Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence.

The effect of exendin-(9-39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine betaTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nM and can be decreased dose dependently by exendin-(9-39). This latter effect was receptor dependent, as a beta-cell line not expressing the GLP-1 receptor was not affected by exendin-(9-39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of betaTC-Tet cells in the presence of exendin-(9-39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, betaTC-Tet cells; it was also seen with nontransformed mouse beta-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9-39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of betaTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of the beta-cells.

The use of proteomics to identify markers associated with metastasis in "Leishmania Viannia" species the role of tryparedoxin peroxidase

RESUME En Amérique Centrale et en Amérique du Sud, la leishmaniose cutanéo-muqueuse (LCM) est provoquée par le protozoaire Leishmania du sous-genre Viannia dont font partie L. (V.) braziliensis, L. (V.) panamensis et L. (V.) guyanensis. Dans la LCM, après guérison apparente de la lésion primitive, des lésions secondaires peuvent apparaître dues à la migration de l'infection à partir du site d'inoculation vers les muqueuses de l'ororhino-pharynx. Ce type de dissémination, communément appelé métastase, peut se produire plusieurs années après la guérison de la lésion cutanée initiale, et est un facteur majeur contribuant à la morbidité associée à la LCM. L'expression reproductible de l'activité métastatique au sein de populations discrètes de leishmanies chez le hamster fournit un modèle expérimental permettant d'étudier le degré de virulence du parasite. Nous avons utilisé des clones de L. (V.) guyanensis présentant des phénotypes stables allant d'un caractère hautement métastatique (M+) à non-métastatique (M-) comme outils pour mettre en évidence des facteurs spécifiques liés à la métastase chez les leishmanies du Nouveau Monde. Des analyses protéomiques comparatives utilisant l'électrophorèse bidimensionnelle sur gel de polyacrylamide couplée à de la spectrométrie de masse ont permis l'identification de plusieurs formes de la tryparedoxine peroxidase (TXNPx) en tant que polypeptides associés au phénotype métastatique. TXNPx, une enzyme de la famille des peroxiredoxines (Prxs), protéines antioxydantes, fonctionne comme la dernière peroxydase d'une cascade d'oxydoréductases qui réduit le peroxyde d'hydrogène aux dépens de NADPH. Toutes les Prxs sont caractérisées par un (1-Cys Prx) ou par deux résidus cystéines (2-Cys Prx), respectivement placés dans un environnement structurel conservé de la protéine et sont centrales dans la réaction catalytique. Des immuno-empreintes (« immunoblotting ») ont révélé que TXNPx est présente sous forme dimérique dans les promastigotes (M+) alors que dans les promastigotes, (M-) TXNPx est présente sous forme monomérique et dimérique. Cette caractéristique spécifique de dimérisation pourrait expliquer les différentes activités enzymatiques observées entre les deux promastigotes (M+) et (M-) en présence de peroxyde d'hydrogène ainsi que leur différence de survie et de charge parasitaire à l'intérieur des macrophages. Par conséquent, le processus métastatique pourrait être lié à la capacité du parasite à échapper efficacement aux défenses microbicides de la cellule hôte. ABSTRACT In South and Central America, protozoan parasites of the Leishmania Viannia subgenus including L. (V.) braziliensis, L. (V.) guyanensis and L. (V). panamensis cause mucocutaneous leishmaniasis (MCL). In MCL, after apparent cure of the primary lesion, secondary lesions may appear in the nasopharyngeal tissues of the infected host due to dissemination of the infection from the inoculation site. This type of dissemination, known as metastasis, can occur several years after healing of the original cutaneous lesion, and is a major contributory factor to the morbidity associated with MCL. The reproducible expression of metastasis by discrete populations of Leishmania parasites in hamsters provides an experimental model to examine the expression of parasite virulence. We used laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) to non-metastatic (M-) as tools for the discovery of specific factors associated with metastasis in New World Leishmania species. Comparative proteome analyses via 2D-electrophoresis (2-DE) coupled with mass spectrometry (MS) enabled the identification of various isoforms of tryparedoxin peroxidase (TXNPx) as polypeptides associated with the metastatic phenotype. TXNPx, an enzyme related to the antioxidant peroxiredoxin family (Prx) functions as the terminal peroxidase of a redox cascade that reduces hydroperoxides by NADPH. All Prxs are characterized by one (1-Cys Prx) or two cysteine residue(s) (2-Cys Prx), respectively, located in a conserved structural environment of the protein which are central for the catalytic reaction. Immunoblotting analysis revealed that, under non-reducing denaturing conditions, TXNPx is present in dimeric forms in (M+) promastigotes, whereas in (M-) promastigotes, both monomeric and dimeric forms are found. This specific dimerization feature may explain the different enzymatic activities of both (M+) and (M-) promastigote parasites in the presence of H2O2 and their difference in survival and parasite load inside macrophages. Therefore, the metastatic process could be related to the ability of the parasite to efficiently evade the microbicidal effect of the host cell.

