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.

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.

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.

Fly Ash Concrete Compressive Strength and Freeze-Thaw Durability, MLR-81-06, 1981

The current study investigated the effect of fly ash class, source and amount on the compressive strength and freeze-thaw durability of fly ash concrete. Concrete aggregates of varying quality were also included as test variables. The current results and those obtained from previous laboratory and field work indicate that compressive strength can·be affected by fly ash class, source and amount while aggregate quality is shown to have no effect on strength. Freeze-thaw durability of fly ash concrete is strongly affected by aggregate quality and to a lesser degree by fly ash class, amount and source.

A Study of the Reliability of the ASTM C-666 Freeze-Thaw Test, MLR-72-01 and R-252, 1972

The Iowa State Highway Commission purchased a Conrad automatic freeze and thaw machine and placed it in operation during October 1961. There were a few problems, but considering, the many electrical and mechanical devices used in the automatic system it has always functioned quite well. Rapid freezing and thawing of 4"x4"xl8" concrete beams has been conducted primarily in accordance with ASTM C-29l (now ASTM C-666 procedure B) at the rate of one beam per day. Over 4000 beams have been tested since 1961, with determination of the resulting durability factors. Various methods of curing were used and a standard 90 day moist cure was selected. This cure seemed to yield durability factors that correlated very well with ratings of coarse aggregates based on service records. Some concrete beams had been made using the same coarse aggregate and the durability factors compared relatively well with previous tests. Durability factors seemed to yield reasonable results until large variations in durability factors were noted from beams of identical concrete mix proportions in research projects R-234 and R-247. This then presents the question "How reliable is the durability as determined by ASTM C-666?" This question became increasingly more important when a specification requiring a minimum durability factor for P.C. concrete made from coarse aggregates was incorporated into the 1972 Standard Specification for coarse aggregates for concrete.

Freeze-Thaw Durability of Concretes with and without Class C Fly Ash, MLR-94-9 Phase I, 1997

The freeze-thaw resistance of concretes was studied. Nine concrete mixes, made with five cements and cement-Class C fly ash combinations, were exposed to freeze-thaw cycling following 110 to 222 days of moist curing. Prior to the freeze-thaw cycling, the specimens were examined by a low-vacuum scanning electron microscope (SEM) for their microstructure. The influence of a wet/dry treatment was also studied. Infilling of ettringite in entrained air voids was observed in the concretes tested. The extent of the infilling depends on the period of moist curing as well as the wet/dry treatment. The concretes with 15% Class C fly ash replacement show more infilling in their air voids. It was found that the influence of the infilling on the freeze-thaw durability relates to the air spacing factor. The greater the spacing factor, the more expansion under the freeze-thaw cycling. The infilling seems to decrease effective air content and to increase effective spacing factor. The infilling also implies that the filled air voids are water-accessible. These might lead to concrete more vulnerable to the freeze-thaw attack. By combining the above results with field observations, one may conclude that the freeze-thaw damage is a factor related to premature deterioration of portland cement concrete pavements in Iowa.

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

Selostus: Perunan ja perunahybridien jäätymisen ja fotoinhibition kestävyys

Freeze-Thaw Durability Testing of Oversanded Bridge Floor Concrete, MLR-95-5, 1995

Due to an equipment malfunction, too much sand was used in the concrete on the bridge floor placed on August 9, 1994, in Washington County, Project No. BRF-22-2(36)38-92. Freeze-thaw durability testing of cores taken from the concrete in question and the other two concretes not in question was performed. The experimental results indicate that the concrete in question is considered at least as durable and resistant to freeze-thaw damage as the concretes which are not in question. The concrete in question can be expected to function properly for the regular service life of the bridge.

Freeze-Thaw Durability of Concretes with Infilling of Ettringite in Voids, Phase 2, MLR-94-9, 1997

Examination of field portland cement concrete cores, from Iowa pavements with premature deterioration, reveals extensive infilling of calcium sulfate aluminum (CSA) compound in their air voids. A previous study (Phase I) has shown some evidence of the correlation between freeze-thaw durability of concretes and ettringite infilling. To further verify the previous observation, a more extensive experimental program was conducted in this Phase 2 study. A total of 101 concrete mixes were examined. Seven cements, six fly ashes, two water reducers and three coarse aggregates were used in the concrete mixes. Specimens were under moist curing for up to 223 days before being subjected to the freeze-thaw cycling. An environmental treatment consisting of three consecutive wet [70 deg F (21 deg C) in distilled water]/dry [120 deg F (49 deg C) in oven] cycles was applied to some specimens. Immediately prior to the freeze-thaw cycling, most specimens were examined by a low-vacuum scanning electron microscope (SEM) for their microstructure. The results obtained further demonstrate the correlation between concrete freeze-thaw response and CSA compound infilling in the air voids. The extent of the infilling depends on the period of moist curing as well as the wet/dry treatment. The extent of the infilling also relates to materials used. Concrete mixes with extensive infilling are more vulnerable to the freeze-thaw attack. Based on the obtained results, material criteria on cements and fly ashes for mainline paving were proposed for minimizing potential infilling of CSA compound in concrete.

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.