In vivo T-lymphocyte tolerance in the absence of thymic clonal deletion mediated by hematopoietic cells.

Thymic negative selection renders the developing T-cell repertoire tolerant to self-major histocompatability complex (MHC)/peptide ligands. The major mechanism of induction of self-tolerance is thought to be thymic clonal deletion, ie, the induction of apoptotic cell death in thymocytes expressing a self-reactive T-cell receptor. Consistent with this hypothesis, in mice deficient in thymic clonal deletion mediated by cells of hematopoietic origin, a twofold to threefold increased generation of mature thymocytes has been observed. Here we describe the analysis of the specificity of T lymphocytes developing in the absence of clonal deletion mediated by hematopoietic cells. In vitro, targets expressing syngeneic MHC were readily lysed by activated CD8(+) T cells from deletion-deficient mice. However, proliferative responses of T cells from these mice on activation with syngeneic antigen presenting cells were rather poor. In vivo, deletion-deficient T cells were incapable of induction of lethal graft-versus-host disease in syngeneic hosts. These data indicate that in the absence of thymic deletion mediated by hematopoietic cells functional T-cell tolerance can be induced by nonhematopoietic cells in the thymus. Moreover, our results emphasize the redundancy in thymic negative selection mechanisms.

Engineering tolerance into transplanted beta cell lines.

In this paper we explore the possibility of improving, by genetic engineering, the resistance of insulin-secreting cells to the metabolic and inflammatory stresses that are anticipated to limit their function and survival when encapsulated and transplanted in a type 1 diabetic environment. We show that transfer of the Bcl-2 antiapoptotic gene, and of genes specifically interfering with cytokine intracellular signaling pathways, greatly improves resistance of the cells to metabolic limitations and inflammatory stresses.

Molecular mechanisms of penicillin-induced killing and penicillin tolerance in Streptococcus gordonii

