Cannabinoid receptor type I modulates alcohol-induced liver fibrosis

The cannabinoid system (CS) is implicated in the regulation of hepatic fibrosis, steatosis and inflammation, with cannabinoid receptors 1 and 2 (CB1 and CB2) being involved in regulation of pro- and antifibrogenic effects. Daily cannabis smoking is an independent risk factor for the progression of fibrosis in chronic hepatitis C and a mediator of experimental alcoholic steatosis. However, the role and function of CS in alcoholic liver fibrosis (ALF) is unknown so far. Thus, human liver samples from patients with alcoholic liver disease (ALD) were collected for analysis of CB1 expression. In vitro, hepatic stellate cells (HSC) underwent treatment with acetaldehyde, Δ9-tetrahydrocannabinol H(2)O(2), endo- and exocannabinoids (2-arachidonoylglycerol (2-AG) and [THC]), and CB1 antagonist SR141716 (rimonabant). In vivo, CB1 knockout (KO) mice received thioacetamide (TAA)/ethanol (EtOH) to induce fibrosis. As a result, in human ALD, CB1 expression was restricted to areas with advanced fibrosis only. In vitro, acetaldehyde, H(2)O(2), as well as 2-AG and THC, alone or in combination with acetaldehyde, induced CB1 mRNA expression, whereas CB1 blockage with SR141716 dose-dependently inhibited HSC proliferation and downregulated mRNA expression of fibrosis-mediated genes PCα1(I), TIMP-1 and MMP-13. This was paralleled by marked cytotoxicity of SR141716 at high doses (5-10 μmol/L). In vivo, CB1 knockout mice showed marked resistance to alcoholic liver fibrosis. In conclusion, CB1 expression is upregulated in human ALF, which is at least in part triggered by acetaldehyde (AA) and oxidative stress. Inhibition of CB1 by SR141716, or via genetic knock-out protects against alcoholic-induced fibrosis in vitro and in vivo.

Novel association to the proprotein convertase PCSK7 gene locus revealed by analysing soluble transferrin receptor (sTfR) levels

The level of body iron storage and the erythropoietic need for iron are indicated by the serum levels of ferritin and soluble transferrin receptor (sTfR), respectively. A meta-analysis of five genome-wide association studies on sTfR and ferritin revealed novel association to the PCSK7 and TMPRSS6 loci for sTfR and the HFE locus for both parameters. The PCSK7 association was the most significant (rs236918, P = 1.1 × 10E-27) suggesting that proprotein convertase 7, the gene product of PCSK7, may be involved in sTfR generation and/or iron homeostasis. Conditioning the sTfR analyses on transferrin saturation abolished the HFE signal and substantially diminished the TMPRSS6 signal while the PCSK7 association was unaffected, suggesting that the former may be mediated by transferrin saturation whereas the PCSK7-associated effect on sTfR generation appears to be more direct.

Bone morphogenetic protein receptor 2 in patients with idiopathic portal hypertension

In idiopathic portal hypertension (IPH) typical vascular lesions are present in the branches of the portal vein or in the perisinusoidal area of the liver. Similar histological alterations have been reported in the pulmonary vasculature of patients with idiopathic pulmonary artery hypertension (IPAH). As IPAH is associated with mutations of the bone morphogenetic protein receptor 2 (BMPR2) gene, the aim of this study was to investigate whether this association might also be found in patients with IPH. Twenty-three samples belonging to 21 unrelated caucasian patients with IPH followed in the hepatic haemodynamic laboratory of the Hospital Clinic in Barcelona were included in the study. All patients were studied for the entire open reading frame and splice site of the BMPR2 gene by direct sequencing and multiple ligation probe amplification (MLPA) in order to detect large deletions/duplications. None of the 23 patients had pulmonary artery hypertension. Four patients presented one single nucleotide polymorphism (SNP) in intron 5, four patients had a SNP in exon 12 and a SNP in exon 1 was found in two cases. Two patients had both intron 5 and exon 12 polymorphisms. All SNPs were previously described. Except for these three SNPs, neither mutations nor rearrangements have been identified in the BMPR2 gene in this population. We did not detect mutations or rearrangements in the coding region of the BMPR2 gene in our patients with IPH. These findings suggest that, in contrast to IPAH, mutations in BMPR2 are not involved in the pathogenesis of IPH.

