Cloning, sequencing and partial functional characterization of the 5' region of the human p75 tumor necrosis factor receptor-encoding gene (TNF-R)

A 1887-bp region at the 5' flank of the human p75 tumor necrosis factor receptor (p75 TNF-R)-encoding gene was found to be active in driving expression of the luc (luciferase-encoding) reporter gene, suggesting that it contains the promoter for the receptor. Rather unexpectedly, a 1827-bp region at the 3' end of the first intron of the p75 TNF-R gene also displayed promoter activity. This activity may be artefactual, reflecting only the presence of an enhancer in this region; yet it also raises the possibility that p75 TNF-R is controlled by more than one promoter and that it encodes various forms of the receptor, or even other proteins. We present here the nucleotide sequences of the 5' flanking and intron regions. Possible implications for the transcriptional regulation of the p75 TNF-R gene are discussed.

A common polymorphism in the NCAN gene is associated with hepatocellular carcinoma in alcoholic liver disease

BACKGROUND & AIMS: The genetic background of alcoholic liver diseases and their complications are increasingly recognized. A common polymorphism in the neurocan (NCAN) gene, which is known to be expressed in neuronal tissue, has been identified as a risk factor for non-alcoholic fatty liver disease (NAFLD). We investigated if this polymorphism may also be related to alcoholic liver disease (ALD) and hepatocellular carcinoma (HCC). METHODS: We analysed the distribution of the NCAN rs2228603 genotypes in 356 patients with alcoholic liver cirrhosis, 126 patients with alcoholic HCC, 382 persons with alcohol abuse without liver damage, 362 healthy controls and in 171 patients with hepatitis C virus (HCV) associated HCC. Furthermore, a validation cohort of 229 patients with alcoholic cirrhosis (83 with HCC) was analysed. The genotypes were determined by LightSNiP assays. The expression of NCAN was studied by RT-PCR and immunofluorescence microscopy. RESULTS: The frequency of the NCAN rs2228603 T allele was significantly increased in patients with HCC due to ALD (15.1%) compared to alcoholic cirrhosis without HCC (9.3%), alcoholic controls (7.2%), healthy controls (7.9%), and HCV associated HCC (9.1%). This finding was confirmed in the validation cohort (15.7% vs. 6.8%, OR=2.53; 95%CI: 1.36-4.68; p=0.0025) and by multivariate analysis (OR=1.840; 95%CI: 1.22-2.78; p=0.004 for carriage of the rs2228603 T allele). In addition, we identified and localised NCAN expression in human liver. CONCLUSIONS: NCAN is not only expressed in neuronal tissue, but also in the liver. Its rs2228603 polymorphism is a risk factor for HCC in ALD, but not in HCV infection.

The role of metabotropic glutamate receptor 5 in the pathogenesis of mood disorders and addiction: combining preclinical evidence with human Positron Emission Tomography (PET) studies.

In the present review, we deliver an overview of the involvement of metabotropic glutamate receptor 5 (mGluR5) activity and density in pathological anxiety, mood disorders and addiction. Specifically, we will describe mGluR5 studies in humans that employed Positron Emission Tomography (PET) and combined the findings with preclinical animal research. This combined view of different methodological approaches-from basic neurobiological approaches to human studies-might give a more comprehensive and clinically relevant view of mGluR5 function in mental health than the view on preclinical data alone. We will also review the current research data on mGluR5 along the Research Domain Criteria (RDoC). Firstly, we found evidence of abnormal glutamate activity related to the positive and negative valence systems, which would suggest that antagonistic mGluR5 intervention has prominent anti-addictive, anti-depressive and anxiolytic effects. Secondly, there is evidence that mGluR5 plays an important role in systems for social functioning and the response to social stress. Finally, mGluR5's important role in sleep homeostasis suggests that this glutamate receptor may play an important role in RDoC's arousal and modulatory systems domain. Glutamate was previously mostly investigated in non-human studies, however initial human clinical PET research now also supports the hypothesis that, by mediating brain excitability, neuroplasticity and social cognition, abnormal metabotropic glutamate activity might predispose individuals to a broad range of psychiatric problems.

The Vacuolar H+-ATPase B1 Subunit Polymorphism p.E161K Associates with Impaired Urinary Acidification in Recurrent Stone Formers

