Different types of cell death are involved in brain damage of methylmalonic aciduria and glutaric aciduria type I

We previously showed that exposure of 3D organotypic rat brain cell cultures to 1mM 2-methylcitrate (2-MCA) or 3-hydroxyglutarate (3- OHGA) every 12h over three days (DIV11-DIV14) results in ammonium accumulation and cell death. The aim of this study was to define the time course (every 24h) of the observed effects. Ammonium in culture medium already increased at DIV12 staying stable on the following days under 3-OHGA exposure, while it increased consecutively up to much higher levels under 2-MCA exposure. Lactate increase and glucose decrease were observed from DIV13 and DIV14, respectively. We conclude that ammonium accumulation precedes alterations of energy metabolism. As observed by immunohistochemistry glial cells were the predominant dying cells. Immunoblotting and immunohistochemistry with cell death specific markers (caspase-3, alpha-fodrin, LC3) showed that 2-MCA exposure significantly increased apoptosis on DIV14, but did not alter autophagy or necrosis. In contrast, 3-OHGA exposure substantially increased necrosis already from DIV13, while no change was observed for apoptosis and autophagy. In conclusion, ammonium accumulation, secondary disturbance of energy metabolism and glial cell death are involved in the neuropathogenesis ofmethylmalonic aciduria and glutaric aciduria type I. Interestingly, brain cells are dying by necrosis under 3-OHGA exposure and by apoptosis under 2-MCA exposure.

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.

Coagulation Factor Xa Activates Thrombin and Signals Ischemic Neuronal Death Via Par-1 and JNK in Ischemic Neural Tissue

Background: The coagulation factor thrombin mediates ischemic neuronal deathand, at a low concentration, induces tolerance to ischemia.We investigated its modeof activation in ischemic neural tissue using an in vitro approach to distinguish therole of circulating coagulation factors from endogenous cerebral mechanisms. Wealso studied the signalling pathway downstream of thrombin in ischemia and afterthrombin preconditioning.Methods: Rat organotypic hippocampal slice cultures to 30 minute oxygen (5%)and glucose (1 mmol/L) deprivation (OGD).Results: Selective factor Xa (FXa) inhibition by fondaparinux during and afterOGD significantly reduced neuronal death in the CA1 after 48 hours. Thrombinactivity was increased in the medium 24 hours after OGD and this increasewas prevented by fondaparinux suggesting that FXa catalyzes the conversion ofprothrombin to thrombin in neural tissue after ischemia in vitro. Treatment withSCH79797, a selective antagonist of the thrombin receptor protease activatedreceptor-1 (PAR-1), significantly decreased neuronal cell death indicating thatthrombin signals ischemic damage via PAR-1. The JNK pathway plays an importantrole in cerebral ischemia and we observed activation of the JNK substrate,c-Jun in our model. Both the FXa inhibitor, fondaparinux and the PAR-1 antagonistSCH79797, decreased the level of phospho-c-Jun Ser73. After thrombin preconditioningc-Jun was activated by phosphorylation in the nuclei of neurons of the CA1.Treatment with a synthetic thrombin receptor agonist resulted in the same c-Junactivation profile and protection against subsequent OGD indicating that thrombinalso signals via PAR-1 and c-Jun in cell protection.Conclusion: These results indicate that FXa activates thrombin in cerebral ischemia,leading via PAR-1 to the activation of the JNK pathway resulting in neuronal death.Thrombin induced tolerance also involves PAR-1 and JNK, revealing commonfeatures in cell death and survival signalling.

