BOLD responses to trigeminal nerve stimulation.

The current study investigates a new model of barrel cortex activation using stimulation of the infraorbital branch of the trigeminal nerve. A robust and reproducible activation of the rat barrel cortex was obtained following trigeminal nerve stimulation. Blood oxygen level-dependent (BOLD) effects were obtained in the primary somatosensory barrel cortex (S1BF), the secondary somatosensory cortex (S2) and the motor cortex. These cortical areas were reached from afferent pathways from the trigeminal ganglion, the trigeminal nuclei and thalamic nuclei from which neurons project their axons upon whisker stimulation. The maximum BOLD responses were obtained for a stimulus frequency of 1 Hz, a stimulus pulse width of 100 μs and for current intensities between 1.5 and 3 mA. The BOLD response was nonlinear as a function of frequency and current intensity. Additionally, modeling BOLD responses in the rat barrel cortex from separate cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO(2)) measurements showed good agreement with the shape and amplitude of measured BOLD responses as a function of stimulus frequency and will potentially allow to identify the sources of BOLD nonlinearities. Activation of the rat barrel cortex using trigeminal nerve stimulation will contribute to the interpretation of the BOLD signals from functional magnetic resonance imaging studies.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

Cellular immune responses and disease control in acute AIDS-associated Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) specific T cell responses and KSHV viremia were analyzed in seven HIV-infected patients with active Kaposi's sarcoma lesions who initiated highly active antiretroviral therapy, and were compared between patients with improved Kaposi's sarcoma and those with progressive Kaposi's sarcoma requiring further systemic chemotherapy. Patients with controlled Kaposi's sarcoma disease demonstrated undetectable Kaposi's sarcoma viremia together with KSHV-specific CD8 T cells secreting interferon-gamma and tumor necrosis factor-alpha, whereas progressors showed increasing viremia with weak or no T-cell responses. These data point toward a potential role of KSHV-specific immunity in the control of AIDS-associated Kaposi's sarcoma.

