Effects of adrenergic blockade on hepatic glucose production during ethanol administration.

Acute ethanol administration stimulates sympathetic nervous system activity. The present study was designed to determine whether this sympathetic activation affects glycogenolysis and total hepatic glucose production (HGP) during ethanol-induced inhibition of gluconeogenesis. Nineteen volunteers participated in four protocols. Two protocols aimed to study--using combined infusion of [6,6-2H2]glucose and [U-13C]glucose, VCO2 and 13CO2 measurements--the effects of ethanol infusion alone (n = 10) or with propranolol (n = 6) or phentolamine infusion (n = 4) on HGP, glucose disposal (Rd), glucose oxidation [13C]Glcox and non-oxidative glucose disposal (NOGD = Rd - [13C]Glcox). The fourth protocol assessed the effects of saline infusion alone on HGP. Using ethanol, HGP decreased by 23%, Rd by 20% and glycaemia by 9% (all P < 0.001); heart rate increased by 10%, whereas blood pressure remained unchanged. The effects were not observed with saline, except a slight (10%) decrease in HGP (P < 0.01 vs. ethanol). Ethanol did not affect [13C]Glcox but decreased NOGD by 73% (P < 0.001). Propranolol or phentolamine did not alter any of the effects of ethanol on glucose metabolism, but decreased mean arterial pressure. Propranolol prevented the ethanol-induced increase in heart rate. In conclusion, ethanol decreased blood glucose by decreasing HGP, presumably by inhibiting gluconeogenesis. Sympathetic activation prevented the decrease in blood pressure produced by ethanol but did not stimulate glycogenolysis.

CD8(+) T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1.

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

Treatment implications of the emerging molecular classification system for melanoma.

Melanoma is an aggressive disease with few standard treatment options. The conventional classification system for this disease is based on histological growth patterns, with division into four subtypes: superficial spreading, lentigo maligna, nodular, and acral lentiginous. Major limitations of this classification system are absence of prognostic importance and little correlation with treatment outcomes. Recent preclinical and clinical findings support the notion that melanoma is not one malignant disorder but rather a family of distinct molecular diseases. Incorporation of genetic signatures into the conventional histopathological classification of melanoma has great implications for development of new and effective treatments. Genes of the mitogen-associated protein kinase (MAPK) pathway harbour alterations sometimes identified in people with melanoma. The mutation Val600Glu in the BRAF oncogene (designated BRAF(V600E)) has been associated with sensitivity in vitro and in vivo to agents that inhibit BRAF(V600E) or MEK (a kinase in the MAPK pathway). Melanomas arising from mucosal, acral, chronically sun-damaged surfaces sometimes have oncogenic mutations in KIT, against which several inhibitors have shown clinical efficacy. Some uveal melanomas have activating mutations in GNAQ and GNA11, rendering them potentially susceptible to MEK inhibition. These findings suggest that prospective genotyping of patients with melanoma should be used increasingly as we work to develop new and effective treatments for this disease.

What influence do anticoagulants have on oral implant therapy? A systematic review.