Mechanisms of functional T-cell deficiency in melanoma patients

SUMMARYAs a result of evolution, humans are equipped with an intricate but very effective immune system with multiple defense mechanisms primarily providing protection from infections. This system comprises various cell types, including T-lymphocytes, which are able to recognize and directly kill infected cells. T-cells are not only able to recognize cells carrying foreign antigens, such as virus-infected cells, but also autologous cells. In autoimmune diseases, e.g. multiple sclerosis, T- cells attack autologous cells and cause the destruction of healthy tissue. To prevent aberrant immune reactions, but also to prevent damage caused by an overreacting immune response against foreign targets, there are multiple systems in place that attenuate T-cell responses.By contrast, anti-self immune responses may be highly welcome in malignant diseases. It has been demonstrated that activated T-cells are able to recognize and lyse tumor cells, and may even lead to successful cure of cancer patients. Through vaccination, and especially with the help of powerful adjuvants, frequencies of tumor-reactive T-cells can be augmented drastically. However, the efficacy of anti-tumor responses is diminished by the same checks and balances preventing the human body from harm induced by overly activated T-cells in infections.In the context of my thesis, we studied spontaneous and vaccination induced T-cell responses in melanoma patients. The aim of my studies was to identify situations of T-cell suppression, and pinpoint immune suppressive mechanisms triggered by malignant diseases. We applied recently developed techniques such as multiparameter flow cytometry and gene arrays, allowing the characterization of tumor-reactive T-cells directly ex vivo. In our project, we determined functional capabilities, protein expression, and gene expression profiles of small numbers of T- cells from metastatic tissue and blood obtained from healthy donors and melanoma patients. We found evidence that tumor-specific T-cells were functionally efficient effector cells in peripheral blood, but severely exhausted in metastatic tissue. Our molecular screening revealed the upregulation of multiple inhibitory receptors on tumor-specific T-cells, likely implied in T-cell exhaustion. Functional attenuation of tumor-specific T-cells via inhibitory receptors depended on the anatomical location and immune suppressive mechanisms in the tumor microenvironment, which appeared more important than self-tolerance and anergy mechanisms. Our data reveal novel potential targets for cancer therapy, and contribute to the understanding of cancer biology.RÉSUMÉAu cours de l'évolution, les êtres humains se sont vus doter d'un système immunitaire complexe mais très efficace, avec de multiples mécanismes de défense, principalement contre les infections. Ce système comprend différents types de cellules, dont les lymphocytes Τ qui sont capables de reconnaître et de tuer directement des cellules infectées. Les cellules Τ reconnaissent non seulement des cellules infectées par des virus, mais également des cellules autologues. Dans le cas de maladies auto-immunes, comme par exemple la sclérose en plaques, les cellules Τ s'attaquent à des cellules autologues, ce qui engendre la destruction des tissus sains. Il existe plusieurs systèmes de contrôle des réponses Τ afin de minimiser les réactions immunitaires aberrantes et d'empêcher les dégâts causés par une réponse immunitaire trop importante contre une cible étrangère.Dans le cas de maladies malignes en revanche, une réponse auto-immune peut être avantageuse. Il a été démontré que les lymphocytes Τ étaient également capables de reconnaître et de tuer des cellules tumorales, pouvant même mener à la guérison d'un patient cancéreux. La vaccination peut augmenter fortement la fréquence des cellules Τ réagissant contre une tumeur, particulièrement si elle est combinée avec des adjuvants puissants. Cependant, l'efficacité d'une réponse antitumorale est atténuée par ces mêmes mécanismes de contrôle qui protègent le corps humain des dégâts causés par des cellules Τ activées trop fortement pendant une infection.Dans le cadre de ma recherche de thèse, nous avons étudié les réponses Τ spontanées et induites par la vaccination dans des patients atteints du mélanome. Le but était d'identifier des conditions dans lesquelles les réponses des cellules Τ seraient atténuées, voire inhibées, et d'élucider les mécanismes de suppression immunitaire engendrés par le cancer. Par le biais de techniques nouvelles comprenant la cryométrie de flux et l'analyse globale de l'expression génique à partir d'un nombre minimal de cellules, il nous fut possible de caractériser des cellules Τ réactives contre des tumeurs directement ex vivo. Nous avons examiné les profiles d'expression de gènes et de protéines, ainsi que les capacités fonctionnelles des cellules Τ isolées à partir de tissus métastatiques et à partir du sang de patients. Nos résultats indiquent que les cellules Τ spécifiques aux antigènes tumoraux sont fonctionnelles dans le sang, mais qu'elles sont épuisées dans les tissus métastatiques. Nous avons découvert dans les cellules Τ antitumorales une augmentation de l'expression des récepteurs inhibiteurs probablement impliqués dans l'épuisement de ces lymphocytes T. Cette expression particulière de récepteurs inhibiteurs dépendrait donc de leur localisation anatomique et des mécanismes de suppression existant dans l'environnement immédiat de la tumeur. Nos données révèlent ainsi de nouvelles cibles potentielles pour l'immunothérapie du cancer et contribuent à la compréhension biologique du cancer.

The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA.

