EFFECT OF HIGHLY ACTIVE ANTIRETROVIRAL THERAPY ON VAGINAL Candida spp. ISOLATION IN HIV-INFECTED COMPARED TO HIV-UNINFECTED WOMEN

 Vulvovaginal candidiasis (VVC) in HIV-infected women contributed to the impairment of their quality of life. The aim of this study was to evaluate the effect of highly active antiretroviral therapy (HAART) use on the vaginal Candida spp. isolation in HIV-infected compared to HIV-uninfected women. This cross-sectional study included 178 HIV-infected (HIV group) and 200 HIV-uninfected women (control) that were studied at the Specialized Assistance Service (SAE) for sexually transmitted diseases (STD)/AIDS of the city of Maringá, Brazil, from April 1 to October 30, 2011. The yeasts were isolated and identified by phenotypic and molecular methods. The in vitro antifungal susceptibility to fluconazole, itraconazole, nystatin and amphotericin B was tested by the reference microdilution method. Higher frequencies of total vaginal Candida spp. isolation were found in the HIV-infected group than in the control group. However, both groups showed a similar frequency of colonization and VVC. Although C. albicans was the most frequent and sensitive to azolics and polyenes in both HIV-infected and uninfected women, the emerging resistance of C. glabrata to amphotericin B in the HIV-infected women was observed. Although higher frequency of vaginal Candida spp. isolation had been observed in the HIV-infected than in HIV-uninfected women, colonization and VVC showed similar frequency in both groups, indicating that HAART appears to protect against vaginal colonization and VVC.

Apresentação Pélvica: Parto Vaginal ou Cesariana?

A apresentação pélvica ocorre em 3% das gravidezes únicas de termo e está associada ao aumento do risco de complicações maternas e fetais. Os autores fizeram uma avaliação retrospectiva de 118 partos pélvicos por via vaginal. Foram considerados os recém-nascidos com pesos compreendidos entre 2000 e 4000 g, de gestação maior ou igual a 34 semanas. Comparou-se a morbilidade materna e do recém-nascido com apresentação pélvica, em 118 partos vaginais e 244 cesarianas. Constatou-se que a morbilidade neonatal major foi significativamente superior no parto vaginal (5,9%), em relação à cesariana (0,4%). A morbilidade materna foi ligeiramente superior na cesariana (3,6%), em relação ao parto vaginal (1,6%), sem significado estatístico.

Characterization of sol-gel matrices with entrapped cutinase

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Ensaio terapêutico na infecção por Giardia muris em camundongo com metronidazole, tinidazole, secnidazole e furazolidone

Foi testado in vivo a sensibilidade de Giardia muris a quatro drogas comumente usadas no tratamento da giardíase humana. Foram utilizados 7 grupos de animais, com 12 camundongos cada, sendo que o grupo controle recebeu apenas solução salina 0,15M (0,5ml/animal). Os demais grupos receberam em dose única: metronidazole e furazolidone (500mg/kg), tinidazole e secnidazole (200mg/kg). A eficácia das drogas foi avaliada através da contagem de cistos nas fezes e pela ausência de trofozoítos no intestino. O metronidazole foi a droga mais eficaz. Os cortes histológicos mostraram diferenças entre o padrão da mucosa intestinal de animais normais e parasitados. No entanto, não se observou diferença entre o padrão de mucosa de animais infectados tratados e não tratados, o que sugere que estas alterações podem ser causadas pelo parasito e não pelas drogas.

Immunoblotting analyses using two-dimensional gel electrophoresis of Trypanosoma cruzi excreted-secreted antigens

Trypanosoma cruzi trypomastigotes excrete-secrete a complex mixture of antigenic molecules. This antigenic mixture denominated trypomastigote excreted-secreted antigens contains a 150-160 kDa band that shows excellent performance in Chagas' disease diagnosis by immunoblotting. The present study partially characterized by two-dimensional gel electrophoresis the immunoreactivity against the 150-160kDa protein using sera samples from chagasic patients in different phases of the disease. Trypomastigote excreted-secreted antigen preparations were subjected to high-resolution two-dimensional (2D) gel electrophoresis followed by immunoblotting with sera from chagasic and non-chagasic patients. The 150-160kDa protein presented four isoforms with isoelectric focusing ranging from 6.2 to 6.7. The four isoforms were recognized by IgM from acute phase and IgG from chronic phase sera of chagasic patients. The 150-160kDa isoform with IF of approximately 6.4 became the immunodominant spot with the progression of the disease. No cross-reactivity was observed with non-chagasic or patients infected with Leishmania sp. In this study we provide basic knowledge that supports the validation of trypomastigote excreted-secreted antigens for serological diagnosis of Chagas' disease.

Paracoccidioidomycosis: no genetic damage in human peripheral blood cells of patients assessed by single-cell gel (comet) assay

Paracoccidioidomycosis is a systemic fungal infection caused by Paracoccidioides brasiliensis. As infectious diseases can cause DNA damage, the authors aimed at analyzing DNA breakage in peripheral blood cells of patients with paracoccidioidomycosis by using the comet assay. The results suggested that paracoccidioidomycosis does not cause genotoxicity.

