Vascular adhesion protein-1 is elevated in primary sclerosing cholangitis, is predictive of clinical outcome and facilitates recruitment of gut-tropic lymphocytes to liver in a substrate-dependent manner

OBJECTIVE: Primary sclerosing cholangitis (PSC) is the classical hepatobiliary manifestation of IBD. This clinical association is linked pathologically to the recruitment of mucosal T cells to the liver, via vascular adhesion protein (VAP)-1-dependent enzyme activity. Our aim was to examine the expression, function and enzymatic activation of the ectoenzyme VAP-1 in patients with PSC.DESIGN: We examined VAP-1 expression in patients with PSC, correlated levels with clinical characteristics and determined the functional consequences of enzyme activation by specific enzyme substrates on hepatic endothelium.RESULTS: The intrahepatic enzyme activity of VAP-1 was elevated in PSC versus immune-mediated disease controls and non-diseased liver (p<0.001). The adhesion of gut-tropic α4β7+lymphocytes to hepatic endothelial cells in vitro under flow was attenuated by 50% following administration of the VAP-1 inhibitor semicarbazide (p<0.01). Of a number of natural VAP-1 substrates tested, cysteamine-which can be secreted by inflamed colonic epithelium and gut bacteria-was the most efficient (yielded the highest enzymatic rate) and efficacious in its ability to induce expression of functional mucosal addressin cell adhesion molecule-1 on hepatic endothelium. In a prospectively evaluated patient cohort with PSC, elevated serum soluble (s)VAP-1 levels predicted poorer transplant-free survival for patients, independently (HR: 3.85, p=0.003) and additively (HR: 2.02, p=0.012) of the presence of liver cirrhosis.CONCLUSIONS: VAP-1 expression is increased in PSC, facilitates adhesion of gut-tropic lymphocytes to liver endothelium in a substrate-dependent manner, and elevated levels of its circulating form predict clinical outcome in patients.</p

Vps34/PI3KC3 deletion in kidney proximal tubules impairs apical trafficking and blocks autophagic flux, causing a Fanconi-like syndrome and renal insufficiency

Kidney proximal tubular cells (PTCs) are highly specialized for ultrafiltrate reabsorption and serve as paradigm of apical epithelial differentiation. Vps34/PI3-kinase type III (PI3KC3) regulates endosomal dynamics, macroautophagy and lysosomal function. However, its in vivo role in PTCs has not been evaluated. Conditional deletion of Vps34/PI3KC3 in PTCs by Pax8-Cre resulted in early (P7) PTC dysfunction, manifested by Fanconi-like syndrome, followed by kidney failure (P14) and death. By confocal microscopy, Vps34∆/∆ PTCs showed preserved apico-basal specification (brush border, NHERF-1 versus Na+/K+-ATPase, ankyrin-G) but basal redistribution of late-endosomes/lysosomes (LAMP-1) and mis-localization to lysosomes of apical recycling endocytic receptors (megalin, cubilin) and apical non-recycling solute carriers (NaPi-IIa, SGLT-2). Defective endocytosis was confirmed by Texas-red-ovalbumin tracing and reduced albumin content. Disruption of Rab-11 and perinuclear galectin-3 compartments suggested mechanistic clues for defective receptor recycling and apical biosynthetic trafficking. p62-dependent autophagy was triggered yet abortive (p62 co-localization with LC3 but not LAMP-1) and PTCs became vacuolated. Impaired lysosomal positioning and blocked autophagy are known causes of cell stress. Thus, early trafficking defects show that Vps34 is a key in vivo component of molecular machineries governing apical vesicular trafficking, thus absorptive function in PTCs. Functional defects underline the essential role of Vps34 for PTC homeostasis and kidney survival

Assessment of measles immunity among infants in Maputo City, Mozambique

<p>Abstract</p> <p>Background</p> <p>The optimum age for measles vaccination varies from country to country and thus a standardized vaccination schedule is controversial. While the increase in measles vaccination coverage has produced significant changes in the epidemiology of infection, vaccination schedules have not been adjusted. Instead, measures to cut wild-type virus transmission through mass vaccination campaigns have been instituted. This study estimates the presence of measles antibodies among six- and nine-month-old children and assesses the current vaccination seroconversion by using a non invasive method in Maputo City, Mozambique.</p> <p>Methods</p> <p>Six- and nine-month old children and their mothers were screened in a cross-sectional study for measles-specific antibodies in oral fluid. All vaccinated children were invited for a follow-up visit 15 days after immunization to assess seroconversion. </p> <p>Results</p> <p>82.4% of the children lost maternal antibodies by six months. Most children were antibody-positive post-vaccination at nine months, although 30.5 % of nine month old children had antibodies in oral fluid before vaccination. We suggest that these pre-vaccination antibodies are due to contact with wild-type of measles virus. The observed seroconversion rate after vaccination was 84.2%. </p> <p>Conclusion</p> <p>These data indicate a need to re-evaluate the effectiveness of the measles immunization policy in the current epidemiological scenario.</p

Proline-rich antimicrobial peptide, PR-39 gene transduction altered invasive activity and actin structure in human hepatocellular carcinoma cells

