Extracellular pH modulates Helicobacter pylori-induced vacuolation and VacA toxin internalization in human gastric epithelial cells

In this study we investigated whether an acidic extracellular pH may inhibit H. pylori-induced internalization of bacterial virulence factors by gastric epithelium, thus preventing ingestion of potentially dangerous luminal contents and resulting cellular damage. The interaction of H. pylori VacA toxin and ammonia (produced by H. pylori urease) with partly polarized gastric MKN 28 cells in culture was investigated at neutral and moderately acidic pH (6.2, compatible with cell viability) by means of neutral red dye uptake and ultrastructural immunocytochemistry. We found that acidic extracellular pH virtually abolished both VacA-dependent and ammonia-dependent cell vacuolation, as shown by the neutral red test, and caused a 50% decrease in VacA internalization into endosomal vesicles and vacuoles, as assessed by quantitation of immunogold particles. In addition, acidic pH blocked endosomal internalization of H. pylori outer membrane vesicles, a convenient indicator of endocytosis. Our data raise the possibility that suppression of gastric acid may increase H. pylori-induced gastric damage by enhancing epithelial internalization of H. pylori virulence factors through activation of endocytosis. Increased transmembrane diffusion of ammonia could also contribute to this process.     

Genetic Predictors of Response to Serotonergic and Noradrenergic Antidepressants in Major Depressive Disorder: A Genome-Wide Analysis of Individual-Level Data and a Meta-Analysis

Background

It has been suggested that outcomes of antidepressant treatment for major depressive disorder could be significantly improved if treatment choice is informed by genetic data. This study aims to test the hypothesis that common genetic variants can predict response to antidepressants in a clinically meaningful way.

Methods and Findings

The NEWMEDS consortium, an academia–industry partnership, assembled a database of over 2,000 European-ancestry individuals with major depressive disorder, prospectively measured treatment outcomes with serotonin reuptake inhibiting or noradrenaline reuptake inhibiting antidepressants and available genetic samples from five studies (three randomized controlled trials, one part-randomized controlled trial, and one treatment cohort study). After quality control, a dataset of 1,790 individuals with high-quality genome-wide genotyping provided adequate power to test the hypotheses that antidepressant response or a clinically significant differential response to the two classes of antidepressants could be predicted from a single common genetic polymorphism. None of the more than half million genetic markers significantly predicted response to antidepressants overall, serotonin reuptake inhibitors, or noradrenaline reuptake inhibitors, or differential response to the two types of antidepressants (genome-wide significance p<5×10⁻⁸). No biological pathways were significantly overrepresented in the results. No significant associations (genome-wide significance p<5×10⁻⁸) were detected in a meta-analysis of NEWMEDS and another large sample (STAR*D), with 2,897 individuals in total. Polygenic scoring found no convergence among multiple associations in NEWMEDS and STAR*D.

Conclusions

No single common genetic variant was associated with antidepressant response at a clinically relevant level in a European-ancestry cohort. Effects specific to particular antidepressant drugs could not be investigated in the current study. Please see later in the article for the Editors'' Summary     

Immunological advantages of everolimus versus cyclosporin A in liver-transplanted recipients, as revealed by polychromatic flow cytometry

Several immunosuppressive drugs with different mechanisms of action are available to inhibit organ rejection after transplant. We analyzed different phenotypic and functional immunological parameters in liver-transplanted patients who received cyclosporin A (CsA) or Everolimus (Evr). In peripheral blood mononuclear cells (PBMC) from 29 subjects receiving a liver transplant and treated with two different immunosuppressive regimens, we analyzed T cell activation and differentiation, regulatory T cells (Tregs) and Tregs expressing homing receptors such as the chemokine receptor CXCR3. T cell polyfunctionality was studied by stimulating cells with the superantigen staphylococcal enterotoxin B (SEB), and measuring the simultaneous production of interleukin (IL)-2 and interferon (IFN)-γ, along with the expression of a marker of cytotoxicity such as CD107a. The analyses were performed by polychromatic flow cytometry before transplantation, and at different time points, up to 220 days after transplant. Patients taking Evr had a higher percentage of total CD4? and na?ve CD4? T cells than those treated with CsA; the percentage of CD8? T cells was lower, but the frequency of na?ve CD8? T cells higher. Patients taking Evr showed a significantly higher percentage of Tregs, and Tregs expressing CXCR3. After stimulation with SEB, CD8? T cells from Evr-treated patients displayed a lower total response, and less IFN-γ producing cells. The effects on the immune system, such as the preservation of the na?ve T cell pool and the expansion of Tregs (that are extremely useful in inhibiting organ rejection), along with the higher tolerability of Evr, suggest that this drug can be safely used after liver transplantation, and likely offers immunological advantages.     

Arginine-Specific Mono ADP-Ribosylation In Vitro of Antimicrobial Peptides by ADP-Ribosylating Toxins

Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and β- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and β- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro. Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues.     