Apoptotic markers in protozoan parasites.

ABSTRACT: The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

Intérêt des marqueurs sanguins dans la prédiction de la toxicité radio-induite [Interest of blood markers in predicting radiation-induced toxicity].

The oncologic outcome and the total dose are highly correlated with the treatment by ionizing radiation. The dose increase (total or per fraction) may provoke late-side effects that are potentially irreversible. The radiation-induced CD8 lymphocyte apoptotic value and the molecular modifications within the lymphocyte are capable of predicting the level of risk of developing late-side effects after curative intent radiotherapy. In this review, we present the different blood assays in this setting and discuss the current possibilities of researches, namely those involving the proteomic process.

Abnormal apical-to-basal transport of dietary ovalbumin by secretory IgA stimulates a mucosal Th1 response.

In celiac disease, enhanced permeability to gliadin peptides can result from their apico-basal transport by secretory immunoglobulin A1 (SIgA1) binding to the CD71 receptor ectopically expressed at the gut epithelial surface. Herein, we have established a mouse model in which there is apico-basal transport of the model antigen ovalbumin (OVA) by specific SIgA1 and have analyzed local T-cell activation. Transgenic DO11.10 mice were grafted with a hybridoma-secreting OVA-specific humanized IgA1, which could bind mouse CD71 and which were released in the intestinal lumen as SIgA. CD71 expression was induced at the gut apical surface by treating the mice with tyrphostin A8. Following gavage of the mice with OVA, OVA-specific CD4(+) T cells isolated from the mesenteric lymph nodes displayed higher expression of the activation marker CD69 and produced more interferon gamma in mice bearing the hybridoma-secreting OVA-specific IgA1, than in ungrafted mice or in mice grafted with an irrelevant hybridoma. These results indicate that the protective role of SIgA1 might be jeopardized in human pathological conditions associated with ectopic expression of CD71 at the gut surface.

Cerebrospinal fluid biomarkers of central nervous system dysfunction in HIV-infected patients