Interpreting Breast International Group (BIG) 1-98: a randomized, double-blind, phase III trial comparing letrozole and tamoxifen as adjuvant endocrine therapy for postmenopausal women with hormone receptor-positive, early breast cancer

The Breast International Group (BIG) 1-98 study is a four-arm trial comparing 5 years of monotherapy with tamoxifen or with letrozole or with sequences of 2 years of one followed by 3 years of the other for postmenopausal women with endocrine-responsive early invasive breast cancer. From 1998 to 2003, BIG -98 enrolled 8,010 women. The enhanced design f the trial enabled two complementary analyses of efficacy and safety. Collection of tumor specimens further enabled treatment comparisons based on tumor biology. Reports of BIG 1-98 should be interpreted in relation to each individual patient as she weighs the costs and benefits of available treatments.

Mutagenesis and computer modeling studies of a GPCR conserved residue W5.43(194) in ligand recognition and signal transduction for CB2 receptor

W5.43(194), a conserved tryptophan residue among G-protein coupled receptors (GPCRs) and cannabinoid receptors (CB), was examined in the present report for its significance in CB2 receptor ligand binding and adenylyl cyclase (AC) activity. Computer modeling postulates that this site in CB2 may be involved in the affinity of WIN55212-2 and SR144528 through aromatic contacts. In the present study, we reported that a CB2 receptor mutant, W5.43(194)Y, which had a tyrosine (Y) substitution for tryptophan (W), retained the binding affinity for CB agonist CP55940, but reduced binding affinity for CB2 agonist WIN55212-2 and inverse agonist SR144528 by 8-fold and 5-fold, respectively; the CB2 W5.43(194)F and W5.43(194)A mutations significantly affect the binding activities of CP55940, WIN55212-2 and SR144528. Furthermore, we found that agonist-mediated inhibition of the forskolin-induced cAMP production was dramatically diminished in the CB2 mutant W5.43(194)Y, whereas W5.43(194)F and W5.43(194)A mutants resulted in complete elimination of downstream signaling, suggesting that W5.43(194) was essential for the full activation of CB2. These results indicate that both aromatic interaction and hydrogen bonding are involved in ligand binding for the residue W5.43(194), and the mutations of this tryptophan site may affect the conformation of the ligand binding pocket and therefore control the active conformation of the wild type CB2 receptor. W5.43(194)Y/F/A mutations also displayed noticeable enhancement of the constitutive activation probably attributed to the receptor conformational changes resulted from the mutations.

Novel positron emission tomography/computed tomography of diffuse parenchymal lung disease combining a labeled somatostatin receptor analogue and 2-deoxy-2[¹⁸F]fluoro-D-glucose