Survival responses in stressed organs

Apoptosis or programmed cell death is a regulated form of cell suicide executed by cysteine proteases, or "caspases", to maintain proper tissue homeostasis in multicellular organisms. Dysregulation of apoptosis leads to pathological complications including cancer, autoimmunity, neurodegenerative, and heart diseases. Beside their known function as the key executioners of apoptotic cell death, caspases were reported to mediate non-apoptotic functions. In this report we study the survival signals conveyed through caspase-3-mediated cleavage of Ras GTPase-activating proteins (RasGAP). Ubiquitously expressed, RasGAP senses caspase activity and controls the cell death/survival switch. RasGAP is cleaved once at low caspase activity and the generated N-terminal fragment (fragment N) induces a survival response by activating Ras/PI3K/Akt pathway. However, high caspase activity associated with increased stress leads to fragment Ν cleavage into fragments that do not mediate any detectable survival signals. In this thesis project we studied the role of fragment Ν in protecting stressed organs as well as in maintenance of their functionality. In response to stress in different organs, we found that mice lacking caspase-3 or unable to cleave RasGAP (Knock-In mice), and therefore unable to generate fragment N, were deficient in Akt activation and experienced increased apoptosis compared to wild-type mice. Augmented tissue damage and organ dysfunction in those mice highlight the importance of fragment Ν in activating Akt-mediated prosurvival pathway and in protection of organs during episodes of stress. In parallel we investigated the role of fragment Ν in regulating the activation of transcription factor NF-kB, a master regulator of inflammation. Sustained NF-kB activation may be detrimental by directly causing apoptosis or leading to a persistent damaging inflammation response. We found that fragment Ν is a potent inhibitor of NF-kB by favoring its nuclear export. Therefore, fragment Ν regulates NF-kB activity and contributes to a controlled response as well as maintenance of homeostasis in stressed cells. Importantly, these findings introduce new insights of how activated caspase-3 acts as a stress intensity sensor that controls cell fate by either initiating a fragment N- dependent cell resistance program or a cell suicide response. This identifies the pivotal role of fragment Ν in protection against patho-physiological damage, and encourages the development of therapies which aim to increase cell resistance to vigorous treatment. - L'apoptose, ou mort cellulaire programmée, est une forme contrôlée de suicide cellulaire exécuté par des protéines appelées caspases, dans le but de maintenir l'homéostasie des tissus sains dans les organismes multicellulaires. Un mauvais contrôle de l'apoptose peut mener à des pathologies comme le cancer, la neurodégénération et les maladies cardiaques et auto-immunes. En dehors de leur rôle connu d'exécutrices de l'apoptose, les caspases ont aussi été identifiées dans d'autres contextes non-apoptotiques. Dans ce projet, nous avons étudié les signaux de survie émis par le résultat du clivage de RasGAP par la caspase-3. Exprimée de façon ubiquitaire, RasGAP est sensible à l'activité de caspase-3 et contrôle la décision de la cellule à entreprendre la mort ou la survie cellulaire. A un taux d'activité faible, la caspase-3 clive RasGAP, ce qui mène à la génération d'un fragment N-terminal, appelé Fragment N, qui induit des signaux de survie via l'activation de la cascade Ras/PI3K/Akt. Cependant, lorsque l'activité de la caspase-3 augmente, le fragment N est clivé, ce qui a pour effet d'éliminer ces signaux de survie. Dans ce travail, nous avons étudié le rôle du Fragment N dans la protection des organes en état de stress et dans le maintien de leur fonctionnalité. En réponse à certains stress, nous avons découvert que les organes de souris n'exprimant pas la caspase-3 ou alors incapables de cliver RasGAP (souris Kl), et de ce fait n'ayant pas la possibilité de générer le Fragment N, perdaient leur faculté d'activer la protéine Akt et démontraient un taux d'apoptose plus élevé que des organes de souris sauvages. Le fait que les organes et tissus de ces souris manifestaient de graves dommages et dysfonctions met en évidence l'importance du Fragment N dans l'activation des signaux de survie via la protéine Akt et dans la neutralisation de l'apoptose induite par la caspase-3. En parallèle, nous avons investigué le rôle du Fragment N dans la régulation de l'activation de NF-kB, un facteur de transcription clé dans l'inflammation. Une activation soutenue de NF-kB peut être délétère par activation directe de l'apoptose ou peut mener à une réponse inflammatoire persistante. Nous avons découvert que le Fragment N, en favorisant l'export de NF-kB depuis le noyau, était capable de l'inhiber très efficacement. Le Fragment N régule donc l'activité de NF-kB et contribue au maintien de l'homéostasie dans des cellules stressées. Ces découvertes aident, de façon importante, à la compréhension de comment l'activation de la caspase-3 agit comme senseur de stress et décide du sort de la cellule soit en initiant une protection par le biais du fragment N, ou en induisant un suicide cellulaire. Cette étude définit le Fragment Ν comme ayant un rôle de pivot dans la protection contre des dommages patho-physiologiques, et ouvre des perspectives de développement de thérapies qui cibleraient à augmenter la résistance à divers traitements.

Cardiorespiratory responses during running and sport-specific exercises in handball players

To determine whether a 4-a-side handball (HB) game is an appropriate aerobic stimulus to reach and potentially enhance maximal oxygen uptake (V O(2)max), and whether heart rate (HR) is a valid index of V O(2) during a handball game. Nine skilled players (21.0+/-2.9 yr) underwent a graded maximal aerobic test (GT) where V O(2)max and HR-V O(2) relationship were determined. V O(2), HR and blood lactate ([La](b)) were recorded during a 2 x 225 s (interspersed with 30s rest) 4-a-side handball game and were compared to those measured during an 480-s running intermittent exercise (IE). Mean V O(2) tended to be higher in handball compared to IE (93.9+/-8.5 vs. 87.6+/-7.4% O(2)max, p=0.06), whereas HR was similar (92.3+/-4.9 vs. 93.9+/-3.9% of the peak of HR, p=0.10). [La](b) was lower for handball than for IE (8.9+/-3.5 vs. 11.6+/-2.1 mmol l(-1), p=0.04). Time spent over 90% of V O(2)max was higher for handball than for IE (336.1+/-139.6s vs. 216.1+/-124.7s; p=0.03). The HR-V O(2) relationship during GT was high (r(2)=0.96, p<0.001) but estimated V O(2) from HR was lower to that measured (p=0.03) in handball, whereas there was no difference in IE. 4-a-side handball game can be used as a specific alternative to IE for enhancing aerobic fitness in handball players. Nevertheless, the accuracy of HR measures for estimating V O(2) during handball is poor.

Adult attachment representations predict cortisol and oxytocin responses to stress.

