Tolerance-inducing immunosuppressive strategies in clinical transplantation: an overview.

The significant development of immunosuppressive drug therapies within the past 20 years has had a major impact on the outcome of clinical solid organ transplantation, mainly by decreasing the incidence of acute rejection episodes and improving short-term patient and graft survival. However, long-term results remain relatively disappointing because of chronic allograft dysfunction and patient morbidity or mortality, which is often related to the adverse effects of immunosuppressive treatment. Thus, the induction of specific immunological tolerance of the recipient towards the allograft remains an important objective in transplantation. In this article, we first briefly describe the mechanisms of allograft rejection and immune tolerance. We then review in detail current tolerogenic strategies that could promote central or peripheral tolerance, highlighting the promises as well as the remaining challenges in clinical transplantation. The induction of haematopoietic mixed chimerism could be an approach to induce robust central tolerance, and we describe recent encouraging reports of end-stage kidney disease patients, without concomitant malignancy, who have undergone combined bone marrow and kidney transplantation. We discuss current studies suggesting that, while promoting peripheral transplantation tolerance in preclinical models, induction protocols based on lymphocyte depletion (polyclonal antithymocyte globulins, alemtuzumab) or co-stimulatory blockade (belatacept) should, at the current stage, be considered more as drug-minimization rather than tolerance-inducing strategies. Thus, a better understanding of the mechanisms that promote peripheral tolerance has led to newer approaches and the investigation of individualized donor-specific cellular therapies based on manipulated recipient regulatory T cells.

Systemic effects of epidural Methylprednisolone injection on glucose tolerance in diabetic patients.

ABSTRACT: BACKGROUND: Several studies have shown that in diabetic patients, the glycemic profile was disturbed after intra-articular injection of corticosteroids. Little is known about the impact of epidural injection in such patients. The goal of this study was double, at first comparing the glycaemic profile in diabetic patients after a unique injection of 80 mg of acetate methylprednisolone either intra-articular or epidural and secondly to compare the amount of systemic diffusion of the drug after both procedures. METHODS: Seventeen patients were included. Glycemic changes were compared in 9 diabetic patients following intra-articular (4 patients) and epidural injections (5 patients). Epidural injections were performed using the sacral route under fluoroscopic control in patients with lumbar spinal stenosis. Diabetes control had to stable for more than 10 days and the renal function to be preserved. Blood glucose was monitored using a validated continuous measuring device (GMS, Medtronic) the day before and for two days following the injection. Results were expressed in the form of daily glycemic profiles and as by mean, peak and minimal values +/ SD. The urinary excretion of methylprednisolone after the 2 routes of injection was analyzed in 8 patients (4 in each group). Urine samples were cropped one hour before the injections, then 4 times during the first day and 3 times a week for 2 weeks. The measurements included the free and conjugated fraction RESULTS: The glycaemic profile remains unchanged with no significant changes in the group of the 5 diabetic patients receiving epidural injections. On the other end, the average peak and mean values were enhanced up to 3 mmol/l above baseline two days after the infiltration in the groups of the 4 diabetic patients infiltrated intra-articular. The mean urinary excretion of the steroid was about ten times higher in the intra-articular versus epidural group: 7000 ng/ml versus 700 ng/ml. Looking at each individual there were marked differences especially after intra-articular injections. CONCLUSION: This is the first study to show that a single epidural steroid injection of 80 mg depot methylprednisolone had no effect on the glycemic control in diabetic patients. The absence of glycemic control changes correlated well with the very low urinary excretion of the drug after epidural injection. Trial registration NCT01420497.

Penicillin tolerance among Beta-hemolytic streptococci and production of the group carbohydrates, hemolysins, hyaluronidases and deoxyribonucleases

Penicillin tolerance among 67 strains of beta-hemolytic streptococci was examined by determining the ratio of the minimal bactericidal concentration to the minimal inhibitory concentration as 32 or greater. Tolerance was demonstrated in 15 group A strains and in 11,7, and 4 of groups B, C and G, respectively. Thereafter the effects of a subminimal inhibitory concentration (1/2MIC) of penicillin on the bacterial products of four tolerant and four nontolerant strains (two of each Lancefield group) were analyzed and compared. The antibiotic caused a marked increase in the expression of the group carbo-hydrates for strains of group B. Penicillin was found to reduce the cell-bound hemolysin activities of the four tolerant strains and to increase the activity of the other (free) form of nontolerant groups A, C and G hemolysins. Penicillin caused an increase in the extracellular hyaluronidase activities of one group A and groups B, C and G streptococci. With added antibiotic the production of deoxyribonuclease by tolerant groups A, C and G was greatly enhanced and that of the group B streptococcus was arrested.