Background :¦In addition to opportunistic infections of the central nervous system (CNS), which are due to immunosuppression related to HIV, the latter virus, itself, can cause neuropathological abnormalities which are located mainly in the basal ganglia and are characterized by microglial giant cells, reactive astrocytosis and perivascular monocytes. This HIV encephalopathy is characterized, clinically, by psycho-motor slowing, memory loss, difficulties in complex tasks requiring executive functions, as well as motor disorders .These cognitive deficits are grouped under the acronym of HIV-associated neurocognitive disorders (HAND). In fact, HANDs are subdivided in three groups in accordance with the severity of the cognitive impairment: Asymptomatic Neurocognitive Impairment (ANI), Mild/moderate Neurocognitive Disorders (MND) and HIV Associated Dementia (HAD).¦While the incidence of HAD has significantly decreased in the era of combined antiretrobiral therapy (cART), the prevalence of milder forms of HIV-associated neurocognitive disorders HAND seem to have increased. There are many potential reasons to explain this state of facts.¦An important question is to understand how soon the brain may be affected by HIV. Since performing a biopsy in these patients is not an issue, the study of the CSF represents the best available way to look at putative biomarkers of inflammation/neurodegeneration in the CNS. Here, we wanted to examined the putative usefulness of different biomarkers as early indicators of anti-retroviral failure at the level of the CNS. We chose to study the CSF levels of:¦Amyloid-β 1-42 (Aβ42), Tau total (tTau), phosphorylated Tau (pTau), Neopterin and S100-β.¦Indeed, these molecules are representative biomarkers of the major cells of the CNS, i.e. neurons,¦macrophages/microglia and astrocytes.¦To examine how sensitive were these CSF biomarkers to indicate CNS insults caused by HIV, we proposed to take advantage of the MOST (Monotherapy Switzerland/Thailand study) study, recently published in AIDS. Thus, we collaborated with Prof. Pietro Vernazza in St-Gall. In MOST study, monotherapy (MT) consisting in ritonavir-boosted lopinavir (LPV/r) was compared to continuous conventional antiretroviral therapy including several molecules, hereafter referred as CT¦Methods :We tested 61 cerebrospinal fluid (CSF) samples from 52 patients enrolled in MOST, including 34 CSF samples of CT and 27 of MT (mean duration on MT: 47+20 weeks) in patients who maintained full VL suppression in blood (<50cps/ml). Using enzyme-linked immunosorbent assay (ELISA), we determined the CSF concentration of S100-beta (astrocytosis), neopterin (microglia, inflammation), total Tau (tTau), phosphorylated Tau (pTau), and amyloid-beta 1-42 (Abeta), the latter three markers indicating neuronal damages. The CSF samples of 37 HIV-negative patients with Alzheimer dementia (AD) served as controls. Results are expressed in pg/ml and reported as median ± interquartile range. Mann Whitney-U test was used to compare the results of a given biomarker between two groups and the Fisher test to compare frequencies.¦Results: We found a higher concentration of S100-beta (570±1132) and neopterin (2.5±2.9) in the CSF of MT versus CT (0±532, p=0.002 and 1.2±2.5, p=0.058, respectively). A cutoff of 940 pg/ml for S100-beta allowed to discriminate MT (11 above versus 16 below) from CT (1 vs 33, p=0.0003). At a lesser extent, a cutoff of 11 pg/ml for neopterin separated MT (4 above versus 23) from CT (0 vs 34, p=0.034) (Figure).¦In AD, tTau was higher (270±414) and Abeta lower (234±328) than in CT (150±153, p=0.0078, and 466±489, p=0.007, respectively). Such as for CT, Abeta was lower in AD than in MT (390±412, p=0.01). However, contrasting with CT, the levels of tTau were not different between AD and MT (199±177, p=0.11). S100b (173±214; p=0.0006) and neopterin (1.1±0.9; p=0.0014) were lower in AD than MT.¦Conclusions: Despite full VL-suppression in blood, HIV monotherapy is sufficient to trigger inflammation and, especially, astrocytosis. CSF markers of patients on CT have the same profile as reported for healthy subjects, suggesting that CT permits a good control of HIV in the brain. Finally, the levels of tTau, which are relatively similar between AD and MT patients, suggest that neurons are damaged during monotherapy.

Description of microsatellite markers and genotyping performances using feathers and buccal swabs for the Ivory gull (Pagophila eburnea).

We report 22 new polymorphic microsatellites for the Ivory gull (Pagophila eburnea), and we describe how they can be efficiently co-amplified using multiplexed polymerase chain reactions. In addition, we report DNA concentration, amplification success, rates of genotyping errors and the number of genotyping repetitions required to obtain reliable data with three types of noninvasive or nondestructive samples: shed feathers collected in colonies, feathers plucked from living individuals and buccal swabs. In two populations from Greenland (n=21) and Russia (Severnaya Zemlya Archipelago, n=21), the number of alleles per locus varied between 2 and 17, and expected heterozygosity per population ranged from 0.18 to 0.92. Twenty of the markers conformed to Hardy-Weinberg and linkage equilibrium expectations. Most markers were easily amplified and highly reliable when analysed from buccal swabs and plucked feathers, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. Although DNA amplification success using single shed feathers was generally high, the genotypes obtained from this type of samples were prone to error and thus need to be amplified several times. The set of microsatellite markers described here together with multiplex amplification conditions and genotyping error rates will be useful for population genetic studies of the Ivory gull.

Comparison of skin microvascular reactivity with hemostatic markers of endothelial dysfunction and damage in type 2 diabetes.