OBJECTIVES: This systematic review aims to assess the risks (both thromboembolic and bleeding) of an oral anticoagulation therapy (OAT) patient undergoing implant therapy and to provide a management protocol to patients under OAT undergoing implant therapy. MATERIAL AND METHODS: Medline, Cochrane Data Base of Systematic Reviews, the Cochrane Central Register of Controlled Trials and EMBASE (from 1980 to December 2008) were searched for English-language articles published between 1966 and 2008. This search was completed by a hand research accessing the references cited in all identified publications. RESULTS: Nineteen studies were identified reporting outcomes after oral surgery procedures (mostly dental extractions in patients on OAT following different management protocols and haemostatic therapies). Five studies were randomized-controlled trials (RCTs), 11 were controlled clinical trials (CCTs) and three were prospective case series. The OAT management strategies as well as the protocols during and after surgery were different. This heterogeneity prevented any possible data aggregation and synthesis. The results from these studies are very homogeneous, reporting minor bleeding in very few patients, without a significant difference between the OAT patients who continue with the vitamin K antagonists vs. the patients who stopped this medication before surgery. These post-operative bleeding events were controlled only with local haemostatic measures: tranexamic acid mouthwashes, gelatine sponges and cellulose gauzes's application were effective. Post-operative bleeding did not correlate with the international normalised ratio (INR) status. In none of the studies was a thromboembolic event reported. CONCLUSIONS: OAT patients (INR 2-4) who do not discontinue the AC medication do not have a significantly higher risk of post-operative bleeding than non-OAT patients and they also do not have a higher risk of post-operative bleeding than OAT patients who discontinue the medication. In patients with OAT (INR 2-4) without discontinuation, topical haemostatic agents were effective in preventing post-operative bleeding. OAT discontinuation is not recommended for minor oral surgery, such as single tooth extraction or implant placement, provided that this does not involve autogenous bone grafts, extensive flaps or osteotomy preparations extending outside the bony envelope. Evidence does not support that dental implant placement in patients on OAT is contraindicated.

The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells.

Susceptibility and development of Th2 cells in BALB/c mice infected with Leishmania major result from early IL-4 production by Vbeta4Valpha8 CD4+ T cells in response to the Leishmania homolog of mammalian RACK1 Ag. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. We further showed that transfer of 10(7) BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 10(7) control BALB/c spleen cells were resistant to infection with L. major and developed a Th1 response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.

Beta-lactams against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) have developed resistance to virtually all non-experimental antibiotics. They are intrinsically resistant to beta-lactams by virtue of newly acquired low-affinity penicillin-binding protein 2A (PBP2A). Because PBP2A can build the wall when other PBPs are blocked by beta-lactams, designing beta-lactams capable of blocking this additional target should help solve the issue. Older molecules including penicillin G, amoxicillin and ampicillin had relatively good PBP2A affinities, and successfully treated experimental endocarditis caused by MRSA, provided that the bacterial penicillinase could be inhibited. Newer anti-PBP2A beta-lactams with over 10-fold greater PBP2A affinities and low minimal inhibitory concentrations were developed, primarily in the cephem and carbapenem classes. They are also very resistant to penicillinase. Most have demonstrated anti-MRSA activity in animal models of infection, and two--the carbapenem CS-023 and the cephalosporin ceftopibrole medocaril--have proceeded to Phase II and Phase III clinical evaluation. Thus, clinically useful anti-MRSA beta-lactams are imminent.

Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells.

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.

An immunomodulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major.

The possible immunomodulatory role of polymorphonuclear leukocytes (PMN) in CD4+ T lymphocyte differentiation in mice was examined by studying the effect of transient depletion of PMN during the early phase after Leishmania major delivery. A single injection of the PMN-depleting NIMP-R14 mAb 6 h before infection with L. major prevented the early burst of IL-4 mRNA transcription otherwise occurring in the draining lymph node of susceptible BALB/c mice. Since this early burst of IL-4 mRNA transcripts had previously been shown to instruct Th2 differentiation in mice from this strain, we examined the effect of PMN depletion on Th subset differentiation at later time points after infection. The transient depletion of PMN in BALB/c mice was sufficient to inhibit Th2 cell development otherwise occurring after L. major infection. Decreased Th2 responses were paralleled with partial resolution of the footpad lesions induced by L. major. Furthermore, draining lymph node-derived CD4+ T cells from PMN-depleted mice remained responsive to IL-12 after L. major infection, unlike those of infected BALB/c mice receiving control Ab. PMN depletion had no effect when the NIMP-R14 mAb was injected 24 h postinfection. The protective effect of PMN depletion was shown to be IL-12 dependent, as concomitant neutralization of IL-12 reversed the protective effect of PMN depletion. These results suggest a role for an early wave of PMN in the development of the Th2 response characteristic of mice susceptible to infection with L. major.

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy.

