A phosphatidylinositol (4,5)-bisphosphate binding site within μ2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within μ2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant μ2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant μ2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to μ2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation

Continuous decline of hepatitis E virus seroprevalence in southern Germany despite increasing notifications, 2003–2015

Hepatitis E virus (HEV) is viewed as an emerging pathogen. Many European countries, including Germany, have observed a steep increase of notified autochthonous hepatitis E cases in recent years. Our study investigated time trends in HEV seroprevalence in southern Germany between 2003 and 2015. A total of 3000 study sera were evenly distributed over sampling years 2003, 2006, 2009, 2012, and 2015, two age groups (20-29 and 30-39 years) and genders and were tested for anti-HEV IgG. Positive samples were quantified. The seroprevalence declined from 32.8% in 2003 over 22.5% in 2006 (p < 0.001) and 22.3% in 2009 to 17.7% and 17.8% in 2012 and 2015. A higher prevalence was found for males (p = 0.018) and the older age group (p < 0.001). Anti-HEV IgG concentrations ranged from 0.22 to 1783.19 WU mL(-1). A higher median concentration (2.41 vs. 1.89 WU mL(-1), p < 0.001) was found in the younger age group. The anti-HEV IgG seroprevalence decreased since 2003 and remains constant at similar to 18% since 2012. A rather low anti-HEV prevalence in young adults is indicative of a susceptible population and denotes a higher risk of HEV infections in this age group in the future. Therefore, reduction of HEV infection sources, close monitoring, and vigilance for proper control measures are warranted

Expression of Human Beta-Defensins in Children with Chronic Inflammatory Bowel Disease

Background: Human beta-defensins (hBDs) are antimicrobial peptides known to play a major role in intestinal innate host defence. Altered mucosal expression of hBDs has been suggested to be implicated in chronic inflammatory bowel disease pathogenesis. However, little is known about expression of these peptides in children. Methods: Intestinal biopsies were obtained from the duodenum (n = 88), terminal ileum (n = 90) and ascending colon (n = 105) of children with Crohn’s disease (n = 26), ulcerative colitis (n = 11) and healthy controls (n = 16). Quantitative realtime (RT) PCR was performed and absolute mRNA copy numbers analyzed for hBD1-3 as well as inflammatory cytokines IL-8 and TNF-alpha. Results: Significant induction of hBD2 and hBD3 was observed in the inflamed terminal ileum and ascending colon of IBD children. In the ascending colon induction of hBD2 was found to be significantly lower in children with Crohn’s disease compared to ulcerative colitis. A strong correlation was found between inducible defensins hBD2 and 3 and the inflammatory cytokines IL-8 and TNF-alpha, both in the terminal ileum and ascending colon. Conclusion: Our study demonstrates distinct changes in hBD expression throughout the intestinal tract of children with IBD

Expression of inducible beta defensins and inflammatory cytokines in colonic biopsies of children with IBD and healthy controls.

<p>Expression of hBD2 (a), hBD3 (b), IL-8 (c) and TNF-alpha (d) was analysed in biopsies obtained from the ascending colon of children with Crohn's disease (CD), ulcerative colitis (UC) and healthy controls (con). Biopsies from CD and UC patients were subdivided into histologically non-inflamed (NI) and inflamed (I). Data is expressed as the mean ± standard error of mean (SEM) of absolute mRNA copy numbers. The number of biopsies analysed for each patient group is stated (n). Values for <i>P</i><0.05 were considered to be statistically significant.</p

Expression of hBDs and inflammatory cytokines in the intestinal mucosa of healthy children.

<p>Biopsies were obtained from duodenum (D), terminal ileum (TI) and ascending colon (AC). Absolute mRNA copy number for hBD1-3 (a-c), IL-8 (d) and TNF-alpha (e) in each biopsy were quantified by real-time PCR using the standard curve method. Expression levels were normalized against the geometrical mean of two selected housekeeping genes (GPDH and β-actin). Data is expressed as the mean ± standard error of mean (SEM). The number of biopsies analysed for each patient group is stated (n). Values for <i>P</i><0.05 were considered to be statistically significant.</p

Expression of hBD1 in mucosal biopsies from IBD children and healthy controls.

<p>Expression of hBD1 in the duodenum (a), terminal ileum (b) and ascending colon (c) was analysed in children with Crohn's disease (CD), ulcerative colitis (UC) and healthy controls (con). For the TI (d) and AC (e), biopsy samples from CD patients were subdivided into histologically non-inflamed (NI) and inflamed (I). Data is expressed as the mean ± standard error of mean (SEM) of absolute mRNA copy numbers. The number of biopsies analysed for each patient group is stated (n). Values for <i>P</i><0.05 were considered to be statistically significant.</p

Expression of inducible beta defensins and inflammatory cytokines in small bowel biopsies of IBD children and healthy controls.

<p>Expression of hBD2 (a and e), hBD3 (b and f), IL-8 (c and g) and TNF-alpha (d and h) was analysed in the duodenum (a–d) and TI (e–h) of children with Crohn's disease (CD), ulcerative colitis (UC) and healthy controls (con). For the TI, biopsy samples from CD patients were subdivided into histologically non-inflamed (NI) and inflamed (I). Data is expressed as the mean ± standard error of mean (SEM) of absolute mRNA copy numbers. The number of biopsies analysed for each patient group is stated (n). Values for <i>P</i><0.05 were considered to be statistically significant.</p

Correlation of intestinal beta-defensin and inflammatory cytokine mRNA expression levels.

<p>Correlation of gene expression between beta-defensins (hBD2 and hBD3) and inflammatory cytokines (IL-8 and TNF-alpha) in the duodenum, terminal ileum and ascending colon was tested by calculating Spearman's rank correlation coefficient (<i>r</i>). The number of biopsies included into calculations is stated (n). Values for <i>P</i><0.05 were considered to be statistically significant.</p