AIM: Patients with non-insulin-dependent diabetes mellitus (NIDDM) are at increased cardiovascular risk due to an accelerated atherosclerotic process. The present study aimed to compare skin microvascular function, pulse wave velocity (PWV), and a variety of hemostatic markers of endothelium injury [von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (t-PA), tissue factor pathway inhibitor (TFPI), and the soluble form of thrombomodulin (s-TM)] in patients with NIDDM. METHODS: 54 patients with NIDDM and 38 sex- and age-matched controls were studied. 27 diabetics had no overt micro- and/or macrovascular complications, while the remainder had either or both. The forearm skin blood flow was assessed by laser-Doppler imaging, which allowed the measurement of the response to iontophoretically applied acetylcholine (endothelium-dependent vasodilation) and sodium nitroprusside (endothelium-independent vasodilation), as well as the reactive hyperemia triggered by the transient occlusion of the circulation. RESULTS: Both endothelial and non-endothelial reactivity were significantly blunted in diabetics, regardless of the presence or the absence of vascular complications. Plasma vWF, TFPI and s-TM levels were significantly increased compared with controls only in patients exhibiting vascular complications. Concentrations of t-PA and PAI-1 were significantly increased in the two groups of diabetics versus controls. CONCLUSION: In NIDDM, both endothelium-dependent and -independent microvascular skin reactivity are impaired, whether or not underlying vascular complications exist. It also appears that microvascular endothelial dysfunction is not necessarily associated in NIDDM with increased circulating levels of hemostatic markers of endothelial damage known to reflect a hypercoagulable state.

Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices.

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants that improve plant nutrient acquisition and promote plant diversity. Although within-species genetic differences among AMF have been shown to differentially affect plant growth, very little is actually known about the degree of genetic diversity in AMF populations. This is largely because of difficulties in isolation and cultivation of the fungi in a clean system allowing reliable genotyping to be performed. A population of the arbuscular mycorrhizal fungus Glomus intraradices growing in an in vitro cultivation system was studied using newly developed simple sequence repeat (SSR), nuclear gene intron and mitochondrial ribosomal gene intron markers. The markers revealed a strong differentiation at the nuclear and mitochondrial level among isolates. Genotypes were nonrandomly distributed among four plots showing genetic subdivisions in the field. Meanwhile, identical genotypes were found in geographically distant locations. AMF genotypes showed significant preferences to different host plant species (Glycine max, Helianthus annuus and Allium porrum) used before the fungal in vitro culture establishment. Host plants in a field could provide a heterogeneous environment favouring certain genotypes. Such preferences may partly explain within-population patterns of genetic diversity.

Cancer testis antigen expression in gastrointestinal stromal tumors: new markers for early recurrence.

Cancer testis antigens (CTAs) are expressed in a variety of malignant tumors but not in any normal adult tissues except germ cells and occasionally placenta. Because of this tumor-associated pattern of expression, CTAs are regarded as potential vaccine targets. The expression of CTAs in gastrointestinal stromal tumors (GIST) has not been analyzed systematically previously. The present study was performed to analyze the expression of CTA in GIST and to determine if CTA expression correlates with prognosis. Thirty-five GIST patients were retrospectively analyzed for their expression of CTAs by immunohistochemistry using the following monoclonal antibodies (mAb/antigen): MA454/MAGE-A1, M3H67/MAGE-A3, 57B/MAGE-A4, CT7-33/MAGE-C1 and E978/NY-ESO-1. Fourteen tumors (40%) expressed 1 or more of the 5 CTAs tested. Fourteen percent (n = 5/35) were positive for MAGE-A1, MAGE-A3 or MAGE-A4, respectively. Twenty-six percent (n = 9/35) stained positive for MAGE-C1 and 20% (n = 7/35) for NY-ESO-1. A highly significant correlation between CTA expression and tumor recurrence risk was observed (71% vs. 29%; p = 0.027). In our study population, the high-risk GIST expressed CTAs more frequently than low-risk GIST (p = 0.012). High-risk GISTs which stained positive for at least 1 CTA, recurred in 100% (n = 25) of the cases. This is the first study analyzing CTA expression in GIST and its prognostic value for recurrence. The CTA staining could add information to the individual patient prognosis and represent an interesting target for future treatment strategies.

Association of alcohol consumption and HIV surrogate markers in participants of the swiss HIV cohort study.

