McCoy cell line as a possible model containing CD4+ receptors for the study of HIV-1 replication

Several studies have recently shown the use of recombinant rabies virus as potential vector-viral vaccine for HIV-1. The sequence homology between gp 120 and rabies virus glycoprotein has been reported. The McCoy cell line has therefore been used to show CD4+ or CD4+ like receptors. Samples of HIV-1 were isolated, when plasma of HIV-1 positive patients was inoculated in the McCoy cell line. The virus infection was then studied during successive virus passages. The proteins released in the extra cellular medium were checked for protein activity, by exposure to SDS Electrophoresis and blotting to nitro-cellulose filter, then reacting with sera of HIV positive and negative patients. Successive passages were performed, and showed viral replication, membrane permeabilization, the syncytium formation, and the cellular lysis (cytopathic effect). Flow cytometry analysis shows clear evidence that CD4+ receptors are present in this cell line, which enhances the likelihood of easy isolation and replication of HIV. The results observed allow the use of this cell line as a possible model for isolating HIV, as well as for carrying out studies of the dynamics of viral infection in several situations, including exposure to drugs in pharmacological studies, and possibly studies and analyses of the immune response in vaccine therapies.

Ciprofloxacin-imprinted polymeric receptors as ionophores for potentiometric transduction

A 3D-mirror synthetic receptor for ciprofloxacin host–guest interactions and potentiometric transduction is presented. The host cavity was shaped on a polymeric surface assembled with methacrylic acid or 2-vinyl pyridine monomers by radical polymerization. Molecularly imprinted particles were dispersed in 2-nitrophenyl octyl ether and entrapped in a poly(vinyl chloride) matrix. The sensors exhibited a near-Nernstian response in steady state evaluations. Slopes and detection limits ranged from 26.8 to 50.0 mV decade−1 and 1.0 × 10−5 to 2.7 × 10−5 mol L−1, respectively. Good selectivity was observed for trimethoprim, enrofloxacin, tetracycline, cysteine, galactose, hydroxylamine, creatinine, ammonium chloride, sucrose, glucose, sulphamerazine and sulfadiazine. The sensors were successfully applied to the determination of ciprofloxacin concentrations in fish and in pharmaceuticals. The method presented offered the advantages of simplicity, accuracy, applicability to colored and turbid samples and automation feasibility, as well as confirming the use of molecularly imprinted polymers as ionophores for organic ion recognition in potentiometric transduction.

Functional and molecular characterization of SR45, a plant-specific factor involved in sugar and stress signaling in Arabidopsis thaliana

Dissertation presented to obtain the Ph.D degree in Molecular Biology

Estratégia Sistematicamente Invasiva nas Síndromes Coronárias Agudas sem Supradesnivelamento do Segmento ST: Será a Idade um Factor Limitante?

Introdução: A estratégia terapêutica sistematicamente invasiva das síndromes coronárias agudas (SCA) é actualmente aceite como segura e eficaz, sendo crescentes as evidências da sua superioridade em relação a uma atitude conservadora. O doente idoso, atendendo à sua maior susceptibilidade, é frequentemente excluído deste tipo de abordagem, o que poderá limitar os potenciais benefícios. Objectivo: Avaliar a influência da idade nas características e evolução clínica dos doentes com SCA tratados segundo uma estratégia invasiva, e se esta limita a sua adopção. Métodos: Estudaram-se retrospectivamente 203 doentes internados por SCA (não seleccionados e consecutivos), considerados de risco intermédio/elevado após estratificação e que efectuaram terapêutica com inibidores das glicoproteínas IIb/IIIa. Destes doentes 45 tinham idade 75 anos e constituíram o grupo intitulado de Idoso, os restantes constituíram o grupo Não Idoso. Foram analisadas e comparadas as características dos dois grupos, a terapêutica realizada e a evolução clínica que apresentaram. Resultados: A percentagem de mulheres no grupo idoso é bastante superior, embora a diferença não atinja significado estatístico. Das outras características estudadas as que apresentam diferenças significativas são a existência de história familiar de doença coronária e o tabagismo, que são menos frequentes entre os idosos. Houve uma tendência não significativa para cateterizar menos os idosos, sendo que os dois grupos são semelhantes em relação à terapêutica de revascularização adoptada. No total as complicações hemorrágicas foram mais frequentes no grupo Idoso, mas a diferença em relação às hemorragias significativas não teve valor estatístico. A mortalidade intra hospitalar foi maior nos idosos, mas diminuiu e não teve significado estatístico quando considerados apenas os doentes cateterizados. Conclusão: Nesta população os idosos tiveram um número maior de complicações hemorrágicas não significativas e a sua maior mortalidade não esteve associada à adopção de uma atitude invasiva. Desta forma sugere-se que a idade, por si só, não limita a adopção de uma estratégia sistematicamente invasiva.