There are many factors contributing to individual variations in the response to stressful experiences. The present study evaluated the patterns of stress responses according to attachment representations in 28 adults from a community sample, plus 46 subjects expected to be particularly sensitive to stress, having been exposed during childhood and/or adolescence to traumatizing events such as abuse or potentially lethal illnesses. Subjects were given the Adult Attachment Interview, which provides attachment classifications, and the Trier Social Stress Test (TSST), involving an experimental psychosocial challenge. Subjective responses to the TSST, as well as saliva samples (assayed for cortisol) and blood plasma samples (assayed for ACTH and oxytocin) were collected before, during and after the stress procedure. The stress responses presented specific patterns according to attachment classifications. Subjects with an autonomous attachment classification reported relatively low subjective stress, they presented a moderate response of the hypothalamic-pituitary-adrenal (HPA) axis (ACTH and cortisol), and a high level of oxytocin. Subjects with a dismissing classification reported a moderate subjective stress, they presented an elevated response of the HPA axis, and moderate levels of oxytocin. Subjects with a preoccupied classification presented moderate levels of subjective stress, and of HPA response, and a relatively low level of oxytocin. Finally, subjects with an unresolved classification reported elevated subjective stress; they presented a suppressed HPA response, and moderate levels of oxytocin. These data support the notion that attachment representations may affect stress responses, and suggest a specific role of oxytocin in both the attachment system and the stress system.

Responses to exercise in normobaric hypoxia: comparison of elite and recreational ski mountaineers.

PURPOSE: Hypoxia is known to reduce maximal oxygen uptake (VO(2max)) more in trained than in untrained subjects in several lowland sports. Ski mountaineering is practiced mainly at altitude, so elite ski mountaineers spend significantly longer training duration at altitude than their lower-level counterparts. Since acclimatization in hypobaric hypoxia is effective, the authors hypothesized that elite ski mountaineers would exhibit a VO2max decrement in hypoxia similar to that of recreational ski mountaineers. METHODS: Eleven elite (E, Swiss national team) and 12 recreational (R) ski mountaineers completed an incremental treadmill test to exhaustion in normobaric hypoxia (H, 3000 m, F(1)O(2) 14.6% ± 0.1%) and in normoxia (N, 485 m, F(1)O(2) 20.9% ± 0.0%). Pulse oxygen saturation in blood (SpO(2)), VO(2max), minute ventilation, and heart rate were recorded. RESULTS: At rest, hypoxic ventilatory response was higher (P < .05) in E than in R (1.4 ± 1.9 vs 0.3 ± 0.6 L · min⁻¹ · kg⁻¹). At maximal intensity, SpO(2) was significantly lower (P < .01) in E than in R, both in N (91.1% ± 3.3% vs 94.3% ± 2.3%) and in H (76.4% ± 5.4% vs 82.3% ± 3.5%). In both groups, SpO(2) was lower (P < .01) in H. Between N and H, VO(2max) decreased to a greater extent (P < .05) in E than in R (-18% and -12%, P < .01). In E only, the VO(2max) decrement was significantly correlated with the SpO(2) decrement (r = .74, P < .01) but also with VO(2max) measured in N (r = .64, P < .05). CONCLUSION: Despite a probable better acclimatization to altitude, VO(2max) was more reduced in E than in R ski mountaineers, confirming previous results observed in lowlander E athletes.

Cardiorespiratory responses to the 30-15 intermittent ice test

PURPOSE: In this study, the authors compared the cardiorespiratory responses between the 30-15 Intermittent Ice Test (30-15(IIT)) and the 30-15 Intermittent Fitness Test (30-15(IFT)) in semiprofessional hockey players. METHODS: Ten players (age 24 ± 6 y) from a Swiss League B team performed the 30-15(IIT) and 30-15(IFT) in random order (13 ± 4 d between trials). Cardiorespiratory variables were measured with a portable gas analyzer. Ventilatory threshold (VT), respiratory-compensation point (RCP), and maximal speeds were measured for both tests. Peak blood lactate ([La(peak)]) was measured at 1 min postexercise. RESULTS: Compared with 30-15(IFT), 30-15(IIT) peak heart rate (HR(peak); mean ± SD 185 ± 7 vs 189 ± 10 beats/min, P = .02) and peak oxygen consumption (VO(2peak)); 60 ± 7 vs 62.7 ± 4 mL/min/kg, P = .02) were lower, whereas [La(peak)] was higher (10.9 ± 1 vs 8.6 ± 2 mmol/L, P < .01) for the 30-15(IIT). VT and RCP values during the 30-15(IIT) and 30-15(IFT) were similar for %HR(peak) (76.3% ± 5% vs 75.5% ± 3%, P = .53, and 90.6% ± 3% vs. 89.8% ± 3%, P = .45) and % VO(2peak) (62.3% ± 5% vs 64.2% ± 6%, P = .46, and 85.9% ± 5% vs 84.0% ± 7%, P = .33). VO(2peak ))(r = .93, P < .001), HR(peak) (r = .86, P = .001), and final velocities (r = .69, P = .029) were all largely to almost perfectly correlated. CONCLUSIONS: Despite slightly lower maximal cardiorespiratory responses than in the field-running version of the test, the on-ice 30-15(IIT) is of practical interest since it is a specific maximal test with a higher anaerobic component.