CD4+CD25-mTGFbeta+ T cells induced by nasal application of ovalbumin transfer tolerance in a therapeutic model of asthma.

Background: Intranasal administration of high amount of allergen was shown to induce tolerance and to reverse the allergic phenotype. However, mechanisms of tolerance induction via the mucosal route are still unclear. Objectives: To characterize the therapeutic effects of intranasal application of ovalbumin (OVA) in a mouse model of bronchial inflammation as well as the cellular and molecular mechanisms leading to protection upon re-exposure to allergen. Methods: After induction of bronchial inflammation, mice were treated intranasally with OVA and re-exposed to OVA aerosols 10 days later. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. The respective role of CD4(+)CD25(+) and CD4(+)CD25(-) T cells in the induction of tolerance was analysed. Results: Intranasal treatment with OVA drastically reduced inflammatory cell recruitment into BALF and bronchial hyperresponsiveness upon re-exposure to allergen. Both OVA- specific-proliferation of T cells, T(h)1 and T(h)2 cytokine production from lung and bronchial lymph nodes were inhibited. Transfer of CD4(+)CD25(-) T cells, which strongly expressed membrane-bound transforming growth factor beta (mTGF beta), from tolerized mice protected asthmatic recipient mice from subsequent aerosol challenges. The presence of CD4(+)CD25(+)(Foxp3(+)) T cells during the process of tolerization was indispensable to CD4(+)CD25(-) T cells to acquire regulatory properties. Whereas the presence of IL-10 appeared dispensable in this model, the suppression of CD4(+)CD25(-)mTGF beta(+) T cells in transfer experiments significantly impaired the down-regulation of airways inflammation. Conclusion: Nasal application of OVA in established asthma led to the induction of CD4(+)CD25(-)mTGF beta(+) T cells with regulatory properties, able to confer protection upon allergen re-exposure.

A single mutation in enzyme I of the sugar phosphotransferase system confers penicillin tolerance to Streptococcus gordonii.

Tolerance is a poorly understood phenomenon that allows bacteria exposed to a bactericidal antibiotic to stop their growth and withstand drug-induced killing. This survival ability has been implicated in antibiotic treatment failures. Here, we describe a single nucleotide mutation (tol1) in a tolerant Streptococcus gordonii strain (Tol1) that is sufficient to provide tolerance in vitro and in vivo. It induces a proline-to-arginine substitution (P483R) in the homodimerization interface of enzyme I of the sugar phosphotransferase system, resulting in diminished sugar uptake. In vitro, the susceptible wild-type (WT) and Tol1 cultures lost 4.5 and 0.6 log(10) CFU/ml, respectively, after 24 h of penicillin exposure. The introduction of tol1 into the WT (WT P483R) conferred tolerance (a loss of 0.7 log(10) CFU/ml/24 h), whereas restitution of the parent sequence in Tol1 (Tol1 R483P) restored antibiotic susceptibility. Moreover, penicillin treatment of rats in an experimental model of endocarditis showed a complete inversion in the outcome, with a failure of therapy in rats infected with WT P483R and the complete disappearance of bacteria in animals infected with Tol1 R483P.

The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium.

The SV channel encoded by the TPC1 gene represents a Ca(2+)- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1, named fou2 for fatty acid oxygenation upregulated 2, results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca2+ signals and cytosolic Ca2+ is required for SV channel function, we here studied the Ca(2+)-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca2+ sensitivity and Ca2+ impermeability. K+ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca2+ reached the 0.1-mm level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca2+ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca2+. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca(2+) before SV channel activity vanishes and K(+) homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca2+ signals resulting in overproduction of jasmonate.