Rat hepatocyte invasion by Listeria monocytogenes and analysis of TNF-alpha role in apoptosis

Listeria monocytogenes, etiological agent of severe human foodborne infection, uses sophisticated mechanisms of entry into host cytoplasm and manipulation of the cellular cytoskeleton, resulting in cell death. The host cells and bacteria interaction may result in cytokine production as Tumor Necrosis Factor (TNF) alpha. Hepatocytes have potential to produce pro-inflammatory cytokines as TNF-alpha when invaded by bacteria. In the present work we showed the behavior of hepatocytes invaded by L. monocytogenes by microscopic analysis, determination of TNF-alpha production by bioassay and analysis of the apoptosis through TUNEL technique. The presence of bacterium, in ratios that ranged from 5 to 50,000 bacteria per cell, induced the rupture of cellular monolayers. We observed the presence of internalized bacteria in the first hour of incubation by electronic microscopy. The levels of TNF-alpha increased from first hour of incubation to sixth hour, ranging from 0 to 3749 pg/mL. After seven and eight hours of incubation non-significant TNF-alpha levels decrease occurred, indicating possible saturation of cellular receptors. Thus, the quantity of TNF-alpha produced by hepatocytes was dependent of the incubation time, as well as of the proportion between bacteria and cells. The apoptosis rate increased in direct form with the incubation time (1 h to 8 + 24 h), ranging from 0 to 43%, as well as with the bacteria : cells ratio. These results show the ability of hepatocyte invasion by non-hemolytic L. monocytogenes, and the main consequences of this phenomenon were the release of TNF-alpha by hepatocytes and the induction of apoptosis. We speculate that hepatocytes use apoptosis induced by TNF-alpha for release bacteria to extracellular medium. This phenomenon may facilitate the bacteria destruction by the immune system.

Um Novo Factor de Risco para Endocardite Infecciosa

A implantação de piercings corporais tem sido uma prática cada vez mais comum nas últimas décadas, sobretudo entre os mais jovens. No entanto, não se trata de um procedimento inócuo, podendo apresentar complicações tão graves como a endocardite infecciosa, que pode surgir em indivíduos com ou sem cardiopatia de base. Neste artigo relatamos o caso de uma endocardite pós piercing numa jovem com pacemaker definitivo, tendo havido necessidade de intervenção cirúrgica. Fazemos igualmente uma revisão dos casos de endocardite pós piercing descritos na literatura. Agora que as recomendações da American Heart Association para a profilaxia de endocardite infecciosa estão mais restritas, discutimos a necessidade de inclusão dos piercings corporais nos procedimentos a merecerem terapêutica profiláctica nos indivíduos de alto risco.

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (> 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.