Abstract The main thesis topic relates to the 'molecular mechanisms of penicillin-induced bacterial death. Indeed, bacteria have developed two principal mechanisms to escape the killing effect of ß-lactam antibiotics: resistance and tolerance. Resistant bacteria are characterized by their ability to grow in the presence of drug concentrations higher than the one inhibiting the growth of susceptible members of the same species. Hence, resistant bacteria have an increased minimal inhibitory concentration (MIC) of the drug. Nevertheless, when exposed to antibiotic concentrations exceeding their new MIC, resistant bacteria remain sensitive to the antibiotic killing effect. In contrast, tolerant bacteria have an unchanged MIC. However, they have a considerably increased ability to survive drug-induced killing, even at concentrations exceeding their MIC by several orders of magnitude. In other words, in the presence of the antibiotic, tolerant bacteria become persister cells which stop growing but are not killed. In the present thesis, it is shown that the survival phenotype of a tolerant Streptococcus gordonii strain depends on two components belonging to sugar metabolism pathways. First, the transcription factor CcpA which mediates a global regulatory mechanism allowing bacteria to utilize the most efficient sugar source for their growth. We show that the inactivation of the ccpA gene leads to a partial loss of penicillin tolerance both in vitro and in a rat model of experimental endocarditis. Second, the Enzyme I of the phosphotransferase system which is involved in the uptake and phosphorylation of sugars. Here, we -show that a single nucleotide mutation in ptsI, the gene encoding the Enzyme I, is sufficient to confer a fully tolerant phenotype in S. gordonii both in vivo and in vivo. The mutation results in a radical proline to arginine substitution in the C-terminal domain of the protein, probably leading to a decrease in its homodimerization and subsequent activity. Taken together our results prove that tolerance is a global survival mechanism linked to sugar metabolism. We hypothesize that, in the presence of the antibiotic, the already altered metabolic processes of the tolerant strain are completely inactivated. Hence, bacteria may enter in a dormant state and become insensitive to the bactericidal effect of ß-lactams, which depends on actively dividing cells. This thesis manuscript also contains two other side-projects. The first one establishes that the ability to form a biofilm is not a requisite for the successful establishment of endocarditis due to S. gordonii. The second one characterizes the S. gordonii a-phosphoglucomutase gene, and shows that its inactivation results in a loss of in vitro fitness and in vivo virulence. Résumé Le sujet principal de cette thèse concerne les mécanismes moléculaires de la mort bactérienne induite par la pénicilline. En effet, les bactéries ont développé deux mécanismes principaux pour échapper à l'effet bactéricide des ß-lactamines : la résistance et la tolérance. Les bactéries résistantes sont caractérisées par leur capacité de croître en présence de concentration d'antibiotiques plus élevées que celles inhibant la croissance des organismes sensibles de la même espèce. Les bactéries résistantes ont donc une augmentation de leur concentration minimale inhibitrice (CMI) à l'antibiotique. Néanmoins, quand elles sont exposées à des concentrations dépassant leur nouvelle CMI, elles restent sensibles à l'effet bactéricide. Au contraire, les bactéries tolérantes ont une CMI inchangée. Toutefois, elles ont une très importante capacité à survivre à l'effet bactéricide des ß-lactamines, ceci même à des concentrations excédant leur CMI de plusieurs ordres de grandeur. En d'autres termes, en présence de l'antibiotique, les bactéries tolérantes deviennent des cellules persistantes qui arrêtent leur croissance mais ne sont pas tuées. Dans la présente thèse, il est montré que le phénotype de survie d'un Streptococcus gordonii tolérant dépend de deux composants appartenant aux voies du métabolisme des sucres. Premièrement, le facteur de transcription CcpA qui contrôle un système global de régulation permettant à la bactérie d'utiliser les sources de sucre les plus efficaces pour sa croissance. Il est montré que l'inactivation du gène ccpA résulte en la perte partielle de la tolérance à la pénicilline aussi bien in vitro que dans un modèle d'endocardite expérimentale chez le rat. Deuxièmement, l'Enzyme I du système de phosphotransfert impliqué dans l'import et la phosphorylation des sucres. Nous montrons qu'une mutation ponctuelle d'un nucléotide dans ptsl, le gène codant pour l'Enzyme I, suffit à complètement conférer un phénotype tolérant chez S. gordonii aussi bien in vitro qu'in vivo. La mutation induit la substitution radicale d'une proline en une arginine dans le domaine C-terminal de la protéine, résultant probablement en une diminution de sa capacité d'homodimérisation et donc d'activité. Dans leur ensemble, nos résultats prouvent que la tolérance est un mécanisme global de survie lié au métabolisme des sucres. Nous présentons l'hypothèse que, en présence de l'antibiotique, les processus métaboliques déjà altérés de la souche tolérante deviennent complètement inactifs. En conséquence, les bactéries entreraient dans un état dormant nonréplicatif, devenant ainsi insensibles à l'effet bactéricide des ß-lactamines qui nécessite des cellules en cours de division active. Le manuscrit de cette thèse contient également deux projets secondaires. Le premier montre que la capacité de former un biofilm n'est pas un prérequis pour le succès de l'initiation de l'endocardite à S. gordonii. Le second caractérise le gène de l'a-phosphoglucomutase de S. gordonii et montre que son inactivation résulte en une perte de fitness in vitro et de virulence in vivo.

Mynodbcsv: Lightweight Zero-Config Database Solution for Handling Very Large CSV Files.

Volumes of data used in science and industry are growing rapidly. When researchers face the challenge of analyzing them, their format is often the first obstacle. Lack of standardized ways of exploring different data layouts requires an effort each time to solve the problem from scratch. Possibility to access data in a rich, uniform manner, e.g. using Structured Query Language (SQL) would offer expressiveness and user-friendliness. Comma-separated values (CSV) are one of the most common data storage formats. Despite its simplicity, with growing file size handling it becomes non-trivial. Importing CSVs into existing databases is time-consuming and troublesome, or even impossible if its horizontal dimension reaches thousands of columns. Most databases are optimized for handling large number of rows rather than columns, therefore, performance for datasets with non-typical layouts is often unacceptable. Other challenges include schema creation, updates and repeated data imports. To address the above-mentioned problems, I present a system for accessing very large CSV-based datasets by means of SQL. It's characterized by: "no copy" approach - data stay mostly in the CSV files; "zero configuration" - no need to specify database schema; written in C++, with boost [1], SQLite [2] and Qt [3], doesn't require installation and has very small size; query rewriting, dynamic creation of indices for appropriate columns and static data retrieval directly from CSV files ensure efficient plan execution; effortless support for millions of columns; due to per-value typing, using mixed text/numbers data is easy; very simple network protocol provides efficient interface for MATLAB and reduces implementation time for other languages. The software is available as freeware along with educational videos on its website [4]. It doesn't need any prerequisites to run, as all of the libraries are included in the distribution package. I test it against existing database solutions using a battery of benchmarks and discuss the results.