A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria.

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant "persister" trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2' Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.

Factors Supporting Cysteine Tolerance and Sulfite Production in Candida albicans.

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.

Effects of immunosuppressive drugs on T helper cells subsets and potential to promote tolerance in a stringent experimental transplantation model.

To achieve the goal of sustained donor-specifi c transplantation (Tx) tolerance, research efforts are now focusing on therapies based on specifi c cell subsets with regulatory properties. We and others have previously highlighted the therapeutic potential of naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTreg) in promoting long-term graft acceptance. Using more stringent experimental Tx models, we were however confronted to limitations. Indeed, while the transfer of antigenspecifi c nTreg promoted long-term MHC-mismatched skin allograft acceptance in lymphopenic mice in the absence of any immunosuppressive drug, allograft survival was only slightly prolonged when nTreg were transferred alone into non-lymphopenic mice. This suggested that in more stringent conditions, adjuvant therapies may be needed to effectively control alloreactive T cells (Teff). Whether and how the expansion of the Treg pool could be best combined with current immunosuppressive regimens in clinical settings remains to be defi ned. In this study, we have used in vitro assays and an in vivo skin Tx model to investigate the effects of various immunosuppressive drugs on the survival, proliferation and effector function of Teff and nTreg in response to alloantigens. Teff proliferation was inhibited in a dose-dependent manner by rapamycin and cyclosporine A, while anti-CD154 mAb only marginally affected Teff survival, proliferation and effector fucntion in vitro. Rapamycin promoted apoptosis of Teff as compared to nTreg that were more resistant in the presence of IL-2. In vivo, the transfer and/or expansion of Treg could be advantageously combined with rapamycin and anti-CD154 mAb treatment to signifi cantly prolong MHC-mismatched skin allografts survival in non-lymphopenic recipients. Taken together our data indicate that immunosuppressive drugs differentially target T-cell subsets and that some regimens could promote Treg expansion while controlling the Teff pool in response to alloantigens.

Exploration of the contribution of isotope ratio mass spectrometry to the investigation of explosives: A study of black powders and ammonium nitrate fertilisers