We prospectively investigated the potential of positron emission tomography (PET) using the somatostatin receptor (SSTR) analogue ⁶⁸Ga-DOTATATE and 2-deoxy-2[¹⁸F]fluoro-D-glucose (¹⁸F-FDG) in diffuse parenchymal lung disease (DPLD). Twenty-six patients (mean age 68.9 ± 11.0 years) with DPLD were recruited for ⁶⁸Ga-DOTATATE and ¹⁸F-FDG combined PET/high-resolution computed tomography (HRCT) studies. Ten patients had idiopathic pulmonary fibrosis (IPF), 12 patients had nonspecific interstitial pneumonia (NSIP), and 4 patients had other forms of DPLD. Using PET, the pulmonary tracer uptake (maximum standardized uptake value [SUV(max)]) was calculated. The distribution of PET tracer was compared to the distribution of lung parenchymal changes on HRCT. All patients demonstrated increased pulmonary PET signal with ⁶⁸Ga-DOTATATE and ¹⁸F-FDG. The distribution of parenchymal uptake was similar, with both tracers corresponding to the distribution of HRCT changes. The mean SUV(max) was 2.2 ± 0.7 for ⁶⁸Ga-DOTATATE and 2.8 ± 1.0 (t-test, p  =  .018) for ¹⁸F-FDG. The mean ⁶⁸Ga-DOTATATE SUV(max) in IPF patients was 2.5 ± 0.9, whereas it was 2.0 ± 0.7 (p  =  .235) in NSIP patients. The correlation between ⁶⁸Ga-DOTATATE SUV(max) and gas transfer (transfer factor of the lung for carbon monoxide [TLCO]) was r  =  -.34 (p  =  .127) and r  =  -.49 (p  =  .028) between ¹⁸F-FDG SUV(max) and TLCO. We provide noninvasive in vivo evidence in humans showing that SSTRs may be detected in the lungs of patients with DPLD in a similar distribution to sites of increased uptake of ¹⁸F-FDG on PET.

Lymphotoxin β receptor signaling promotes development of autoimmune pancreatitis

Little is known about the pathogenic mechanisms of autoimmune pancreatitis (AIP), an increasingly recognized, immune-mediated form of chronic pancreatitis. Current treatment options are limited and disease relapse is frequent. We investigated factors that contribute to the development of AIP and new therapeutic strategies.

Human epidermal growth factor receptor-2 gene amplification in gastric cancer using tissue microarray technology

To assess human epidermal growth factor receptor-2 (HER2)-status in gastric cancer and matched lymph node metastases by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH).

Epidermal growth factor receptor, phosphatidylinositol-3-kinase catalytic subunit/PTEN, and KRAS/NRAS/BRAF in primary resected esophageal adenocarcinomas: loss of PTEN is associated with worse clinical outcome

Alterations of the epidermal growth factor receptor (EGFR) can be observed in a significant subset of esophageal adenocarcinomas (EACs), and targeted therapy against EGFR may become an interesting approach for the treatment of these tumors. Mutations of KRAS, NRAS, BRAF, and phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) and deregulation of PTEN expression influence the responsiveness against anti-EGFR therapy in colorectal carcinomas. We investigated the prevalence of these events in a collection of 117 primary resected EACs, correlated the findings with EGFR expression and amplification, and determined their clinicopathologic impact. KRAS mutations were detected in 4 (3%) of 117 tumors (3× G12D and 1 G12V mutation). One tumor had a PIK3CA E545K mutation. Neither NRAS nor BRAF mutations were detected. Sixteen (14%) of 117 cases were negative for PTEN expression, determined by immunohistochemistry. Loss of PTEN was observed predominantly in advanced tumor stages (P = .004). There was no association between PTEN and EGFR status. Loss of PTEN was associated with shorter overall and disease-free survival (P < .001 each) and also an independent prognostic factor in multivariate analysis (P = .015). EGFR status had no prognostic impact in this case collection. In summary, loss of PTEN can be detected in a significant subset of EAC and is associated with an aggressive phenotype. Therefore, PTEN may be useful as a prognostic biomarker. In contrast, mutations of RAS/RAF/PIK3CA appear only very rarely, if at all, in EAC. A possible predictive role of PTEN in anti-EGFR treatment warrants further investigations, whereas determination of RAS/RAF/PIK3CA mutations may only have a minor impact in this context.