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase.

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

Assessing small non-zero perceptions of chance: The case of H1N1 (swine) flu risks

Feelings of invulnerability, seen in judgments of 0% risk, can reflect misunderstandings of risk and risk behaviors, suggesting increased need for risk communication. However, judgments of 0% risk may be given by individuals who feel invulnerable, and by individuals who are rounding from small non-zero probabilities. We examined the effect of allowing participants to give more precise responses in the 0-1% range on the validity of reported probability judgments. Participants assessed probabilities for getting H1N1 influenza and dying from it conditional on infection, using a 0-100% visual linear scale. Those responding in the 0-1% range received a follow-up question with more options in that range. This two-step procedure reduced the use of 0% and increased the resolution of responses in the 0-1% range. Moreover, revised probability responses improved predictions of attitudes and self-reported behaviors. Hence, our two-step procedure allows for more precise and more valid measurement of perceived invulnerability. [Authors]

Malignant lymphoma cells induce tolerance of T cells

Inhibitory receptors are involvedin the induction of T cell dysfunctionand exhaustion in chronic viral infectionsand in tumors. In the presentstudy, we analyzed the expressionpattern of 3 different inhibitory receptors(PD-1, Lag-3, 2B4) in a murine Bcell lymphoma model. Furthermore,we functionally characterized CD8+T cells expressing inhibitory receptorsfor cytokine production and proliferation.Expansion and secretion ofpro-inflammatory cytokines of CD8+T cells from lymphoma-bearing E-myc mice were significantly reducedcompared to the healthy controls.Similarly, expansion and effectorfunction of CD8+ TCR transgenic(p14) Tcells specific for the gp-33 antigenof lymphocytic choriomeningitisvirus (LCMV) was reduced inlymphoma-bearing E-myc mice afteractivation with LCMV. The functionalimpairment of CTL in the presenceof lymphoma was reversible aftertransfer to naive C57BL/6 recipients.In vitro co-culture experimentsrevealed that the proliferation ofanti-CD3-activated CD8+ T cellsfrom WT mice was significantly inhibitedby CD19+ lymphoma cellsfrom E-myc mice, whereas no inhibitionwas observed after co-culturewith normal B cells. Supernatants ofin vitro cultured lymphoma B cellsand blood sera from lymphoma-bearingE-myc mice significantly reducedT cell proliferation in vitro, ascompared to supernatants from normalB cells cultures or sera of healthyanimals. These experiments indicatethat the lymphoma B cells inactivateCTL by a soluble factor. Expressionanalysis of different important immunologicalcytokines revealed that themacrophage migration inhibitory factor(MIF) is selectively overexpressedin malignant B cells. This finding wasconfirmed by analyzing MIF proteinin culture supernatants and in celllysates. Therefore, lymphoma B cellsmay reduce T cell function and suppresslymphoma surveillance by secretionof MIF.

Freezing and low temperature photoinhibition tolerance in cultivated potato and potato hybrids

Selostus: Perunan ja perunahybridien jäätymisen ja fotoinhibition kestävyys

Flooding tolerance and cell wall alterations in maize mesocotyl during hypoxia

This research aimed to characterize the tolerance to flooding and alterations in pectic and hemicellulose fractions from mesocotyl of maize tolerant to flooding when submitted to hypoxia. In order to characterize tolerance seeds from maize cultivars Saracura BRS-4154 and BR 107 tolerant and sensitive to low oxygen levels, respectively, were set to germinate. Plantlet survival was evaluated during five days after having been submitted to hypoxia. After fractionation with ammonium oxalate 0.5% (w/v) and KOH 2M and 4M, Saracura BRS-4154 cell wall was obtained from mesocotyl segments with different damage intensities caused by oxygen deficiency exposure. The cell wall fractions were analyzed by gel filtration and gas chromatography, and also by Infrared Spectrum with Fourrier Transformation (FTIR). The hypoxia period lasting three days or longer caused cell lysis and in advanced stages plant death. The gelic profile from pectic, hemicellulose 2M and 4M fractions from samples with translucid and constriction zone showed the appearance of low molecular weight compounds, similar to glucose. The main neutral sugars in pectic and hemicellulose fractions were arabinose, xilose and mannose. The FTIR spectrum showed a gradual decrease in pectic substances from mesocotyl with normal to translucid and constriction appearance respectively.

