Bcl-X-L translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

Background. The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X-L, pro-apoptotic Bax and Bad), Methods. Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) Or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (West ern immunoblots, densitometry, immunoelectron microscopy). Results. Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X-L and Bax, but not Bcl-2 or Bad, was identified in control distal cells, Bcl-X-L and Bax had nonsignificant increases (P > 0.05) in these cells. Bcl-2, Bax, and Bcl-X-L, but not Bad, were endogenously expressed in control proximal cells. Bcl-X-L was significantly decreased in treated proximal cultures (P < 0.05), with Bas and Bcl-2 having nonsignificant increases (P > 0.05). Immunoelectron microscopy localization indicated that control and treated hut surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X-L from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-XL expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. Conclusion. The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X-L in proximal cells, as well as translocation of Bcl-X-L protein to mitochondria within the surviving distal cells.

Replication of bovine papillomavirus type 1 (BPV-1) DNA in Saccharomyces cerevisiae following infection with BPV-1 virions

Saccharomyces cerevisiae protoplasts exposed to bovine papillomavirus type 1 (BPV-1) virions demonstrated uptake of virions on electron microscopy. S. cerevisiae cells looked larger after exposure to BPV-1 virions, and cell wall regeneration was delayed. Southern blot hybridization of Hirt DNA from cells exposed to BPV-1 virions demonstrated BPV-1 DNA, which could be detected over 80 days of culture and at least 13 rounds of division. Two-dimensional gel analysis of Hirt DNA showed replicative intermediates, confirming that the BPV-1 genome was replicating within S. cerevisiae. Nicked circle, linear, and supercoiled BPV-1 DNA species were observed in Hirt DNA preparations from S. cerevisiae cells infected for over 50 days, and restriction digestion showed fragments hybridizing to BPV-1 in accord with the predicted restriction map for circular BPV-1 episomes. These data suggest that BPV-1 can infect S. cerevisiae and that BPV-1 episomes can replicate in the infected S. cerevisiae cells.

An atomistic simulation of solid state sintering using Monte Carlo methods

This paper presents results on the simulation of the solid state sintering of copper wires using Monte Carlo techniques based on elements of lattice theory and cellular automata. The initial structure is superimposed onto a triangular, two-dimensional lattice, where each lattice site corresponds to either an atom or vacancy. The number of vacancies varies with the simulation temperature, while a cluster of vacancies is a pore. To simulate sintering, lattice sites are picked at random and reoriented in terms of an atomistic model governing mass transport. The probability that an atom has sufficient energy to jump to a vacant lattice site is related to the jump frequency, and hence the diffusion coefficient, while the probability that an atomic jump will be accepted is related to the change in energy of the system as a result of the jump, as determined by the change in the number of nearest neighbours. The jump frequency is also used to relate model time, measured in Monte Carlo Steps, to the actual sintering time. The model incorporates bulk, grain boundary and surface diffusion terms and includes vacancy annihilation on the grain boundaries. The predictions of the model were found to be consistent with experimental data, both in terms of the microstructural evolution and in terms of the sintering time. (C) 2002 Elsevier Science B.V. All rights reserved.

Current concepts in central nervous system regeneration

A dictum long-held has stated that the adult mammalian brain and spinal cord are not capable of regeneration after injury. Recent discoveries have, however, challenged this dogma. In particular, a more complete understanding of developmental neurobiology has provided an insight into possible ways in which neuronal regeneration in the central nervous system may be encouraged. Knowledge of the role of neurotrophic factors has provided one set of strategies which may be useful in enhancing CNS regeneration. These factors can now even be delivered to injury sites by transplantation of genetically modified cells. Another strategy showing great promise is the discovery and isolation of neural stem cells from adult CNS tissue. It may become possible to grow such cells in the laboratory and use these to replace injured or dead neurons. The biological and cellular basis of neural injury is of special importance to neurosurgery, particularly as therapeutic options to treat a variety of CNS diseases becomes greater. (C) 2002 Published by Elsevier Science Ltd.

Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum

Background The ability of T cells, acting independently of antibodies, to control malaria parasite growth in people has not been defined. If such cell-mediated immunity was shown to be effective, an additional vaccine strategy could be pursued. Our aim was to ascertain whether or not development of cell-mediated immunity to Plasmodium falciparum blood-stage infection could be induced in human beings by exposure to malaria parasites in very low density. Methods We enrolled five volunteers from the staff at our research institute who had never had malaria. We used a cryopreserved inoculum of red cells infected with P falciparum strain 3D7 to give them repeated subclinical infections of malaria that we then cured early with drugs, to induce cell-mediated immune responses. We tested for development of immunity by measurement of parasite concentrations in the blood of volunteers by PCR of the multicopy gene STEVOR and by following up the volunteers clinically, and by measuring antibody and cellular immune responses to the parasite. Findings After challenge and a extended period without drug cure, volunteers were protected against malaria as indicated by absence of parasites or parasite DNA in the blood, and absence of clinical symptoms. Immunity was characterised by absence of detectable antibodies that bind the parasite or infected red cells, but by the presence of a proliferative T-cell response, involving CD4+ and CD8+ T cells, a cytokine response, consisting of interferon gamma but not interleukin 4 or interleukin 10, induction of high concentrations of nitric oxide synthase activity in peripheral blood mononuclear cells, and a drop in the number of peripheral natural killer T cells. Interpretation People can be protected against the erythrocytic stage of malaria by a strong cell-mediated immune response, in the absence of detectable parasite-specific antibodies, suggesting an additional strategy for development of a malaria vaccine.

Trichostome ciliates from Australian marsupials. I. Bandia gen nov (Litostomatea : Amylovoracidae)

A new genus of amylovoracid ciliates, Bandia gen.nov., is described. They are endosymbiotic/endocommensal in the stomachs of macropodid marsupials. Six new species, B. beveridgei, B. equimontanensis, B. tammar, B. deveneyi, B. cribbi and B. smalesae, are described from Setonix brachyurus, Petrogale assimilis, Macropus eugenii, M. robustus, M. parryi and M. agilis respectively. The gross morphology of Bandia is similar to that of Bitricha, with holotrichous somatic ciliation in two fields, longitudinal dorsal and oblique ventral. The somatic kineties are arranged in groups between non-ciliated. major interkinetal ridges; the groups of kineties thus give the cell a banded appearance. Several species are bimorphic, one form holotrichous and the other with a glabrous right body groove which appears to be derived from an ingrowth of one of the major interkinetal ridges. The groove may function in attachment either in sequestration or conjugation. The ultrastructure of the somatic kineties and the oral structures is similar to that of Amylovorax. Bandia also has unique ultrastructural features associated with the major interkinetal ridges, right body groove and a karyophore. Morphological evolution within the Amylovoracidae may have proceeded from simple forms such as Amylovorax via a process of cellular torsion and/or oral migration to forms similar to Bitricha and by further torsion and cellular elaboration to Bandia.

Ação mediada em aulas de Biologia : um enfoque a partir dos conceitos de fotossíntese e respiração celular

Esta pesquisa teve como objetivo investigar o papel da ação mediada na configuração dos sentidos que perpassam um processo de interação discursiva em uma sala de aula de Biologia, da 1ª série do ensino médio, de uma escola pública estadual do município de Vitória-ES, durante aulas que visavam abordar os conceitos de fotossíntese e respiração celular. Para isso, apoiamo-nos na tese de que a ação mediada qualificada como organizada, dialógica, compreensiva e interativa favorece a formação desses conceitos científicos por alunos de ensino médio. Assim, partimos de uma concepção teórico-metodológica ancorada na matriz histórico-cultural. A pesquisa-ação crítico-colaborativa foi utilizada como aporte metodológico e os instrumentos de coleta de dados se basearam em observações do cotidiano escolar e da sala de aula com registros em diários de campo, filmagem das aulas em vídeo, questionários, provas escritas dos alunos e entrevistas reflexivas. As análises dos dados se basearam na análise microgenética proposta por Vigotski, complementada com uma análise compreensiva ancorada nas ideias de Bakhtin. Dois grandes eixos de análise foram delimitados: 1 - a ação mediada no trabalho com os alunos; 2 - os enunciados e sentidos produzidos durante o processo da pesquisa na perspectiva da professora. Os resultados revelam evidências de que a ação mediada qualificada como intencional, organizada, dialógica, compreensiva e interativa favorece a formação dos conceitos científicos de fotossíntese e respiração celular por alunos do ensino médio, promovendo um ensino fecundo, na concepção defendida por Vigotski. Os resultados indicam também que o processo de pesquisa-ação crítico- colaborativa apresentou resultados positivos no que concerne à formação continuada da professora de Biologia. Concluímos que se fazem necessários investimentos em programas de formação de professores que procuram articular escola e universidade, integrando formação inicial e continuada de professores. Para isso, defendemos que esse processo de formação seja feito dentro de uma perspectiva crítica e colaborativa, baseada em uma ação mediada intencional e dialógica que favoreça o desenvolvimento de um ensino fecundo

