985 resultados para In vivo–in vitro correlation


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We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels - hitherto observed in all experiments especially in those using HMC - CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continous fibroplastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background

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This study investigates in vitro growth of human urinary tract smooth muscle cells under static conditions and mechanical stimulation. The cells were cultured on collagen type I- and laminin-coated silicon membranes. Using a Flexcell device for mechanical stimulation, a cyclic strain of 0-20% was applied in a strain-stress-time model (stretch, 104 min relaxation, 15 s), imitating physiological bladder filling and voiding. Cell proliferation and alpha-actin, calponin, and caldesmon phenotype marker expression were analyzed. Nonstretched cells showed significant better growth on laminin during the first 8 days, thereafter becoming comparable to cells grown on collagen type I. Cyclic strain significantly reduced cell growth on both surfaces; however, better growth was observed on laminin. Neither the type of surface nor mechanical stimulation influenced the expression pattern of phenotype markers; alpha-actin was predominantly expressed. Coating with the extracellular matrix protein laminin improved in vitro growth of human urinary tract smooth muscle cells.

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In this work we have studied the modifications in the biological properties of Trypanosoma cruzi when the parasite is maintained for a long time in axenic culture. The studies were done with a clone from an avirulent strain (Dm30L) and a non-cloned virulent strain (EP) of T. cruzi. Both parasiteswere maintained, for at least three years, by successive triatomine/mouse alternate passage (control condition), or by serial passage in axenic medium (culture condition), or only in the mouse (mouse condition). The comparison between parasites of culture and control condition showed that metacyclogenesis capacity was reduced in the former and that the resulting metacyclics displayed an attenuatedvirulence. In order to compare the virulence of metacyclics from the urine of the insect vector, Rhodnius prolixus were infected by artificial feeding with parasites of the control or culture condition. After three triatomine/triatomine passages, there was observed an almost identical biological behavior for these parasites, hence indicating that the maintenance of T. cruzi for a long time in axenic culture affects the differentiation capacity and the virulence of the parasite. Additionally, it was demonstrated that it is possible to maintain T. cruzi exclusively through passages in the invertebrate host.

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Mycophenolic acid, a selective inhibitor of the de novo synthesis of guanosine nucleotides in T and B lymphocytes, has been proposed to inhibit human immunodeficiency virus (HIV) replication in vitro by depleting the substrate (guanosine nucleotides) for reverse transcriptase. Here we show that mycophenolic acid induced apoptosis and cell death in a large proportion of activated CD4+ T cells, thus indicating that it may inhibit HIV infection in vitro by both virological mechanisms and immunological mechanisms (depletion of the pool of activated CD4+ T lymphocytes). Administration of mycophenolate mophetil, the ester derivate of mycophenolic acid, to HIV-infected subjects treated with anti-retroviral therapy and with undetectable viremia resulted in the reduction of the number of dividing CD4 + and CD8+ T cells and in the inhibition of virus isolation from purified CD4+ T-cell populations. Based on these results, the potential use of mycophenolate mophetil in the treatment of HIV infection deserves further investigation in controlled clinical trials.

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Sera from 29 individuals residing in a malaria-endemic region of Colombia were evaluated by an inhibition assay for their capacity to retard the growth of Plasmodium falciparum in vitro. The inhibitory activity was found to be independent of antibody activity. Furthermore, the degree of inhibition of parasite development was variable, depending on the parasite isolate used for the assay and the season of malaria transmission. We selected sera with high inhibitory activity and carried out partial analytical characterization by anion exchange fast protein liquid chromatography (FPLC) to identify the chemical nature of the inhibitory factor(s). The results suggested that the in vitro inhibitory activity might result from the additive effect of different molecules. It appears that these molecules could be non-specifically induced by stimulation of the immune system, they seem to play a role in the immunity to malaria.

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ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

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We have shown previously that HLA-A*0201 melanoma patients can frequently develop a CTL response to the cancer testis antigen NY-ESO-1. In the present study, we have analyzed in detail the relative antigenicity and in vitro immunogenicity of natural and modified NY-ESO-1 peptide sequences. The results of this analysis revealed that, although suboptimal for binding to the HLA-A*0201 molecule, peptide NY-ESO-1 157-165 is, among natural sequences, very efficiently recognized by specific CTL clones derived from three melanoma patients. In contrast, peptides NY-ESO-1 157-167 and NY-ESO-1 155-163, which bind very strongly to HLA-A*0201, are recognized less efficiently. In agreement with previous data, substitution of peptide NY-ESO-1 157-165 COOH-terminal C with various other amino acids resulted in a significantly increased binding to HLA-A*0201 molecules as well as in an increased CTL recognition, although variable at the clonal level. Among natural peptides, NY-ESO-1 157-165 and NY-ESO-1 157-167 exhibited good in vitro immunogenicity, whereas peptide NY-ESO-1 155-163 was poorly immunogenic. The fine specificity of interaction between peptide NY-ESO-1 C165A, HLA-A*0201, and T-cell receptor was analyzed at the molecular level using a series of variant peptides containing single alanine substitutions. The findings reported here have significant implications for the formulation of NY-ESO-1-based vaccines as well as for the monitoring of either natural or vaccine-induced NY-ESO-1-specific CTL responses in cancer patients.