PPARs as drug targets to modulate inflammatory responses?

The three peroxisome proliferator-activated receptors (PPARs) isotypes (PPAR alpha, beta/delta and gamma) belong to the nuclear hormone receptor family. During the last decade, they have been identified as anti-inflammatory transcription factors. Part of this regulation antiinflammatory is mediated through negative interference between PPARs and other nuclear factors such as NFkB, AP-1 and C/EBP, which regulate innate as well as adaptative immunity. In addition, the PPARs control the functions of macrophages, B cells and T cells. In this review, we summarise the pathways through which the PPARs control inflammatory responses. We also discuss the potential utilisation of PPAR specific ligands in the treatment of inflammatory diseases, such as inflammatory bowel diseases, atherosclerosis, Parkinson's and Alzheimer's diseases.

Jasmonates and related oxylipins in plant responses to pathogenesis and herbivory.

The vigorous production of oxygenated fatty acids (oxylipins) is a characteristic response to pathogenesis and herbivory, and is often accompanied by the substantial release of small and reactive lipid-fragmentation products. Some oxylipins, most notably those of the jasmonate family, have key roles as potent regulators. Recent advances have been made in understanding oxylipin-regulated signal transduction in response to attack. Much jasmonate signaling takes place via a genetically defined signal network that is linked to the ethylene, auxin, and salicylic acid signal pathways, but a second aspect of jasmonate signaling is emerging. Some jasmonates and several newly discovered cyclopentenone lipids can activate or repress gene expression through the activities of a conserved electrophilic atom group.

Thogoto virus infection induces sustained type I interferon responses that depend on RIG-I-like helicase signaling of conventional dendritic cells.

Type I interferon (IFN-α/β) induction upon viral infection contributes to the early antiviral host defense and ensures survival until the onset of adaptive immunity. Many viral infections lead to an acute, transient IFN expression which peaks a few hours after infection and reverts to initial levels after 24 to 36 h. Robust IFN expression often is conferred by specialized plasmacytoid dendritic cells (pDC) and may depend on positive-feedback amplification via the type I IFN receptor (IFNAR). Here, we show that mice infected with Thogoto virus (THOV), which is an influenza virus-like orthomyxovirus transmitted by ticks, mounted sustained IFN responses that persisted up to 72 h after infection. For this purpose, we used a variant of THOV lacking its IFN-antagonistic protein ML, an elongated version of the matrix (M) protein [THOV(ΔML)]. Of note, large amounts of type I IFN were also found in the serum of mice lacking the IFNAR. Early IFN-α expression seemed to depend on Toll-like receptor (TLR) signaling, whereas prolonged IFN-α responses strictly depended on RIG-I-like helicase (RLH) signaling. Unexpectedly, THOV(ΔML)-infected bone marrow-derived pDC (BM-pDC) produced only moderate IFN levels, whereas myeloid DC (BM-mDC) showed massive IFN induction that was IPS-1-dependent, suggesting that BM-mDC are involved in the massive, sustained IFN production in THOV(ΔML)-infected animals. Thus, our data are compatible with the model that THOV(ΔML) infection is sensed in the acute phase via TLR and RLH systems, whereas at later time points only RLH signaling is responsible for the induction of sustained IFN responses.