Caracterização de cepas de referência de Leptospira sp utilizando a técnica de pulsed field gel electrophoresis

INTRODUÇÃO: A leptospirose é uma zoonose endêmica, mundialmente distribuída, causada por bactérias do gênero Leptospira. Este gênero compreende espécies patogênicas e saprofíticas, com mais de 200 sorovares distintos, dificultando sua caracterização. A técnica de pulsed field gel electrophoresis tem sido empregada como uma ferramenta para auxiliar nesta caracterização. Os objetivos deste trabalho foram padronizar a técnica de PFGE, determinar os perfis moleculares das cepas de referência utilizadas pelo Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial de Saúde para Leptospirose e criar um banco de dados com estes perfis. MÉTODOS: Foram analisadas, por PFGE, dezenove cepas utilizando a enzima de restrição NotI. RESULTADOS: Cada cepa apresentou um perfil único que pode ser considerado como uma identidade genômica específica, com exceção dos sorovares Icterohaemorrhagiae e Copenhageni, cujos perfis foram indistinguíveis. CONCLUSÕES: Dessa forma, foi possível a criação de um banco de perfis moleculares que está sendo utilizado no Laboratório para a comparação e identificação de cepas isoladas de quadros clínicos.

Sol-gel entrapped biosystems: enzyme(s) and whole cells

In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells. CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor β-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC). The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to “in-cell” Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.

In vitro mechanical fatigue behavior of poly-ε-caprolactone macroporous scaffolds for cartilage tissue engineering: Influence of pore filling by a poly(vinyl alcohol) gel

Polymeric scaffolds used in regenerative therapies are implanted in the damaged tissue and subjected to repeated loading cycles. In the case of articular cartilage engineering, an implanted scaffold is typically subjected to long term dynamic compression. The evolution of the mechanical properties of the scaffold during bioresorption has been deeply studied in the past, but the possibility of failure due to mechanical fatigue has not been properly addressed. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. In this work fatigue studies of polycaprolactone scaffolds were carried by subjecting the scaffold to repeated compression cycles in conditions simulating the scaffold implanted in the articular cartilage. The behaviour of the polycaprolactone sponge with the pores filled with a poly(vinyl alcohol) gel simulating the new formed tissue within the pores was compared with that of the material immersed in water. Results were analyzed with Morrow’s criteria for failure and accurate fittings are obtained just up to 200 loading cycles. It is also shown that the presence of poly(vinyl alcohol) increases the elastic modulus of the scaffolds, the effect being more pronounced with increasing the number of freeze/thawing cycles.

Extração e análise eletroforética em gel de poliacrilamida (SDS-PAGE) de proteínas totais de folhas e raízes de Piper tuberculatum

O Estado do Pará é o principal produtor brasileiro de pimenta-do-reino (Piper nigrum Link), entretanto a sua produção tem sido bastante afetada pela doença conhecida como fusariose. O Fusarium solani f. sp. piperis é o agente causador desta doença que afeta o sistema radicular da planta, causando o apodrecimento das raízes e a queda das folhas levando à morte da planta. Algumas piperáceas nativas da região amazônica, entre elas a espécie Piper tuberculatum Jacq., têm se mostrado resistentes à infecção pelo F. solani f. sp. piperis, e desta forma têm sido utilizadas em estudos de interação planta-patógeno. Neste trabalho foram avaliadas cinco condições de extração de proteínas com o objetivo de selecionar tampões adequados para a extração de proteínas totais de folhas e raízes de P. tuberculatum. Os tampões utilizados para a extração de proteínas de raízes e folhas foram: tampão salino, tampão sacarose, tampão glicerol, tampão uréia e tampão fosfato de sódio. As análises quantitativas mostraram que os tampões sacarose, glicerol e uréia foram mais eficientes na extração de proteínas de folhas e raízes. Análises de SDS-PAGE mostraram padrões diferenciados de bandas em extratos protéicos de folhas e raízes obtidos com os diferentes tampões. Os resultados obtidos neste trabalho contribuem para a identificação de tampões de extração adequados para a obtenção de amostras de proteínas totais em estudos de interação P. tuberculatum - F. solani f. sp. piperis.