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The Amazon region of Brazil is an area of great interest because of the large distribution of hepatitis B virus in specific Western areas. Seven urban communities and 24 Indian groups were visited in a total of 4,244 persons. Each individual was interviewed in order to obtain demographic and familial information. Whole blood was collected for serology and genetic determinations. Eleven genetic markers and three HBV markers were tested. Among the most relevant results it was possible to show that (i) there was a large variation of previous exposure to HBV in both urban and non-urban groups ranging from 0 to 59.2%; (ii) there was a different pattern of epidemiological distribution of HBV that was present even among a same linguistic Indian group, with mixed patterns of correlation between HBsAg and anti-HBs and (iii) the prevalence of HBV markers (HBsAg and anti-HBs) were significantly higher (P=0.0001) among the Indian population (18.8%) than the urban groups (12.5%). Its possible that the host genetic background could influence and modulate the replication of the virus in order to generate HB carrier state.

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During the first steps of reverse transcription of the retroviral genome, sequences present at the extremities of the RNA are used to reconstitute a host cell PolII promoter. The assembly of the promoter occurs by template switching, which takes advantage of a direct repeat at the ends of the RNA molecule. These steps are catalysed by the viral reverse transcriptase, which carries an intrinsic RNaseH activity that is probably also involved therein. To study the role of the RNaseH activity in this first template-switching event, an in vitro system has been developed based on primer extensions of synthetic RNAs. When an RNA was reverse transcribed with wild-type reverse transcriptase in the presence of a second RNA the 3' part of which was repeated at the 5' end of the first one, extension products could be observed corresponding to a chimeric cDNA comprising both RNA species. This template switching could not be detected when a mutant reverse transcriptase lacking the RNaseH activity was used. The results show that the RNaseH activity is needed to remove the 5' RNA sequences from the cDNA:RNA hybrid thereby enabling its translocation to another RNA containing an appropriate complementary target sequence.

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Biotecnologia Vegetal de materials de treball que incorporin les TICs. El material elaborat ha estat un llibre electrònic utilitzable tant on-line (web) com off-line (CD). Els materials generats com a resultes d’aquest projecte han estat situats en un servidor de la UdL accessible des de l’exterior de la Universitat. Aquest llibre electrònic és d’accés obert i es pot consultar on-line a l’URL (http://sakai.udl.es/cursos/76304/indexC.htm). El llibre electrònic conté un total de 98 Mb d’informació hipermèdia i hipertexual distribuïda en més de 380 arxius dels quals 50 són pàgines html, 10 arxius doc, 10 arxius pdf, 258 imatges fixes i 8 arxius de vídeo en format flash (swf). L’accés al llibre electrònic es realitza a través d’una pantalla inicial que dóna pas a un menú que distribueix els materials en 7 apartats. Els textos estan acompanyats de gran quantitat d’imatges fotogràfiques, gràfics, esquemes, imatges infogràfiques i videoclips generats de novo per aquest projecte. En dissenyar la web s’han tingut en compte criteris de confiabilitat, accessibilitat i usabilitat. El primer disseny de la web ha estat validat per un panel d’usuaris i utilitzat posteriorment amb alumnes durant el curs 2007-2008. Les observacions i suggeriments fets per aquests ja han estat incorporats en aquest document final. Una primera enquesta de satisfacció realitzada amb aquest alumnat permet concloure, a títol provisional donat lo reduït de la població enquestada, que l’alumnat mostra un grau de coneixement de les eines TIC suficient i que consideren positiva la incorporació de materials multimèdia com a recurs educatiu. A més a més, la realització d’aquest projecte i la seva aplicació a l’aula ha estat presentada en dues ponències al “Congreso Internacional de Docencia y Innovación Universitaria”.

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The production of interleukin 2 (IL-2) by peripheral blood mononuclear cells, from patients with different clinical forms of Chagas disease and healthy controls, was evaluated after stimulation with Trypanosoma cruzi antigen, PPD and PHA. PHA induced higher production of IL-2 in infected patients than healthy controls. No diferences were found between infected groups. With PPD the trend was similar, the only difference was that asymptomatic infected patients (INF) showed higher levels of IL-2 production than patients with cardiomyopathy (CDM). With T. cruzi antigen, most patients showed little or no IL-2 production at 24 hr, a peak at 48 hr and an abrupt fall at 72 hr. A similar pattern of IL- 2 production was observed in INF and CDM. To evaluate the physiologic relevance of the deficit in IL-2 production, we studied the effect of non-mitogenic concentratios of IL-2 in the proliferative response to specific antigens. The addition of IL-2 only enhanced the proliferative response of CDM patients. These observations suggest that patients suffering Chagas' disease, particularly CDM, have a significant reduction in the capacity to produce IL-2. These findings could be of importance in the pathogenesis of Chagas' disease.

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Une étude rétrospective de tous les cydes de fécondation in vitro (FIV) et d'injection intracytoplasmique de spermatozoïdes (ICSI) effectuée de janvier à décembre 1997 à l'Unité de médecine de la reproduction du CHUV est présentée. Chez les patientes jeunes (< 35 ans), les transferts d'embryons frais et cryoconservés obtenus après FIV ou ICSI donnent des taux cumulés de grossesse similaires. En conclusion, si une stratégie de sélection adéquate entre les deux techniques est pratiquée, les taux de succès en terme de grossesses évolutives et d'accouchements par FIV ou ICSI sont comparables. L'ICSI ne doit pas être utilisée en dehors de ses indications spécifiques.