The RuvB protein is induced in Escherichia coli as part of the SOS response to DNA damage. It is required for genetic recombination and the postreplication repair of DNA. In vitro, the RuvB protein promotes the branch migration of Holliday junctions and has a DNA helicase activity in reactions that require ATP hydrolysis. We have used electron microscopy, image analysis, and three-dimensional reconstruction to show that the RuvB protein, in the presence of ATP, forms a dodecamer on double-stranded DNA in which two stacked hexameric rings encircle the DNA and are oriented in opposite directions with D6 symmetry. Although helicases are ubiquitous and essential for many aspects of DNA repair, replication, and transcription, three-dimensional reconstruction of a helicase has not yet been reported, to our knowledge. The structural arrangement that is seen may be common to other helicases, such as the simian virus 40 large tumor antigen.

Stochastic models and numerical algorithms for a class of regulatory gene networks.

Regulatory gene networks contain generic modules, like those involving feedback loops, which are essential for the regulation of many biological functions (Guido et al. in Nature 439:856-860, 2006). We consider a class of self-regulated genes which are the building blocks of many regulatory gene networks, and study the steady-state distribution of the associated Gillespie algorithm by providing efficient numerical algorithms. We also study a regulatory gene network of interest in gene therapy, using mean-field models with time delays. Convergence of the related time-nonhomogeneous Markov chain is established for a class of linear catalytic networks with feedback loops.

Spatial organization of nucleotide excision repair proteins after UV-induced DNA damage in the human cell nucleus.

Nucleotide excision repair (NER) is an evolutionary conserved DNA repair system that is essential for the removal of UV-induced DNA damage. In this study we investigated how NER is compartmentalized in the interphase nucleus of human cells at the ultrastructural level by using electron microscopy in combination with immunogold labeling. We analyzed the role of two nuclear compartments: condensed chromatin domains and the perichromatin region. The latter contains transcriptionally active and partly decondensed chromatin at the surface of condensed chromatin domains. We studied the distribution of the damage-recognition protein XPC and of XPA, which is a central component of the chromatin-associated NER complex. Both XPC and XPA rapidly accumulate in the perichromatin region after UV irradiation, whereas only XPC is also moderately enriched in condensed chromatin domains. These observations suggest that DNA damage is detected by XPC throughout condensed chromatin domains, whereas DNA-repair complexes seem preferentially assembled in the perichromatin region. We propose that UV-damaged DNA inside condensed chromatin domains is relocated to the perichromatin region, similar to what has been shown for DNA replication. In support of this, we provide evidence that UV-damaged chromatin domains undergo expansion, which might facilitate the translocation process. Our results offer novel insight into the dynamic spatial organization of DNA repair in the human cell nucleus.

Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression.

Hypertension and chronic kidney disease (CKD) are complex traits representing major global health problems. Multiple genome-wide association studies have identified common variants in the promoter of the UMOD gene, which encodes uromodulin, the major protein secreted in normal urine, that cause independent susceptibility to CKD and hypertension. Despite compelling genetic evidence for the association between UMOD risk variants and disease susceptibility in the general population, the underlying biological mechanism is not understood. Here, we demonstrate that UMOD risk variants increased UMOD expression in vitro and in vivo. Uromodulin overexpression in transgenic mice led to salt-sensitive hypertension and to the presence of age-dependent renal lesions similar to those observed in elderly individuals homozygous for UMOD promoter risk variants. The link between uromodulin and hypertension is due to activation of the renal sodium cotransporter NKCC2. We demonstrated the relevance of this mechanism in humans by showing that pharmacological inhibition of NKCC2 was more effective in lowering blood pressure in hypertensive patients who are homozygous for UMOD promoter risk variants than in other hypertensive patients. Our findings link genetic susceptibility to hypertension and CKD to the level of uromodulin expression and uromodulin's effect on salt reabsorption in the kidney. These findings point to uromodulin as a therapeutic target for lowering blood pressure and preserving renal function.

Expérience de dix ans de réanimation chirurgicale d'un centre hospitalier universitaire. Détermination d'un critère d'identification des patients à risque de décéder de coagulopathie irréversible [10 years experience in surgical resuscitation at a university hospital center. Determination of a criterion for identifying patients at risk for fatal irreversible coagulopathy]

The authors evaluated ten years of surgical reanimation in the University Centre of Lausanne (CHUV). Irreversible coagulopathy (IC) is the predominant cause of death for the polytraumatized patient. Acidosis, hypothermy, and coagulation troubles are crucial elements of this coagulopathy. The authors looked for a criterion allowing the identification of dying of IC. In a retrospective study, laboratory results of pH, TP, PTT, thrombocyte count and the need for blood transfusion units were checked for each major step of the primary evaluation and treatment of the polytraumatized patients. These results were considered as critical according to criteria of the literature (30). The authors conclude that the apparation of a third critical value may be useful to identify the polytraumatized patient at risk of dying of IC status. This criterion may also guide the trauma team in selecting a damage control surgical approach (DCS). This criterion was then introduced into an algorithm involving the Emergency Department, the operating room and the Intensive Care Unit. This criterion is a new tool to address the patient at the crucial moment to the appropriate hospital structure.

