caracterização de biotérios, legislação e padrões de biossegurança / Animal housing: characterization, legislation and biosafety standards

O emprego de animais de laboratório em conjunto com estudos realizados em humanos fornece uma base para a compreensão de vários processos fisiológicos e patológicos importantes. Além disso, os resultados de estudos experimentais possibilitam uma melhor compreensão e segurança para o desenvolvimento de técnicas cirúrgicas na clínica médica. Por se tratar de material biológico vivo, devemos garantir sua integridade física levando-se em consideração as contaminações microbiológicas, a genética, a nutrição ea correta manipulação, a fim de se evitar que ocorram conclusões inválidas nos experimentos ou que se aumente desnecessariamente o número de animais utilizados. Em paralelo às preocupações e legislações sobre o uso de animais de laboratório, desenvolveu-se também a preocupação com o bem estar e a segurança das pessoas que manuseiam os animais de laboratório, uma vez que estas correm riscos de adquirirem doenças ocupacionais pela presença de contaminações zoonóticas ou por desenvolvimento de alergias. A prevenção requer a aplicação de modernos avanços tecnológicos no desenho do biotério e nas rotinas de trabalho. Infelizmente poucos estabelecimentos do país apresentam recursos humanos com formação apropriada e infra-estrutura básica de pesquisa que inclua os centros de criação de animais de laboratório, equivalentes àqueles existentes nos Estados Unidos e Europa.

Bioprospecção de atividade antioxidante e antimicrobiana da casca de Stryphnodendron adstringens (Mart) Coville (Leguminosae-Mimosoidae) / Bioprospection of antioxidant and antimicrobial activities in the bark of Stryphnodendron adstringens (Mart)Coville (Leguminosae-Mimosoidae)

O presente estudo teve por objetivo avaliar a classe de metabólitos secundários responsável pela atividade antioxidante e antimicrobiana de extratos da casca de Stryphnodendron adstringens (Mart.) Coville(Leguminosae-Mimosoidae), planta amplamente utilizada medicinalmente pela população. Foram obtidos extratos da casca utilizando etanol 50%, etanol 70%, acetona:água (7:3, v/v) e clorofórmio. A bioprospecção da atividade antioxidante foi realizada por meio de cromatografia em camada delgada revelada com 2,2-difenil-1-picrilhidrazila (DPPH) e a capacidade de sequestro de radicais livres utilizando DPPH foi quantificada por espectrofotometria. A bioprospecção da atividade antibacteriana foi realizada por meio de difusão em ágar e bioautografia. A cromatografia revelada com DPPH revelou atividade antioxidante na região onde foram identificadas as manchas referentes aos derivados de taninos e o extrato clorofórmico foi o que apresentou menor capacidade de sequestro de radicais livres. A atividade antimicrobiana foi comprovada para os extratos polares pela formação de halos de inibição de crescimento bacteriano e abioautografia revelou atividade na região onde foram identificadas as manchas de derivados tânicos. Assim, foi determinado que extratos da casca de S. adstringens apresentaram atividades antioxidante e antimicrobiana devido aos metabólitos secundários derivados da classe de taninos, que são os principais constituintes desta droga vegetal, de acordo com a literatura.

Analysis of susceptibility profile of Pseudomonas spp. and prevalence of bacterial samples from the surfaces of dental consulting-rooms

The aim of this research was to evaluate the susceptibility profile of Pseudomonas spp. and the prevalence of bacterial samples isolated from horizontal surfaces surrounding wash-basins used by dentists in several adjoined consulting-rooms, at points next to and at a distance from the basin, before and after surgical procedures. Our results showed a high percentage of Gram-positive cocci and Gram-negative bacilli; 34.66% were Staphylococcus spp. and 30.12% were non-fermentative Gram-negative bacilli among which Pseudomonas spp. (40.90%) was the commonest genus. Analysis of the susceptibility profile of Pseudomonas spp. isolates by determining the minimal inhibitory concentration (MIC) of 14 antibiotics showed a great variation among the strains and high rates of resistance to cefazolin, ceftazidime and aztreonan. Of the 14 antibiotics tested, 59.03% were found to be active against all the environmental isolates. Strains were resistant to aztreonan (62.82%), while susceptibility to third generation cephalosporins was variable

Antimycobacterial physalins from Physalis angulata L. (Solanaceae).