BACKGROUND: Alcohol consumption may affect the course of HIV infection and/or antiretroviral therapy (ART). The authors investigated the association between self-reported alcohol consumption and HIV surrogate markers in both treated and untreated individuals. DESIGN: Prospective cohort study. METHODS: Over a 7-year period, the authors analyzed 2 groups of individuals in the Swiss HIV Cohort Study: (1) ART-naïve individuals remaining off ART and (2) individuals initiating first ART. For individuals initiating first ART, time-dependent Cox proportional hazards models were used to assess the association between alcohol consumption, virological failure, and ART interruption. For both groups, trajectories of log-transformed CD4 cell counts were analyzed using linear mixed models with repeated measures. RESULTS: The authors included 2982 individuals initiating first ART and 2085 ART naives. In individuals initiating first ART, 241 (8%) experienced virological failure. Alcohol consumption was not associated with virological failure. ART interruption was noted in 449 (15%) individuals and was more prevalent in severe compared with none/light health risk drinkers [hazard ratio: 2.24, 95% confidence interval: 1.42 to 3.52]. The association remained significant even after adjusting for nonadherence. The authors did not find an association between alcohol consumption and change in CD4 cell count over time in either group. CONCLUSIONS: No effect of alcohol consumption on either virological failure or CD4 cell count in both groups of ART-initiating and ART-naive individuals was found. However, severe drinkers were more likely to interrupt ART. Efforts on ART continuation should be especially implemented in individuals reporting high alcohol consumption.

Markers of atherosclerosis and of inflammation for prediction of coronary heart disease in older adults

BACKGROUND: Several markers of atherosclerosis and of inflammation have been shown to predict coronary heart disease (CHD) individually. However, the utility of markers of atherosclerosis and of inflammation on prediction of CHD over traditional risk factors has not been well established, especially in the elderly. METHODS: We studied 2202 men and women, aged 70-79, without baseline cardiovascular disease over 6-year follow-up to assess the risk of incident CHD associated with baseline noninvasive measures of atherosclerosis (ankle-arm index [AAI], aortic pulse wave velocity [aPWV]) and inflammatory markers (interleukin-6 [IL-6], C-reactive protein [CRP], tumor necrosis factor-a [TNF-a]). CHD events were studied as either nonfatal myocardial infarction or coronary death ("hard" events), and "hard" events plus hospitalization for angina, or the need for coronary-revascularization procedures (total CHD events). RESULTS: During the 6-year follow-up, 283 participants had CHD events (including 136 "hard" events). IL-6, TNF-a and AAI independently predicted CHD events above Framingham Risk Score (FRS) with hazard ratios [HR] for the highest as compared with the lowest quartile for IL-6 of 1.95 (95%CI: 1.38-2.75, p for trend<0.001), TNF-a of 1.45 (95%CI: 1.04-2.02, p for trend 0.03), of 1.66 (95%CI: 1.19-2.31) for AAI £0.9, as compared to AAI 1.01-1.30. CRP and aPWV were not independently associated with CHD events. Results were similar for "hard" CHD events. Addition of IL-6 and AAI to traditional cardiovascular risk factors yielded the greatest improvement in the prediction of CHD; C-index for "hard"/total CHD events increased from 0.62/0.62 for traditional risk factors to 0.64/0.64 for IL-6 addition, 0.65/0.63 for AAI, and 0.66/0.64 for IL-6 combined with AAI. Being in the highest quartile of IL-6 combined with an AAI £ 0.90 or >1.40 yielded an HR of 2.51 (1.50-4.19) and 4.55 (1.65-12.50) above FRS, respectively. With use of CHD risk categories, risk prediction at 5 years was more accurate in models that included IL-6, AAI or both, with 8.0, 8.3 and 12.1% correctly reclassified respectively. CONCLUSIONS: Among older adults, markers of atherosclerosis and of inflammation, particularly IL-6 and AAI, are independently associated with CHD. However, these markers only modestly improve cardiovascular risk prediction beyond traditional risk factors. Acknowledgments: This study was supported by Contracts NO1-AG-6-2101, NO1-AG-6- 2103, and NO1-AG-6-2106 of the National Institute on Aging. This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.