The increasing number of bomb attacks involving improvised explosive devices, as well as the nature of the explosives, give rise to concern among safety and law enforcement agencies. The substances used in explosive charges are often everyday products diverted from their primary licit applications. Thus, reducing or limiting their accessibility for prevention purposes is difficult. Ammonium nitrate, employed in agriculture as a fertiliser, is used worldwide in small and large homemade bombs. Black powder, dedicated to hunting and shooting sports, is used illegally as a filling in pipe bombs causing extensive damage. If the main developments of instrumental techniques in explosive analysis have been constantly pushing the limits of detection, their actual contribution to the investigation of explosives in terms of source discrimination is limited. Forensic science has seen the emergence of a new technology, isotope ratio mass spectrometry (IRMS), that shows promising results. Its very first application in forensic science dates back to 1979. Liu et al. analysed cannabis plants coming from different countries [Liu et al. 1979]. This preliminary study highlighted its potential to discriminate specimens coming from different sources. Thirty years later, the keen interest in this new technology has given rise to a flourishing number of publications in forensic science. The countless applications of IRMS to a wide range of materials and substances attest to its success and suggest that the technique is ready to be used in forensic science. However, many studies are characterised by a lack of methodology and fundamental data. They have been undertaken in a top-down approach, applying this technique in an exploratory manner on a restricted sampling. This manner of procedure often does not allow the researcher to answer a number of questions, such as: do the specimens come from the same source, what do we mean by source or what is the inherent variability of a substance? The production of positive results has prevailed at the expense of forensic fundamentals. This research focused on the evaluation of the contribution of the information provided by isotopic analysis to the investigation of explosives. More specifically, this evaluation was based on a sampling of black powders and ammonium nitrate fertilisers coming from known sources. Not only has the methodology developed in this work enabled us to highlight crucial elements inherent to the methods themselves, but also to evaluate both the longitudinal and transversal variabilities of the information. First, the study of the variability of the profile over time was undertaken. Secondly, the variability of black powders and ammonium nitrate fertilisers within the same source and between different sources was evaluated. The contribution of this information to the investigation of explosives was then evaluated and discussed. --------------------------------------------------------------------------------------------------- Le nombre croissant d'attentats à la bombe impliquant des engins explosifs artisanaux, ainsi que la nature des charges explosives, constituent une préoccupation majeure pour les autorités d'application de la loi et les organismes de sécurité. Les substances utilisées dans les charges explosives sont souvent des produits du quotidien, détournés de leurs applications licites. Par conséquent, réduire ou limiter l'accessibilité de ces produits dans un but de prévention est difficile. Le nitrate d'ammonium, employé dans l'agriculture comme engrais, est utilisé dans des petits et grands engins explosifs artisanaux. La poudre noire, initialement dédiée à la chasse et au tir sportif, est fréquemment utilisée comme charge explosive dans les pipe bombs, qui causent des dommages importants. Si les développements des techniques d'analyse des explosifs n'ont cessé de repousser les limites de détection, leur contribution réelle à l'investigation des explosifs est limitée en termes de discrimination de sources. Une nouvelle technologie qui donne des résultats prometteurs a fait son apparition en science forensique: la spectrométrie de masse à rapport isotopique (IRMS). Sa première application en science forensique remonte à 1979. Liu et al. ont analysé des plants de cannabis provenant de différents pays [Liu et al. 1979]. Cette étude préliminaire, basée sur quelques analyses, a mis en évidence le potentiel de l'IRMS à discriminer des spécimens provenant de sources différentes. Trente ans plus tard, l'intérêt marqué pour cette nouvelle technologie en science forensique se traduit par un nombre florissant de publications. Les innombrables applications de l'IRMS à une large gamme de matériaux et de substances attestent de son succès et suggèrent que la technique est prête à être utilisée en science forensique. Cependant, de nombreuses études sont caractérisées par un manque de méthodologie et de données fondamentales. Elles ont été menées sans définir les hypothèses de recherche et en appliquant cette technique de façon exploratoire sur un échantillonnage restreint. Cette manière de procéder ne permet souvent pas au chercheur de répondre à un certain nombre de questions, tels que: est-ce que deux spécimens proviennent de la même source, qu'entend-on par source ou encore quelle est l'intravariabilité d'une substance? La production de résultats positifs a prévalu au détriment des fondamentaux de science forensique. Cette recherche s'est attachée à évaluer la contribution réelle de l'information isotopique dans les investigations en matière d'explosifs. Plus particulièrement, cette évaluation s'est basée sur un échantillonnage constitué de poudres noires et d'engrais à base de nitrate d'ammonium provenant de sources connues. La méthodologie développée dans ce travail a permis non seulement de mettre en évidence des éléments cruciaux relatifs à la méthode d'analyse elle-même, mais également d'évaluer la variabilité de l'information isotopique d'un point de vue longitudinal et transversal. Dans un premier temps, l'évolution du profil en fonction du temps a été étudiée. Dans un second temps, la variabilité du profil des poudres noires et des engrais à base de nitrate d'ammonium au sein d'une même source et entre différentes sources a été évaluée. La contribution de cette information dans le cadre des investigations d'explosifs a ensuite été discutée et évaluée.

Immunological tolerance to pig-serum partially inhibits the formation of septal fibrosis of the liver in Capillaria hepatica-infected rats

Systhematized septal fibrosis of the liver can be induced in rats either by repeated intraperitoneal injections of pig-serum or by Capillaria hepatica infection. The relationship between these two etiological factors, as far as hepatic fibrosis is concerned, is not known, and present investigation attempts to investigate it. C. hepatica-induced septal fibrosis of the liver was considerably inhibited in rats previously rendered tolerant to pig-serum. Pig-serum-tolerant rats developed antibodies against pig-serum when infected with C. hepatica, but this did not happen when the infection occurred in normal rats. On the other hand, anti-C. hepatica antibodies failed to recognize any epitope in pig-serum, by Western blot. However, no evidence of an immunological cross reactivity was found, at least at the humoral level. Alternatively, cell-mediated mechanisms may be involved, and further investigations are warranted.

Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance.

Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.