Crude extracts and fractions from aerial parts of Physalis angulata have been bioassayed for antimycobacterial activity. Fraction A1-29-12 containing physalins B, F and D exhibited a minimum inhibitory concentration value (MIC) against Mycobacterium tuberculosis H(37)Rv strain of 32 microg/mL. Purified physalin B and physalin D were also tested showing MIC values against Mycobacterium tuberculosis H(37)Rv strain of > 128 microg/mL and 32 microg/mL respectively, suggesting that physalin D plays a relevant role in the antimycobacterial activity displayed. Structural elucidation of both physalins D and B was based on detailed (13)C and (1)H NMR spectral analysis with the aid of 2D-correlation spectroscopy ((1)H-(1)H, COSY, HSQC and HMBC). The assignment of the (13)C chemical shift for physalin D is reported here for the first time.

In vitro antimycobacterial activities of Physalis angulata L.

The HIV-tuberculosis co-infection has caused an impact on tuberculosis epidemiology all over the world and the efficacies of the therapeutic schemes traditionally prescribed in the treatment of tuberculosis, such as isoniazid, rifampicin and pyrazinamide, have decreased due to the appearance of multidrug-resistant M. tuberculosis strains (MDR). This work is part of research on natural antimicrobial agents from plant extracts through bioassay-guided fractionation, by in vitro determination of the minimum inhibitory concentration (MIC) using the microdilution method with Alamar blue oxidation-reduction dye. Crude CHCl3 Physalis angulata extracts and physalin-containing fractions displayed antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium malmoense and Mycobacterium intracellulare.

Interleukin-5 modulates interleukin-8 secretion in eosinophilic inflammation.

Serum and BALF (bronchoalveolar lavage fluid) IL-8 levels and serum levels were investigated in Toxocara canis infected guinea-pigs and the role of IL-5 as a modulator of cytokine secretion was studied. Serum levels increased early in infected animals, exceeding control levels 4 h after infection, peaked between days 6 and 18, and continued to exceed control levels after 48 days of infection. Serum and BALF IL-8 levels showed the same profile as blood eosinophilia, increasing 6 days post-infection and peaking between days 18 and 24. Treatment of infected animals with anti-IL-5 Ab suppressed eosinophilia with a parallel increase in blood IL-8 levels, whereas no change was found in levels. To support our in vivo observation we carried out experiments in vitro using guinea-pig LPS-stimulated adherent peritoneal cells which release large amounts of IL-8 into the supernatants. When rIL-5 was added to LPS-stimulated cells, 65% inhibition of IL-8 release into the supernatants was observed. Pre-incubation of cells with anti-IL-5 Ab prevented the inhibition of IL-8 release into the supernatants induced by rIL-5. Our results demonstrate for the first time that TNF-alpha and IL-8 are released concomitant with or after IL-5 in the eosinophilic inflammation induced by T. canis. Moreover, in addition to showing that IL-5 is fundamental for the induction of blood eosinophilia, the present results suggest that this cytokine may play a new biological role by acting as modulator of IL-8 secretion.

Characterization of T cells that confer a high degree of protective immunity against tuberculosis in mice after vaccination with tumor cells expressing mycobacterial hsp65.

Mice vaccinated by injection with tumor cells expressing the Mycobacterium leprae gene for hsp65 acquire a remarkably high degree of protection against challenge with Mycobacterium tuberculosis. We used limiting-dilution analysis to assess the frequency of CD4+ CD8- and CD4- CD8+ splenocytes responding to mycobacterial hsp65 in such vaccinated mice. Cells of both phenotypes were present at very high and equal frequencies (approximately 1:100). Vaccination with live Mycobacterium bovis BCG also increased the frequencies of both phenotypes of hsp65-reactive cells equally (to approximately 1:2,500), whereas vaccination procedures that were not protective, with either dead BCG, hsp65 protein in incomplete Freund's adjuvant, or hsp65 mixed with tumor cells, resulted in preferential increase in CD4+ CD8- cells. Twelve CD4+ CD8- and twelve CD4- CD8+ hsp65-responsive T-cell clones were obtained and characterized. All showed conventional antigen recognition via major histocompatibility complex class II and class I pathways but differed in secretion of gamma interferon and interleukin 4 and cytotoxicity. In tests of antimycobacterial activity against M. tuberculosis, both in infected macrophages in vitro and by adoptive transfer of protection with T-cell clones injected into irradiated mice, the most effective clones were the most cytotoxic and secretion of gamma interferon made only a secondary contribution.

Differential correlation between interleukin patterns in disseminated and chronic human paracoccidioidomycosis.