Antifungal activity of Imidazolidin-4-ones of the antimalarial primaquine

The 5-Isopropyl-3-[4-(6-methoxy-quinolin-8-ylamino)-pentyl]-2,2-dimethyl-imidazolidin-4-one (ValPQacet) was sinthesized through acylation of the anti-malarial primaquine with α-valine and subsequent reaction of the resulting -aminoamide with propanone (Sheme 1).Imidazolidin-4-ones of the anti malarial primaquine are being sinthesized to develop new variants in order to improve more effective treatments against malaria . Recently it has been observed that primaquine derivates could have effect in a new kind of yeast . To study the fungicidal activity against Candida albicans, Candida tropicalis, Issatchenkia orientalis, Sacharomyces cerevisae, the ValPQacet was put in the form of the hydrochloride salt. The minimal inhibitory concentration (MIC) could be determined for all yeast in the concentration range assayed. Also was determined MIC’s of primaquine hydrochloride salt for all yeast, and this shows that the parent drug is less active than our compound. Further studies are being performed to determine viability and cellular injury with this drugs.

Ecosapentanoic acid (EPA) does not affect cell kinetics in peripheral lymphocites from patients with Crohn's disease (CD) according to IL6 174G/C polymorphism

Rationale: Omega 3 fatty acids have been shown to be of potential benefit in patients with CD. The aim of the present study was to evaluate whether EPA can modulate the inflammatory response according to different genotypes of IL6G174G/C polymorphism. Methods: Peripheral blood cells were collected from CD patients with different genotypes for IL6 174G/C (GG, n = 16, GC, n = 8, CC, n = 7), and lymphocytes were established in culture media. Replicates with the addition of EPA (25 mM) were analysed in a period of 24h, 48h and 72h. Expression of IL6 e a PGE2 was assessed by ELISA. Apoptosis and cellular proliferation was determined by flow cytometry.

Immunopathology of human schistosomiasis mansoni: I. Immunomodulatory influences on T cell function

Cell mediated immune response was studied in patients with recent and chronic Schistosoma mansoni infection. Precultured peripheral mononuclear cells showed significantly higher responses to S. mansoni adult worm antigen (SAWA) when compared to fresh cell preparations. The addition of each patient serum to the precultured cells reactions to SAWA or recall antigens demonstrated a strong inhibitory serum action, which was also noted on allogeneic cells derived from healthy subjects. The CD4 subset was the main responding cell to SAWA being this reactivity highly suppressed by the presence of the monocyte macrophage accessory cells. We stressed the simultaneous inhibitory action of humoral and cellular factors on the specific cell response to S. mansoni.

Reduced schedule of human anti rabies immunization with Fuenzalida & Palacios vaccine

The present study evaluates the humoral and cellular immune responses in 35 volunteers submited to short antirabies vaccination schedules with the Fuenzalida & Palacios vaccine based on the administration of doses on non consecutive days. The volunteers were divided into two groups. The first group received a total number of five doses given on days 0, 4, 7, 20 and 35. The other group received four doses, the first one being a double dose given on day 0 and than three other single doses on days 7, 20 and 35. The evaluation of humoral immune response was carried out by serum neutralization (SN) and indirect immunofluorescense (IIF) tests, while the cellular immune response was evaluated by lymphoblastic transformation assay (LTA) and skin test (ST). According to our results these reduced schedules elicited early and effective humoral and cellulafimmune responses to rabies antigen suggesting that new reduced schedules should be extensively studied in order to give the proper bases to the proposition of changes in the current long-term schedule.