MHC class I-related antigen-processing machinery component defects in feline mammary carcinoma

Defects in HLA class I antigen-processing machinery (APM) component expression and/or function are frequent in human tumors. These defects may provide tumor cells with a mechanism to escape from recognition and destruction by HLA class I antigen-restricted, tumor antigen-specific cytotoxic T cells. However, expression and functional properties of MHC class I antigens and APM components in malignant cells in other animal species have been investigated to a limited extent. However, this information can contribute to our understanding of the mechanisms underlying the association of MHC class I antigen and APM component defects with malignant transformation of cells and to identify animal models to validate targeted therapies to correct these defects. To overcome this limitation in the present study, we have investigated the expression of the catalytic subunits of proteasome (Y, X, and Z) and of immunoproteasome (LMP2, LMP7, and LMP10) as well as of MHC class I heavy chain (HC) in 25 primary feline mammary carcinomas (FMCs) and in 23 matched healthy mammary tissues. We found a reduced expression of MHC class I HC and of LMP2 and LMP7 in tumors compared with normal tissues. Concordantly, proteasomal cleavage specificities in extracts from FMCs were different from those in healthy tissues. In addition, correlation analysis showed that LMP2 and LMP7 were concordantly expressed in FMCs, and their expression was significantly correlated with that of MHC class I HC. The abnormalities we have found in the APM in FMCs may cause a defective processing of some tumor antigens.     

HERVs Expression in Autism Spectrum Disorders

Background

Autistic Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental disorder, resulting from complex interactions among genetic, genomic and environmental factors. Here we have studied the expression of Human Endogenous Retroviruses (HERVs), non-coding DNA elements with potential regulatory functions, and have tested their possible implication in autism.

Methods

The presence of retroviral mRNAs from four HERV families (E, H, K and W), widely implicated in complex diseases, was evaluated in peripheral blood mononuclear cells (PBMCs) from ASD patients and healthy controls (HCs) by qualitative RT-PCR. We also analyzed the expression of the env sequence from HERV-H, HERV-W and HERV-K families in PBMCs at the time of sampling and after stimulation in culture, in both ASD and HC groups, by quantitative Real-time PCR. Differences between groups were evaluated using statistical methods.

Results

The percentage of HERV-H and HERV-W positive samples was higher among ASD patients compared to HCs, while HERV-K was similarly represented and HERV-E virtually absent in both groups. The quantitative evaluation shows that HERV-H and HERV-W are differentially expressed in the two groups, with HERV-H being more abundantly expressed and, conversely, HERV-W, having lower abundance, in PBMCs from ASDs compared to healthy controls. PMBCs from ASDs also showed an increased potential to up-regulate HERV-H expression upon stimulation in culture, unlike HCs. Furthermore we report a negative correlation between expression levels of HERV-H and age among ASD patients and a statistically significant higher expression in ASD patients with Severe score in Communication and Motor Psychoeducational Profile-3.

Conclusions

Specific HERV families have a distinctive expression profile in ASD patients compared to HCs. We propose that HERV-H expression be explored in larger samples of individuals with autism spectrum in order to determine its utility as a novel biological trait of this complex disorder.     

Use of national and international growth charts for studying height in European children: development of up-to-date European height-for-age charts

Background

Growth charts based on data collected in different populations and time periods are key tools to assess children’s linear growth. We analyzed the impact of geographic factors and the secular trend on height-for-age charts currently used in European populations, developed up-to-date European growth charts, and studied the effect of using different charts in a sample of growth retarded children.

Methods and Findings

In an international survey we obtained 18 unique national height-for-age charts from 28 European countries and compared them with charts from the World Health Organization (WHO), Euro-Growth reference, and Centers of Disease Control and Prevention (CDC). As an example, we obtained height data from 3,534 children with end-stage renal disease (ESRD) from 13 countries via the ESPN/ERA-EDTA registry, a patient group generally suffering from growth retardation. National growth charts showed a clear secular trend in height (mean height increased on average 0.6 cm/decade) and a North-South height gradient in Europe. For countries without a recent (>1990) national growth chart novel European growth charts were constructed from Northern and Southern European reference populations, reflecting geographic height differences in mean final height of 3.9 cm in boys and 3.8 cm in girls. Mean height SDS of 2- to 17-year-old ESRD patients calculated from recent national or derived European growth charts (−1.91, 95% CI: −1.97 to −1.85) was significantly lower than when using CDC or WHO growth charts (−1.55, 95% CI: −1.61 to −1.49) (P<0.0001).

Conclusion

Differences between height-for-age charts may reflect true population differences, but are also strongly affected by the secular trend in height. The choice of reference charts substantially affects the clinical decision whether a child is considered short-for-age. Therefore, we advocate using recent national or European height-for-age charts derived from recent national data when monitoring growth of healthy and diseased European children.     

Evaluation of gene, protein and neurotrophin expression in the brain of mice exposed to space environment for 91 days

Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occurred only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity.     

Improvement of tuberculosis laboratory capacity on pemba island, zanzibar: a health cooperation project

Low-income countries with high Tuberculosis burden have few reference laboratories able to perform TB culture. In 2006, the Zanzibar National TB Control Programme planned to decentralize TB diagnostics. The Italian Cooperation Agency with the scientific support of the "L. Spallanzani" National Institute for Infectious Diseases sustained the project through the implementation of a TB reference laboratory in a low-income country with a high prevalence of TB. The implementation steps were: 1) TB laboratory design according to the WHO standards; 2) laboratory equipment and reagent supplies for microscopy, cultures, and identification; 3) on-the-job training of the local staff; 4) web- and telemedicine-based supervision.From April 2007 to December 2010, 921 sputum samples were received from 40 peripheral laboratories: 120 TB cases were diagnosed. Of all the smear-positive cases, 74.2% were culture-positive. During the year 2010, the smear positive to culture positive rate increased up to 100%.In March 20, 2010 the Ministry of Health and Social Welfare of Zanzibar officially recognized the Public Health Laboratory- Ivo de Carneri as the National TB Reference Laboratory for the Zanzibar Archipelago.An advanced TB laboratory can represent a low cost solution to strengthen the TB diagnosis, to provide capacity building and mid-term sustainability.