Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis.

Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Approximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U.S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8+ cells, interferon-gamma recovery and reduction of lung injury.

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-gamma recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-alpha. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-gamma and to restrict the growth of bacilli.

Automação do exame de urina: comparação do Urisys 2400 com a rotina manual (Microscopia do Sedimento Urinário) / Automation of urine examination: a comparison of Urisys 2400 system with the routine manual process (Urinary ediment Microscopy)

O Urisys 2400 é um sistema totalmente automatizado que fornece resultados semi-quantitativos para leucócitos, eritrócitos, nitrito, proteína, glicose, corpos cetônicos, urobilinogênio, bilirrubina, pH e cor atraves do uso de tiras reagentes, pelo método da reflectância, e, aspecto s densidade através de colorimetria e célula de fluxo (refratometria). O processo é automatizado desde a identificação das amostras por leitura de código de barras até a pipetagem e medida de áreas reagentes. Os resultados podem ser transmitidos para uma impressora e ou para um sistema informatizado (interfaceamento). Fez-se um estudo de 3958 amostras de urina submetidas ao exame de rotina com o Urisys 2400, sendo que 2450 (61,89%) apresentaram exame físico-químico normal, número normal de leucócitos e eritrócitos. Estas amostras foram avaliadas relacionando os achados microscópicos do sedimento após centrifugação padronizada, enumerando leucócitos e eritrócitos e comparando-os com os resultados obtidos com o Urisys 2400. Houve uma concordância de 99,88% entre as duas metodologias. Os resultados apresentados sugerem que o sistema automatizado de exame de urina pode ser umaopção eficaz e segura para o laboratório de análises reduzir o tempo de análise, não havendo a necessidade da execução da sedimentoscopia nas urinas sem anormalidades físico-químicas.

Genetic aspects and microenvironment affect expression of CD18 and VLA-4 in experimental tuberculosis.

Control of infection by Mycobacterium tuberculosis is dependent on macrophage activation and efficient migration of effector T-cell populations. Lymphocyte differentiation is associated with changes in cell surface phenotype and alterations in the migratory pattern of these cells. In this study, we investigated the expression of adhesion receptors involved in activation and migration process in experimental tuberculosis. We observed that susceptible BALB/c mice infected with virulent M. tuberculosis by intraperitoneal route presented downmodulation of very late antigen 4 (VLA-4) and unchanged levels of CD18 and CD44hi on peritoneal lymphocytes. On the other hand, lymphocytes from resistant C57BL/6 mice infected by the same route showed unchanged levels of VLA-4 and upregulation of CD18 and CD44hi. However, when BALB/c mice were infected by intratracheal route, lung lymphocytes presented a different pattern of CD18, CD44hi and VLA-4 expression from that observed on peritoneal cells, characterized by unchanged levels of VLA-4 and upregulation of CD18 and CD44hi- coincidentally the same phenotype found on peritoneal cells from C57BL/6. These results suggest that susceptibility and resistance to M. tuberculosis infection, depending on the experimental model, are related to the expression of CD18, CD44hi and VLA-4. Moreover, the microenvironment at the site of infection seems to differentially regulate the expression of these receptors. Thus, the up- or downmodulation of these adhesion receptors is probably associated with differential recruitment of T cells at the site of infection, which may or may not mediate protection in experimental tuberculosis.

Role of trehalose dimycolate in recruitment of cells and modulation of production of cytokines and NO in tuberculosis.

Mice treated with viable Mycobacterium tuberculosis with no glycolipid trehalose dimycolate (TDM) on the outer cell wall (delipidated M. tuberculosis) by intraperitoneal or intratracheal inoculation presented an intense recruitment of polymorphonuclear cells into the peritoneal cavity and an acute inflammatory reaction in the lungs, respectively. In addition, lung lesions were resolved around the 32nd day after intratracheal inoculation. TDM-loaded biodegradable poly-DL-lactide-coglycolide microspheres as well as TDM-coated charcoal particles induced an intense inflammatory reaction. In addition, high levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), IL-12, IL-10, gamma interferon (IFN-gamma), and IL-4 production were detected in lung cells, and nitric oxide (NO) production was high in culture supernatants of bronchoalveolar lavage cells. These in vivo data were confirmed by in vitro experiments using peritoneal macrophages cultured in the presence of TDM adsorbed onto coverslips. High levels of IFN-gamma, IL-6, TNF-alpha, IL-12, IL-10, and NO were detected in the culture supernatants. Our results suggest that TDM contributes to persistence of infection through production of cytokines, which are important for the recruitment of inflammatory cells and maintenance of a granulomatous reaction. In addition, our findings are important for a better understanding of the immunostimulatory activity of TDM and its possible use as an adjuvant in experiments using DNA vaccine or gene therapy against tuberculosis.

Avaliação da soroconversão pós vacina recombinante de hepatite B em alunos do curso de enfermagem da Universidade do Oeste do Paraná / Evaluation of the seroconversion rate after hepatitis B recombinant vaccine given to nursing students at the Universidade do Oeste do Paraná

Profissionais e estudantes da área de saúde (PEAS), são grupos de risco suscetíveis à contaminação com o vírus da hepatite B (VHB). A vacinação sistemática é altamente recomendada, no entanto os exames sorológicos pré e pós vacinas não são indicados por alguns serviços de saúde pública no Brasil. Neste estudo foram avaliados os resultados dos marcadores sorológicos de 63 alunos do curso de enfermagem da Universidade do Oeste do Paraná, pré e pós vacina contra a hepatite B. Nos resultados das análises pré vacina, foram encontrados 11 (17,5%) com um ou mais marcadores positivos para o HBV, assim distribuídos: 06 (9,5%) anti HBc positivo, 01 (1,6%) anti HBc e anti HBe positivo, 02 (3,2%) anti HBc, anti HBe e anti HBs positivo, 02 (3,2%) anti HBc, anti HBe e HBs Ag positivo. Os demais 52 (82,5%) todos os marcadores foram negativos. Após a terceira dose da vacina, a taxa de soroconversão foi de 80,7%. Os autores concluem que para populações de risco onde as taxas de soroconversão são baixas, é prudente a realização de testes pré e pós vacina para o VHB.