Comparison study of procedures for aspiration of biopsy samples

Introduction: The samples obtained from fine needle aspiration in liquid base cytology (FNAC) are often limited by scarce cellularity compared to the amount of colloid and presence of blood. Accordingly, it was important to test alternative technical procedures so as to maximize the cellularity of each sample. Objective: To compare the morphological features and cellularity of the three procedures in the FNAC cytodiagnosis of the thyroid. Methods: A total of 31 cases were each subjected to a cell block and ThinPrep preparation as well as a routine smear. The observation and analysis was performed using an optical microscope. Cytological diagnosis of each cell block case was objectively analysed for cellularity, presence of background and cellular preservation. Each smear and ThinPrep case was analysed for the presence or absence of cells. The data was analysed with Microsoft Excel (Office 2010) and SPSS (Statistical Package of Social Science) version 15.0 for Windows. Results: Of 31 cases, only 20 had thyroid cells in the cell block and ThinPrep preparations, however, all smear cases contained thyroid cells. Some background was found in 30 Cell block cases with only 5 of these containing well preserved cells for cytodiagnosis. Conclusions: As indicated by the results, smear is the most appropriate procedure for FNAC of the thyroid.

Cyclophosphamide effect on coccidioidomycosis in the rat

Coccidioidomycosis is a systemic mycosis, endemic in arid areas of the American continent. The rat was employed as an experimental host, since it had been shown to reproduce human lesions and present a chronic course of disease with granulomas mainly restricted to lungs. Given the influence of immunosuppressive therapy on the clinical course of human coccidioidomycosis, we studied the effect of cyclophosphamide (CY) in the experimental rat model. Accordingly, animals were inoculated with 400 Coccidioides immitis arthroconidia of the Acosta strain, by intracardiacal route. As single CY doses failed to alter the course of disease, three schedules were used: A) 4 daily doses of 20 mg/kg each, prior to C. immitis inoculation; B) 4 similar daily doses after infection; and C); 6 doses of 20 mg/kg each, given from day +1 to +4, then on days +8 and +9, post infection (pi), taking day 0 as the time of fungal inoculation. The first two schedules inhibited antibody formation up to day 28 pi, without modifying cellular response to coccidioidin as measured by foodpad swelling. Initially, there was greater fungal spread than in controls receiving C. immitis alone, which proved self-limiting in the latter. In contrast, schedule C led to 559r mortality, with both humoral and cellular response abrogation, accompanied by extensive C. immitis dissemination. Histology disclosed significant alterations, such as the persistence of primary infection sporangia, corresponding to the acute stage of coccidioidomycosis in the absence of granuloma development. Therefore, the observed depression in cellular immunity seems responsible for the lack of inflammatory reaction capable of restricting sporangia proliferation in tissues which, in turn, enhances pathogen spread and mortality rate.

Evaluation of different immunization protocols with P. brasiliensis antigens in Guinea pigs

The objective of the present study was to develop an efficient and reproducible protocol of immunization of guinea pigs with P. brasiliensis antigens as an animal model for future studies of protective immunity mechanisms. We tested three different antigens (particulate, soluble and combined) and six protocols in the presence and absence of Freund's complete adjuvant and with different numbers of immunizing doses and variable lenght of time between the last immunizing dose and challenge. The efficacy of the immunizing protocol was evaluated by measuring the humoral and cellular anti-P. brasiliensis immune response of the animals, using immuno-diffusion, skin test and macrophage migration inhibition test. It was observed that: 1. Three immunizing doses of the antigens induced a more marked response than two doses; 2. The highest immune response was obtained with the use of Freund's complete adjuvant; 3. Animals challenged a long time (week 6) after the last immunizing dose showed good anti-P. brasiliensis immune response; 4. The particulate antigen induced the lowest immune response. The soluble and the combined antigens were equally efficient in raising good humoral and cellular anti-P. brasiliensis immune response

Immune response in mice immunized with acidic antigenic fractions from Trypanosoma cruzi cytosol

The humoral and cellular immune responses as well as the resistance to infection with bloodstream forms of T. cruzi were studied in mice immunized with acidic antigenic fractions from parasite cytosol, F III and F IV, plus Bordetella pertussis as adjuvant. The immunization with F III induced positive ITH and DTH responses to homologous antigens. In mice immunized with F IV, the ITH was negative and four out of six animals presented positive DTH reactions. In both groups of mice the analysis of IgG aginst T. cruzi showed that the major isotype elicited was IgG1. Specific IgE was also detected in sera from F III immunized mice, thus confirming the presence of homocytothropic antibodies. The parasitemias reached by F III and F IV immunized mice after challenge were lower than those of the controls showing in this way a partial protection against the acute infection. The histological studies of heart and skeletal muscle performed two months after the infection revealed variable mononuclear infiltration in all infected mice despite immunization.