Tolerance to flooding in five Brachiaria brizantha accessions

Some physiological and morphological responses of five Brachiaria brizantha accessions (BRA000591 cultivar Marandu, BRA003441, BRA002844, BRA004308 and BRA004391) were compared for plants grown in pots under flooding and well-drained conditions for 14 days. Flooding caused a significant reduction in leaf dry mass production in all accessions, but, for root biomass, no differences between treatments could be detected in BRA003441 and BRA004391. No adventitious root production was observed in flooded BRA003441; all other accessions produced adventitious roots when flooded. Relative growth rate was reduced by flooding only in BRA000591 and BRA004308. Leaf elongation rate was reduced by flooding in all accessions, however, more severely in BRA003441. Net photosynthesis was reduced by flooding in all accessions, but with less intensity in BRA004391. For all accessions, there was a close relationship between net photosynthesis and stomatal conductance under flooding. The five accessions tested differed in tolerance to flooding. BRA004391 was the most tolerant. Accession BRA003441 was the most sensitive, followed by BRA000591 cultivar Marandu. Accessions BRA002844 and BRA004308 were classified as intermediate in flooding tolerance.

Genetic analysis of aluminum tolerance in Brazilian barleys

Aluminum (Al) toxicity is a major factor limiting barley growth in acid soils, and genotypes with adequate level of tolerance are needed for improving barley adaptation in Brazil. To study the inheritance of Al tolerance in Brazilian barleys, cultivars Antarctica 1, BR 1 and FM 404 were crossed to sensitive Kearney and PFC 8026, and intercrossed. Parental, F1, F2 and F6 generations were grown in nutrient solution containing 0.03, 0.05 and 0.07 mM of Al and classified for tolerance by the root tip hematoxylin staining assay. Tolerant by sensitive F2 progenies segregated three tolerant to one sensitive, fitting the 3:1 ratio expected for a single gene. The F6 populations segregated one tolerant to one sensitive also fitting a monogenic ratio. The F2 seedlings from crosses among tolerant genotypes scored the same as the parents. Since the population size used would allow detection of recombination as low as 7%, the complete absence of Al sensitive recombinants suggests that tolerance in these cultivars is most probably, controlled by the same gene. Thus, the potential for improving Al tolerance through recombination of these genotypes is very low and different gene sources should be evaluated.

Impaired glucose tolerance and diabetes in obesity: a 6-year follow-up study of glucose metabolism.

To investigate the time course of glucose metabolism in obesity 33 patients (21 to 69 years old; body mass index [BMI], 25.7 to 53.3 kg/m2) with different degrees of glucose intolerance or diabetes who had been studied initially and 6 years later were submitted to the same 100-g oral glucose tolerance test (OGTT) with indirect calorimetry. From a group of 13 obese subjects with normal glucose tolerance (NGT), four developed impaired glucose tolerance (IGT); from a group of nine patients with IGT, three developed non-insulin-dependent diabetes mellitus (NIDDM); five of six obese NIDDM subjects with high insulin response developed NIDDM with low insulin response. Five patients had diabetes with hypoinsulinemia initially. As previously seen in a cross-sectional study, the 3-hour glucose storage measured by continuous indirect calorimetry remained unaltered in patients with IGT, whereas it decreased in NIDDM patients. A further decrease in glucose storage was observed with the lowering of the insulin response in the previously hyperinsulinemic diabetics. These results confirm cross-sectional studies that suggest successive phases in the evolution of obesity to diabetes: A, NGT; B, IGT (the hyperglycemia normalizing the glucose storage over 3 hours); C, diabetes with increased insulin response, where hyperglycemia does not correct the resistance to glucose storage anymore; and D, diabetes with low insulin response, with a low glucose storage and an elevated fasting and postload glycemia.