Influence of GABA(A) receptor α subunit isoforms on the benzodiazepine binding site

Classical benzodiazepines, such as diazepam, interact with α(x)β(2)γ(2) GABA(A) receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands. We studied here the interaction of the covalently reacting diazepam analogue 3-NCS with α(1)S204Cβ(2)γ(2), α(1)S205Cβ(2)γ(2) and α(1)T206Cβ(2)γ(2) and with receptors containing the homologous mutations in α(2)β(2)γ(2), α(3)β(2)γ(2), α(5)β(1/2)γ(2) and α(6)β(2)γ(2). The interaction was studied using radioactive ligand binding and at the functional level using electrophysiological techniques. Both strategies gave overlapping results. Our data allow conclusions about the relative apposition of α(1)S204Cβ(2)γ(2), α(1)S205Cβ(2)γ(2) and α(1)T206Cβ(2)γ(2) and homologous positions in α(2), α(3), α(5) and α(6) with C-atom adjacent to the keto-group in diazepam. Together with similar data on the C-atom carrying Cl in diazepam, they indicate that the architecture of the binding site for benzodiazepines differs in each GABA(A) receptor isoform α(1)β(2)γ(2), α(2)β(2)γ(2), α(3)β(2)γ(2), α(5)β(1/2)γ(2) and α(6)β(2)γ(2).

The cannabinoid CB1 receptor antagonists rimonabant (SR141716) and AM251 directly potentiate GABA(A) receptors

Rimonabant (SR141716) and the structurally related AM251 are widely used in pharmacological experiments as selective cannabinoid receptor CB(1) antagonists / inverse agonists. Concentrations of 0.5-10 µM are usually applied in in vitro experiments. We intended to show that these drugs did not act at GABA(A) receptors but found a significant positive allosteric modulation instead.

Marked global reduction in mGluR5 receptor binding in smokers and ex-smokers determined by [11C]ABP688 positron emission tomography

Nicotine addiction is a major public health problem, resulting in primary glutamatergic dysfunction. We measured the glutamate receptor binding in the human brain and provided direct evidence for the abnormal glutamate system in smokers. Because antagonism of the metabotropic glutamate receptor 5 (mGluR5) reduced nicotine self-administration in rats and mice, mGluR5 is suggested to be involved in nicotine addiction. mGluR5 receptor binding specifically to an allosteric site was observed by using positron emission tomography with [(11)C]ABP688. We found a marked global reduction (20.6%; P < 0.0001) in the mGluR5 distribution volume ratio (DVR) in the gray matter of 14 smokers. The most prominent reductions were found in the bilateral medial orbitofrontal cortex. Compared with 14 nonsmokers, 14 ex-smokers had global reductions in the average gray matter mGluR5 DVR (11.5%; P < 0.005), and there was a significant difference in average gray matter mGluR5 DVR between smokers and ex-smokers (9.2%; P < 0.01). Clinical variables reflecting current nicotine consumption, dependence and abstinence were not correlated with mGluR5 DVR. This decrease in mGluR5 receptor binding may be an adaptation to chronic increases in glutamate induced by chronic nicotine administration, and the decreased down-regulation seen in the ex-smokers could be due to incomplete recovery of the receptors, especially because the ex-smokers were abstinent for only 25 wk on average. These results encourage the development and testing of drugs against addiction that directly target the glutamatergic system.

Opioids trigger alpha 5 beta 1 integrin-mediated monocyte adhesion

Inflammatory reactions involve a network of chemical and molecular signals that initiate and maintain host response. In inflamed tissue, immune system cells generate opioid peptides that contribute to potent analgesia by acting on specific peripheral sensory neurons. In this study, we show that opioids also modulate immune cell function in vitro and in vivo. By binding to its specific receptor, the opioid receptor-specific ligand DPDPE triggers monocyte adhesion. Integrins have a key role in this process, as adhesion is abrogated in cells treated with specific neutralizing anti-alpha5beta1 integrin mAb. We found that DPDPE-triggered monocyte adhesion requires PI3Kgamma activation and involves Src kinases, the guanine nucleotide exchange factor Vav-1, and the small GTPase Rac1. DPDPE also induces adhesion of pertussis toxin-treated cells, indicating involvement of G proteins other than Gi. These data show that opioids have important implications in regulating leukocyte trafficking, adding a new function to their known effects as immune response modulators.