Mutations in the vacuolar–type H+-ATPase B1 subunit gene ATP6V1B1 cause autosomal–recessive distal renal tubular acidosis (dRTA). We previously identified a single-nucleotide polymorphism (SNP) in the human B1 subunit (c.481G.A; p.E161K) that causes greatly diminished pump function in vitro. To investigate the effect of this SNP on urinary acidification, we conducted a genotype-phenotype analysis of recurrent stone formers in theDallas and Bern kidney stone registries. Of 555 patients examined, 32 (5.8%) were heterozygous for the p.E161K SNP, and the remaining 523 (94.2%) carried two wild–type alleles. After adjustment for sex, age, body mass index, and dietary acid and alkali intake, p.E161K SNP carriers had a nonsignificant tendency to higher urinary pH on a random diet (6.31 versus 6.09; P=0.09). Under an instructed low–Ca and low–Na diet, urinary pH was higher in p.E161K SNP carriers (6.56 versus 6.01; P,0.01). Kidney stones of p.E161K carriers were more likely to contain calcium phosphate than stones of wild-type patients. In acute NH4Cl loading, p.E161K carriers displayed a higher trough urinary pH (5.34 versus 4.89; P=0.01) than wild-type patients. Overall, 14.6% of wild-type patients and 52.4% of p.E161K carriers were unable to acidify their urine below pH 5.3 and thus, can be considered to have incomplete dRTA. In summary, our data indicate that recurrent stone formers with the vacuolar H+-ATPase B1 subunit p.E161K SNP exhibit a urinary acidification deficit with an increased prevalence of calcium phosphate– containing kidney stones. The burden of E161K heterozygosity may be a forme fruste of dRTA.

Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro

BACKGROUND Platelet-rich concentrates are used as a source of growth factors to improve the healing process. The diverse preparation protocols and the gaps in knowledge of their biological properties complicate the interpretation of clinical results. QUESTIONS/PURPOSES In this study we aimed to (1) analyze the concentration and kinetics of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF), leukocyte- and platelet-rich plasma (L-PRP), and natural blood clot during in vitro culture; (2) investigate the migration of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as a functional response to the factors released; and (3) uncover correlations between individual growth factors with the initial platelet/leukocyte counts or the induced cell migration. METHODS L-PRF, L-PRP, and natural blood clot prepared from 11 donors were cultured in vitro for 28 days and media supernatants collected after 8 hours and 1, 3, 7, 14, and 28 days. Released transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), insulin growth factor (IGF-1), platelet-derived growth factor AB (PDGF-AB), and interleukin-1β (IL-1β) were measured in the supernatants with enzyme-linked immunosorbent assay. Migration of MSC and HUVEC induced by the supernatants was evaluated in Boyden chambers. RESULTS More TGF-ß1 was released (mean ± SD in pg/mL of blood) from L-PRF (37,796 ± 5492) compared with L-PRP (23,738 ± 6848; p < 0.001) and blood clot (3739 ± 4690; p < 0.001), whereas more VEGF and IL-1ß were released from blood clot (1933 ± 704 and 2053 ± 908, respectively) compared with both L-PRP (642 ± 208; p < 0.001 and 273 ± 386; p < 0.001, respectively) and L-PRF (852 ± 376; p < 0.001 and 65 ± 56, p < 0.001, respectively). No differences were observed in IGF-1 and PDGF-AB released from any of the concentrates. TGF-β1 release peaked at Day 7 in L-PRF and at 8 hours and Day 7 in L-PRP and 8 hours and Day 14 in blood clot. In all concentrates, main release of VEGF occurred between 3 and 7 days and of IL-1β between Days 1 and 7. IGF-1 and PDGF-AB were released until Day 1 in L-PRP and blood clot, in contrast to sustained release over the first 3 days in L-PRF. The strongest migration of MSC occurred in response to L-PRF, and more HUVEC migration was seen in L-PRF and blood clot compared with L-PRP. TGF-β1 correlated with initial platelet counts in L-PRF (Pearson r = 0.66, p = 0.0273) and initial leukocyte counts in L-PRP (Pearson r = 0.83, p = 0.0016). A positive correlation of IL-1β on migration of MSC and HUVEC was revealed (Pearson r = 0.16, p = 0.0208; Pearson r = 0.31, p < 0.001). CONCLUSIONS In comparison to L-PRP, L-PRF had higher amounts of released TGF-β1, a long-term release of growth factors, and stronger induction of cell migration. Future preclinical studies should confirm these data in a defined injury model. CLINICAL RELEVANCE By characterizing the biologic properties of different platelet concentrates in vitro, we may gain a better understanding of their clinical effects and develop guidelines for specific future applications.

Transforming growth factor β2 (TGF-β2)-induced connective tissue growth factor (CTGF) expression requires sphingosine 1-phosphate receptor 5 (S1P5) in human mesangial cells

Transforming growth factor β2 (TGF-β2) is well known to stimulate the expression of pro-fibrotic connective tissue growth factor (CTGF) in several cell types including human mesangial cells. The present study demonstrates that TGF-β2 enhances sphingosine 1-phosphate receptor 5 (S1P5) mRNA and protein expression in a time and concentration dependent manner. Pharmacological and siRNA approaches reveal that this upregulation is mediated via activation of classical TGF-β downstream effectors, Smad and mitogen-activated protein kinases. Most notably, inhibition of Gi with pertussis toxin and downregulation of S1P5 by siRNA block TGF-β2-stimulated upregulation of CTGF, demonstrating that Gi coupled S1P5 is necessary for TGF-β2-triggered expression of CTGF in human mesangial cells. Overall, these findings indicate that TGF-β2 dependent upregulation of S1P5 is required for the induction of pro-fibrotic CTGF by TGF-β. Targeting S1P5 might be an attractive novel approach to treat renal fibrotic diseases.