Avaliação do efeito da combinação de pectina, gelatina e alginato de sódio sobre as características de gel de fruta estruturada a partir de "mix" de polpa de cajá e mamão, por meio da metodologia de superfície de resposta

Este trabalho teve por objetivo estabelecer procedimento tecnológico para produção de fruta estruturada a partir de "mix" de polpa de cajá e mamão, procurando unir as propriedades sensoriais de cada uma das frutas e potencializar as características funcionais do produto final. Avaliou-se o efeito da combinação de pectina, gelatina e alginato de sódio, via metodologia de superfície de resposta, nas características do gel de fruta. As polpas de mamão e cajá e os estruturados obtidos foram caracterizados com relação aos compostos funcionais, avaliando-se o teor de taninos e carotenóides totais, além das análises de composição centesimal, pH, acidez titulável, sólidos solúveis, açúcares, atividade de água, carboidratos e valor energético total. Os resultados obtidos através do planejamento experimental indicam que para o estruturado misto de cajá e mamão, somente o aumento da concentração de gelatina afeta a firmeza do produto final. Os estruturados de frutas desenvolvidos apresentaram boa aceitação sensorial para todos os atributos avaliados. Com relação à intenção de compra, 70% dos provadores responderam que provavelmente ou certamente, comprariam o estruturado misto de cajá e mamão se o encontrassem à venda.

Quadro citológico vaginal, concentração plasmática de progesterona durante a gestação e medidas fetais em paca (Cuniculus paca Linnaeus, 1766)

Em 27 pacas (Cuniculus paca Linnaeus, 1766) objetivou-se descrever aos 30, 60 e 90 dias (D) de prenhez diagnosticada por ultrassonografia (US), os tipos celulares do epitélio vaginal em esfregaços vaginais, relatar as condições de abertura da vulva e as características do muco vaginal, determinar a concentração plasmática de progesterona (P4) por radioimunoensaio, e ainda, mensurar por ultrassonografia (US) o diâmetro biparietal (DBP) fetal aos 60 e 90 dias de prenhez. No D30, 40% das amostras exibiram células (com características estrogênicas) superficiais e presença de núcleos nus. Nos D60 e D90, células parabasais, intermediárias, superficiais e naviculares estavam presentes nas mesmas proporções, mas células endocervicais foram descritas em apenas 73,9% e 69% das amostras daqueles dias, respectivamente. No D30 a maior proporção de células naviculares e superficiais diferiu (p < 0,05) em relação aos outros tipos celulares presentes. O muco vaginal apresentou-se cristalino e fluido em 100% e em 70% das fêmeas nos D30 e D60, respectivamente. Observou-se o vestíbulo vaginal aberto em torno de 50% das fêmeas em todos os dias de exames. Valores mínimos detectáveis de P4 foram obtidos em 72% e em 83% das fêmeas, enquanto que as médias das medidas dos DBP foram 1,25 cm (± 0,16) e 2,34 cm (± 0,25) nos D60 e D90, respectivamente. O quadro citológico vaginal nos D30, D60 e D90 e o DBP fornecem elementos que contribuem para diagnóstico de gestação em pacas. A concentração de P4 demonstra a necessidade de maiores estudos da endocrinologia da gestação em pacas.

Bacterial vaginosis biofilms: challenges to current therapies and emerging solutions

Bacterial vaginosis (BV) is the most common genital tract infection in women during their reproductive years and it has been associated with serious health complications, such as preterm delivery and acquisition or transmission of several sexually transmitted agents. BV is characterized by a reduction of beneficial lactobacilli and a significant increase in number of anaerobic bacteria, including Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp., Bacteroides spp. and Prevotella spp.. Being polymicrobial in nature, BV etiology remains unclear. However, it is certain that BV involves the presence of a thick vaginal multi-species biofilm, where G. vaginalis is the predominant species. Similar to what happens in many other biofilm-related infections, standard antibiotics, like metronidazole, are unable to fully eradicate the vaginal biofilm, which can explain the high recurrence rates of BV. Furthermore, antibiotic therapy can also cause a negative impact on the healthy vaginal microflora. These issues sparked the interest in developing alternative therapeutic strategies. This review provides a quick synopsis of the currently approved and available antibiotics for BV treatment while presenting an overview of novel strategies that are being explored for the treatment of this disorder, with special focus on natural compounds that are able to overcome biofilm-associated antibiotic resistance.

Investigating a galactomannan gel obtained from Cassia grandis seeds as immobilizing matrix for Cramoll lectin

Characterization, with emphasis on the rheological properties, of Cassia grandis seeds galactomannan gel containing immobilized Cramoll 1,4 is presented. The gels, with and without immobilized Cramoll 1,4, were evaluated along time by rheometry, pH, color, microbial contamination and lectin hemagglutinating activity (HA). Rheological determinations confirmed the gels to be very stable up to 30 days with variations occurring after this period. Rheological data also showed that the gel/Cramoll 1,4 immobilizing matrix loses its elastic modulus substantially after 60 days. Both gels presented no microbial contamination as well as a pH close to neutral. Colorimetric parameters demonstrated the gels transparency with occasional yellowness. The opacity of the galactomannan gel did not change significantly along the study; the same did not occur for the gel with immobilized Cramoll 1,4 as a statistically significant reduction of its opacity was observed. In what concerns immobilized Cramoll 1,4HA, up to 90% of its initial HA was maintained after 20 days, with a decrease to 60% after 60 days. These results combined with the thickening and stabilizing characteristics of the galactomannan gel make this gel a promising immobilizing matrix for Cramoll 1,4 that can be further exploited for clinical and cosmetic applications.