PR-39 is an endogenous proline-rich antimicrobial peptide which induces the synthesis of syndecan-1, a transmembrane heparan sulphate proteoglycan involved in cell-to-matrix interactions and wound healing. Previously, we revealed that the expression of syndecan-1 was reduced in human hepatocellular carcinomas with high metastatic potential and speculated that syndecan-1 played an important role in inhibition of invasion and metastasis. It is assumed that a modification of this process with PR-39 and syndecan-1 may result in a new strategy by which it can inhibit the invasion and metastasis. Therefore, we transduced a gene of PR-39 into human hepatocellular carcinoma cell line HLF, which shows a low expression of syndecan-1 and a high in vitro invasive activity, and examined whether this procedure could reduce the invasive activity of tumour cells. In two transfectants with PR-39 gene, the syndecan-1 expression was induced and the invasive activity in type I collagen-coated chamber was inhibited. Moreover, these transfectants showed the suppression of motile activity assayed by phagokinetic tracks in addition to the disorganization of actin filaments observed by a confocal imaging system. In contrast, five transfectants with syndecan-1 gene in the HLF cells revealed suppression of invasive activity but did not alter the motile activity and actin structures of the cell. These results suggest that PR-39 has functions involved in the suppression of motile activity and alteration of actin structure on human hepatocellular carcinoma cells in addition to the suppression of invasive activity which might result from the induction of syndecan-1 expression. © 1999 Cancer Research Campaig

Microarray profiling for differential gene expression in PMSG-hCG stimulated preovulatory ovarian follicles of Chinese Taihu and Large White sows

<p>Abstract</p> <p>Background</p> <p>The Chinese Taihu is one of the most prolific pig breeds in the world, which farrows at least five more piglets per litter than Western pig breeds partly due to a greater ovulation rate. Variation of ovulation rate maybe associated with the differences in the transcriptome of Chinese Taihu and Large White ovaries. In order to understand the molecular basis of the greater ovulation rate of Chinese Taihu sows, expression profiling experiments were conducted to identify differentially expressed genes in ovarian follicles at the preovulatory stage of a PMSG-hCG stimulated estrous cycle from 3 Chinese Taihu and 3 Large White cycling sows by using the Affymetrix Porcine Genechip™.</p> <p>Results</p> <p>One hundred and thirty-three differentially expressed genes were identified between Chinese Taihu and Large White sows by using Affymetrix porcine GeneChip (<it>p </it>≤ 0.05, Fold change ≥ 2 or ≤ 0.5). Gene Ontology (GO) analysis revealed that these genes belonged to the class of genes that participated in regulation of cellular process, regulation of biological process, biological regulation, developmental process, cell communication and signal transduction and so on. Significant differential expression of 6 genes including <it>WNT10B </it>and <it>DKK2 </it>in the WNT signaling pathway was detected. Real-time RT-PCR confirmed the expression pattern in seven of eight selected genes. A search of chromosomal location revealed that 92 differentially expressed transcripts located to the intervals of quantitative trait loci (QTLs) for reproduction traits. Furthermore, SNPs of two differentially expressed genes- <it>BAX </it>and <it>BMPR1B </it>were showed to be associated with litter size traits in Large White pigs and Chinese DIV line pigs (<it>p </it>≤ 0.1 or <it>p </it>≤ 0.05).</p> <p>Conclusions</p> <p>Our study detected many genes that showed differential expression between ovary follicles of two divergent breeds of pigs. Genes involved with regulation of cellular process, regulation of biological process, in addition to several genes not previously associated with ovarian physiology or with unknown function, were differentially expressed between two breeds. The suggestive or significant associations of <it>BAX </it>and <it>BMPR1B </it>gene with litter size indicated these genetic markers had the potentials to be used in pig industry after further validation of their genetic effects. Taken together, this study reveals many potential avenues of investigation for seeking new insights into ovarian physiology and the genetic control of reproduction.</p

Phosphorylation of Kif26b Promotes Its Polyubiquitination and Subsequent Proteasomal Degradation during Kidney Development

Kif26b, a member of the kinesin superfamily proteins (KIFs), is essential for kidney development. Kif26b expression is restricted to the metanephric mesenchyme, and its transcription is regulated by a zinc finger transcriptional regulator Sall1. However, the mechanism(s) by which Kif26b protein is regulated remain unknown. Here, we demonstrate phosphorylation and subsequent polyubiquitination of Kif26b in the developing kidney. We find that Kif26b interacts with an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) in developing kidney. Phosphorylation of Kif26b at Thr-1859 and Ser-1962 by the cyclin-dependent kinases (CDKs) enhances the interaction of Kif26b with Nedd4. Nedd4 polyubiquitinates Kif26b and thereby promotes degradation of Kif26b via the ubiquitin-proteasome pathway. Furthermore, Kif26b lacks ATPase activity but does associate with microtubules. Nocodazole treatment not only disrupts the localization of Kif26b to microtubules but also promotes phosphorylation and polyubiquitination of Kif26b. These results suggest that the function of Kif26b is microtubule-based and that Kif26b degradation in the metanephric mesenchyme via the ubiquitin-proteasome pathway may be important for proper kidney development

Gene Expression Profiling of Preovulatory Follicle in the Buffalo Cow: Effects of Increased IGF-I Concentration on Periovulatory Events

The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development