BACKGROUND: The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with Crohn's disease (CD), ulcerative colitis (UC), type-I diabetes, and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen-activated protein kinase (MAPK) signaling and cytokine secretion in human THP-1 monocytes and intestinal epithelial cells (IEC). Here, we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide-oligomerization domain 2 (NOD2) ligand, muramyl-dipeptide (MDP). MATERIALS AND METHODS: Genomic DNA samples from 343 CD and 663 non-IBD control patients (male and female) from a combined German, Swiss, and Polish cohort were genotyped for the presence of the PTPN2 SNPs, rs2542151, and rs1893217. PTPN2-variant rs1893217 was introduced into T(84) IEC or THP-1 cells using a lentiviral vector. RESULTS: We identified a novel association between the genetic variant, rs1893217, located in intron 7 of the PTPN2 gene and CD. Human THP-1 monocytes carrying this variant revealed increased MAPK activation as well as elevated mRNA expression of T-bet transcription factor and secretion of interferon-γ in response to the bacterial wall component, MDP. In contrast, secretion of interleukin-8 and tumor necrosis factor were reduced. In both, T(84) IEC and THP-1 monocytes, autophagosome formation was impaired. CONCLUSIONS: We identified a novel CD-associated PTPN2 variant that modulates innate immune responses to bacterial antigens. These findings not only provide key insights into the effects of a functional mutation on a clinically relevant gene, but also reveal how such a mutation could contribute to the onset of disease.

Meédicaments: une cause sous-estimée d'hypertension artérielle [Drugs: an underestimated cause of arterial hypertension].

In Switzerland, as in other Occidental countries, the prevalence of arterial hypertension (AHT) in the adult population is around 30-40%. Among the causes of secondary AHT, drug induced hypertension is sometimes omitted. Many molecules can induce AHT or worsen it due to an interaction with anti hypertensive drugs. Among these, NSAIDs and anti depressants, widely prescribed, should be used with caution, particularly in patients at risk, namely: those with preexisting AHT, the elderly, or patients suffering from kidney disease, diabetes, and/or heart failure. Increases in blood pressure have also been described with anti-vascular endothelial growth factor (VEGF) drugs, used in the treatment of (metastatic) cancer. A thorough anamnesis of drugs, including over the counter ones, should be performed in every hypertensive patient, and can avoid cumbersome and unnecessary investigations and therapy.

Impact, challenges and perspectives of Euromelanoma, a pan-European campaign of skin cancer prevention.

The serotonin-2A receptor (5-HT(2A)R) has been implicated in the pathogenesis of schizophrenia and related inhibitory gating and behavioral inhibition deficits of schizophrenia patients. The hallucinogen psilocybin disrupts automatic forms of sensorimotor gating and response inhibition in humans, but it is unclear so far whether the 5-HT(2A)R or 5-HT(1A)R agonist properties of its bioactive metabolite psilocin account for these effects. Thus, we investigated whether psilocybin-induced deficits in automatic and controlled inhibition in healthy humans could be attenuated by the 5-HT(2A/2C)R antagonist ketanserin. A total of 16 healthy participants received placebo, ketanserin (40 mg p.o.), psilocybin (260 μg/kg p.o.), or psilocybin plus ketanserin in a double-blind, randomized, and counterbalanced order. Sensorimotor gating was measured by prepulse inhibition (PPI) of the acoustic startle response. The effects on psychopathological core dimensions and behavioral inhibition were assessed by the altered states of consciousness questionnaire (5D-ASC), and the Color-Word Stroop Test. Psilocybin decreased PPI at short lead intervals (30 ms), increased all 5D-ASC scores, and selectively increased errors in the interference condition of the Stroop Test. Stroop interference and Stroop effect of the response latencies were increased under psilocybin as well. Psilocybin-induced alterations were attenuated by ketanserin pretreatment, whereas ketanserin alone had no significant effects. These findings suggest that the disrupting effects of psilocybin on automatic and controlled inhibition processes are attributable to 5-HT(2A)R stimulation. Sensorimotor gating and attentional control deficits of schizophrenia patients might be due to changes within the 5-HT(2A)R system.