Serotype and mating type characterization of Cryptococcus neoformans by Multiplex PCR

Cryptococcus neoformans is an encapsulated yeast, etiological agent of cryptococcosis. The species is commonly associated with pigeon droppings and plant materials. The aim of the present work was to verify the presence of the yeast in pigeon droppings, and to identify the isolates obtained in serotypes and mating types (MAT). Ten samples of pigeon droppings were collected in the rural area of the city of Alfenas, Brazil. Samples were inoculated in agar Niger medium for fungal isolation and 22 isolates with characteristics of C. neoformans were obtained. The serotypes and MAT were determined by multiplex PCR using specific primers. Serotypes were also determined by using the Kit Crypto Check. Among the 22 samples evaluated, eight were identified as C. neoformans by classic identification tests. These samples were characterized as serotype A by the Kit Crypto check and as serotype A MAT alpha by the multiplex PCR. The present study reinforces the evidence that pigeon droppings are a reservoir for C. neoformans and confirms the prevalence of C. neoformans var. grubii (Aalpha) among environmental isolates. It also demonstrates that multiplex PCR is an acceptable alternative for serotype analysis because it reduces the costs for each reaction and analyses serotype and MAT simultaneously.

Deficiência de Factor XI

A deficiência de factor XI é uma doença hematológica rara na população em geral. Pode manifestar-se apenas como complicação hemorrágica no doente submetido a cirurgia electiva. Os autores descrevem o caso clínico de uma mulher de 59 anos, que apresenta um quadro de hemorragia vaginal abundante 10 dias após ter sido submetida a histerectomia vaginal com McCall, plastia anterior e posterior. Salientam a importância da avaliação analítica pré-operatória, em especial o estudo da coagulação, e descrevem a abordagem e o tratamento da deficiência de factor XI.

Aerobic Exercise Does Not Predict Brain Derived Neurotrophic Factor And Cortisol Alterations in Depressed Patients

The pathophysiology of depression is related to neurobiological changes that occur in the monoamine system, hypothalamic-pituitary-adrenal axis, neurogenesis system and the neuroimmune system. In recent years, there has been a growing interest in the research of the effects of exercise on brain function, with a special focus on its effects on brainderived neurotrophic factor (BDNF), cortisol and other biomarkers. Thus, the aim of this study is to present a review investigating the acute and chronic effects of aerobic exercise on BDNF and cortisol levels in individuals with depression. It was not possible to establish an interaction between aerobic exercise and concentration of BDNF and cortisol, which may actually be the result of the divergence of methods, such as type of exercises, duration of the sessions, and prescribed intensity and frequency of sessions.

Decreased Vascular Endothelial Growth Factor (VEGF) Expression in Focal Segmental Glomeruloesclerosis Lesions of Patients Under Sirolimus

Background: Proteinuria (PT) with SRL appears not only after conversion from a calcineurin inhibitor (CI), but also in de novo patients. The PT may be related to a hemodynamic effect of CI withdrawal or to a direct effect of SRL in glomerulus (GL). Recently an association between PT in SRL patients and FSGS lesions has been described. It is also known that SRL decrease VEGF synthesis and experimental data suggest that VEGF is essential to podocyte survival and differentiation. Aim: To determine if glomerular lesions and PT in SRL patients could be related to altered glomerular VEGF expression. Material and methods: We evaluated glomerular VEGF expression in 10 biopsies: A-allograft kidney in backtable (n=3); B-native normal kidney (n=1); C-native kidney with FSGS lesions (n=2); D-allograft kidney with FSGS lesions from proteinuric patients under SRL after conversion from CI (n=3); E-allograft kidney in proteinuric patient under SRL with a membranous glomerulonephritis (n=1). We employed indirect immunohistochemistry in paraffin-embedded sections using a mouse monoclonal antibody against human VEGF-C1 (Santa Cruz). Results: The controls biopsies (A; B) showed normal global VEGF expression, with strong podocyte staining. The VEGF expression in the group C was similar to the controls, although no FSGS lesions were observed in the stained GL. The group D showed normal VEGF expression in the apparently normal GL, hypertrophied podocytes with reduction of VEGF in anomalous GL, and no staining in slcerotic lesions. We observed a gradual reduction of VEGF expression with progressive dedifferentiation of podocytes. In the group E the VEGF was globally reduced, with some hypertrophied podocytes expressing decreased VEGF. Conclusion: We confirmed the diminished VEGF expression in injured podocytes of SRL patients.This decreased expression may result from a direct effect of SRL and precede the appearance of FSGS lesions and PT. Further studies are needed with greater number of cases and controls, including early biopsies of patients under SRL.