Deleterious Actions Of Vegf On The Blood Retinal Barrier And Photoreceptor Survival In The Light-damage Model

Purpose: The retinal balance between pro- and anti-angiogenic factors is critical for angiogenesis control, but is also involved in cell survival. We previously reported upregulation of VEGF and photoreceptor (PR) cell death in the Light-damage (LD) model. Preliminary results showed that anti-VEGF can rescue PR from cell death. Thus, we investigated the role of VEGF on the retina and we herein described the effect of anti-VEGF antibody delivered by lentiviral gene transfer in this model.Methods: To characterize the action of VEGF during the LD, we exposed Balb/c mice subretinally injected with LV-anti-VEGF, or not, to 5'000 lux for 1h. We next evaluated the retinal function, PR survival and protein expression (VEGF, VEGFR1/2, Src, PEDF, p38MAPK, Akt, Peripherin, SWL-opsin) after LD. We analyzed Blood retinal barrier (BRB) integrity on flat-mounted RPE and cryosections stained with β-catenin, ZO-1, N-cadherin and albumin.Results: Results indicate that the VEGF pathway is modulated after LD. LD leads to extravascular albumin leakage and BRB breakdown: β-catenin, ZO-1 and N-cadherin translocate to the cytoplasm of RPE cells showing loss of cell cohesion. This phenomenon is in adequacy with the VEGF time-course expression. Assessment of the retinal function reveals that PR rescue correlates with the level of LV-anti-VEGF expression. Rhodopsin content was higher in the LV-anti-VEGF group than in controls and measures of the ONL thickness indicate that LV-anti-VEGF preserves by 82% the outer nuclear layer from degeneration. Outer segments (OS) appeared well organized with an appropriate length in the LV-anti-VEGF group compared to controls, and the expression of SWL-opsin is maintained in the OS without being mislocalized as in the LV-GFP group. Finally, LV-anti-VEGF treatment prevents BRB breakdown and maintained RPE cell integrity.Conclusions: This study involves VEGF in LD and highlights the prime importance of the BRB integrity for PR survival. Taken together, these results show that anti-VEGF is neuroprotective in this model and maintains functional PR layer in LD-treated mice.

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.

Endogenous protease nexin-1 protects against cerebral ischemia.

The serine protease thrombin plays a role in signalling ischemic neuronal death in the brain. Paradoxically, endogenous neuroprotective mechanisms can be triggered by preconditioning with thrombin (thrombin preconditioning, TPC), leading to tolerance to cerebral ischemia. Here we studied the role of thrombin's endogenous potent inhibitor, protease nexin-1 (PN-1), in ischemia and in tolerance to cerebral ischemia induced by TPC. Cerebral ischemia was modelled in vitro in organotypic hippocampal slice cultures from rats or genetically engineered mice lacking PN-1 or with the reporter gene lacZ knocked into the PN-1 locus PN-1HAPN-1-lacZ/HAPN-1-lacZ (PN-1 KI) exposed to oxygen and glucose deprivation (OGD). We observed increased thrombin enzyme activity in culture homogenates 24 h after OGD. Lack of PN-1 increased neuronal death in the CA1, suggesting that endogenous PN-1 inhibits thrombin-induced neuronal damage after ischemia. OGD enhanced β-galactosidase activity, reflecting PN-1 expression, at one and 24 h, most strikingly in the stratum radiatum, a glial cell layer adjacent to the CA1 layer of ischemia sensitive neurons. TPC, 24 h before OGD, additionally increased PN-1 expression 1 h after OGD, compared to OGD alone. TPC failed to induce tolerance in cultures from PN-1(-/-) mice confirming PN-1 as an important TPC target. PN-1 upregulation after TPC was blocked by the c-Jun N-terminal kinase (JNK) inhibitor, L-JNKI1, known to block TPC. This work suggests that PN-1 is an endogenous neuroprotectant in cerebral ischemia and a potential target for neuroprotection.