In an attempt to understand better the immunoregulatory disorders in paracoccidioidomycosis (PCM), the possible correlation between interleukin pattern, lymphoproliferation, C-reactive protein (CRP) and specific antibody levels was investigated in the polarized clinical forms of this disease. We studied 16 PCM patients, eight with the disseminated disease (four under treatment and four non-treated) and eight with the chronic disease. The patients with disseminated disease exhibited high antibody titres specific to Paracoccidioides brasiliensis antigen compared with patients with the chronic form of disease. Tumour necrosis factor (TNF), IL-1, IL-6 and CRP in the serum of non-treated disseminated PCM patients were increased, which correlated positively with the low mitogenic response of peripheral blood mononuclear cells (PBMC) to phytohaemagglutinin (PHA) (P < 0.01) and with the high antibody titres (P < 0.001) of these patients. Moreover, we found in the disseminated PCM patients positive correlations between IL-1 and IL-6 (P = 0.0007); IL-1 and TNF (P = 0.0045); IL-1 and IL-6 with the high antibody titres (P = 0.0834 and P = 0.0631, respectively); IL-1, IL-6 and TNF with CRP levels. By contrast, no correlations were found with those interleukins in the treated disseminated and chronic patients or in controls. It was interesting to find an inverse correlation between IL-4 and antibody production in non-treated disseminated PCM (r = -0.4770); moreover, a significant correlation (P = 0.0820) was found in chronic PCM patients with respect to the low level of either IL-4 and antibody titres against fungus antigen. Chronic PCM patients also had IL-2 levels inversely correlated with antibody production (r = -0.6313; P = 0.0628). Inverse correlations were also observed between IL-2 and IL-6 levels in non-treated disseminated patients (P = 0.0501) and between IL-2 and IL-4 in chronic patients (P = 0.0131). The inflammatory cytokines might have a pivotal role in the genesis and in control of some aspects of the disease, such as granulomatous reaction, hypergammaglobulinaemia and depression of T cell-mediated immunity in PCM.

Protection against tuberculosis by passive transfer with T-cell clones recognizing mycobacterial heat-shock protein 65.

We have previously shown that mice vaccinated by injection with J774 macrophage-like tumour cells that expressed Mycobacterium leprae heat-shock protein (hsp) 65 as a transgene had acquired a remarkably high degree of protection against subsequent challenge with virulent M. tuberculosis. We show here that antigen-specific T cells cloned from spleens of such vaccinated animals can transfer a high level of protection to non-vaccinated recipients. The most efficient cells were of T-cell receptor (TCR) alpha beta+ and CD4- CD8+ type and specifically lysed mycobacteria-infected macrophages. These findings are consistent with the importance for protective immunity of engaging the endogenous antigen-presenting pathway to bias the immune response towards a cytolytic action against a mycobacterial antigen that is expressed at the surface of infected macrophages. TCR gamma delta+ and TCR alpha beta+ cells interacted synergistically.

Effects of cell wall deficiency on the synthesis of polysaccharide-degrading exoenzymes: a study on mycelial and wall-less phenotypes of the fz; sg; os-1 ('slime') triple mutant of Neurospora crassa.

The production of exoenzymes which degrade cellulose, polygalacturonic acid and xylan was studied in mycelial and wall-less phenotypic derivatives of Neurospora crassa obtained by vegetative selection applied to a single fz;sg;os-1 ('slime'-like) segregant (strain RCP-3) of a cross 'slime' x wild type. The unrelated stable 'slime' strain FGSC 1118 was also studied. The synthesis of polysaccharide-degrading enzymes was normally induced by polysaccharidic substrates and was sensitive to carbon-catabolite repression for both mycelium-forming phenotypes (mycelial intermediate and spheroplast-hyphal intermediate) of strain RCP-3. The stable 'slime' from RCP-3 produced cellulose-degrading activity and xylan-degrading activity constitutively but was fully sensitive to glucose repression. The stable 'slime' RCP-3 did not synthesize polygalacturonic-acid-degrading activity, even in the presence of inducers. For the stable 'slime' FGSC 1118, all of the polysaccharide-degrading activities were produced constitutively and were markedly resistant to glucose repression. The possible epigenetic origin of the different properties of stable 'slimes' RCP-3 and FGSC 1118 is considered. These results may relate to the role of the cell surface in the processing of regulatory signals which control the adaptation of the fungal cell to the nutritional environment.