Impact of Sirtuin 2 knockout on innate immune responses

Purpose/Objective: Histone deacetylases (HDACs) deacetylate histones and transcriptional regulators thereby affecting numerous biological functions. Seven mammalian sirtuins (SIRT1-7) constitute the NAD-dependent class III subfamily of HDACs. Sirtuins are the center of great interest due to their regulatory role in the control of metabolism, ageing and age-related diseases. Up to now, little is known about the influence of sirtuins on immune responses, and nothing about the role of SIRT2. The aim of the study was to analyze the influence of SIRT2 knockout on immune cell development and innate immune responses in vitro and in vivo. Materials and methods: SIRT2 germline knockout were produced on a C57BL/6J background. The cellularity of thymus and spleen was assessed by flow cytometry (n = 3). Bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) and splenocytes were stimulated with LPS, Pam3CSK4 lipopeptide, CpG ODN, E. coli, S. aureus, TSST-1, SEB, anti-CD3+ CD28 and concanavalin A (n = 3_8). TNF, IL-2, IL-6, IL-12p40 and IFNc production, SIRT1_7 and CD40 expression, and proliferation were quantified by real time-PCR, ELISA, flow cytometry and H3-thymidine incorporation. Mice (n = 6_16) were challenged with LPS, TNF/D-galactosamine, E. coli and K. pneumonia titrated to cause either mild or severe infections or shock. Blood was collected to quantify cytokines and bacteria. Mortality was checked regularly. Results: SIRT2 is the most expressed sirtuin in macrophages and myeloid DCs. To test whether SIRT2 impacts on innate immune responses, we generated SIRT2 germline knockout mice. SIRT2-/- mice born at the expected Mendelian ratio and develop normally. The proportions and absolute numbers of DN1-4, DP and SP thymocytes, and of T-cells (DN and SP, naı¨ve and memory), B-cells (immature and mature), DCs (cDCs and pDCs) and granulocytes in the spleen are similar in SIRT2+/+ and SIRT2-/- mice. SIRT2+/+ and SIRT2-/- BMDMs, BMDCs and splenocytes produce cytokines (RNA and protein), upregulate CD40, and proliferate to the same extent. SIRT2+/+ and SIRT2-/- mice respond similarly (cytokine blood levels, bacterial counts and mortality) to non-severe and lethal endotoxemia, E. coli peritonitis, K. pneumonia pneumonia and TNF-induced shock. Conclusions: SIRT2 knockout has no dramatic impact on the development of immune cells and on innate immune responses in vitro and in vivo. Considering that SIRT2 may participate to control metabolic homeostasis, we are currently assessing the impact of SIRT2 deficiency on innate immune responses under metabolic stress.

Combined Use of Mycobacterium tuberculosis-Specific CD4 and CD8 T-Cell Responses Is a Powerful Diagnostic Tool of Active Tuberculosis.

Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infection.

Therapeutic Blockade of LIGHT Interaction With Herpesvirus Entry Mediator and Lymphotoxin β Receptor Attenuates In Vivo Cytotoxic Allogeneic Responses.

BACKGROUND: Tumor necrosis factor/tumor necrosis factor receptor superfamily members conform a group of molecular interaction pathways of essential relevance during the process of T-cell activation and differentiation toward effector cells and particularly for the maintenance phase of the immune response. Specific blockade of these interacting pathways, such as CD40-CD40L, contributes to modulate the deleterious outcome of allogeneic immune responses. We postulated that antagonizing the interaction of LIGHT expression on activated T cells with its receptors, herpesvirus entry mediator and lymphotoxin β receptor, may decrease T cell-mediated allogeneic responses. METHODS: A flow cytometry competition assay was designed to identify anti-LIGHT monoclonal antibodies capable to prevent the interaction of mouse LIGHT with its receptors expressed on transfected cells. An antibody with the desired specificity was evaluated in a short-term in vivo allogeneic cytotoxic assay and tested for its ability to detect endogenous mouse LIGHT. RESULTS: We provide evidence for the first time that in mice, as previously described in humans, LIGHT protein is rapidly and transiently expressed after T-cell activation, and this expression was stronger on CD8 T cells than on CD4 T cells. Two anti-LIGHT antibodies prevented interactions of mouse LIGHT with its two known receptors, herpesvirus entry mediator and lymphotoxin β receptor. In vivo administration of anti-LIGHT antibody (clone 10F12) ameliorated host antidonor short-term cytotoxic response in wild type B6 mice, although to a lesser extent than that observed in LIGHT-deficient mice. CONCLUSION: The therapeutic targeting of LIGHT may contribute to achieve a better control of cytotoxic responses refractory to current immunosuppressive drugs in transplantation.