NPY regulates catecholamine secretion from human adrenal chromaffin cells.

The aim of the present work was to find out whether NPY synthesized in human adrenal chromaffin cells controls in an autocrine/paracrine fashion the release of catecholamines by these cells. Accordingly, the constitutive and regulated release of both NPY and catecholamines was measured simultaneously in cultured human chromaffin cells. In addition, by using both RT-PCR and a combination of specific agonists and antagonists, we characterized the expression of NPY receptors on these cells as well as their pharmacology. Our results were as follows. 1) Human chromaffin cells constitutively secrete NPY. 2) Nicotine elicits a rapid increase in the release of both catecholamines and NPY; this release of NPY is more sustained than that of catecholamines. 3) RT-PCR shows expression of Y1, Y2, Y4, and Y5 receptor mRNA by chromaffin cells; these receptors are functional, as various receptor specific agonists elicit an increase in intracellular calcium. 4) Peptide YY, in contrast to NPY, is not able to stimulate the release of catecholamines. This finding was corroborated by the observation that no receptor-specific antagonists were able to reduce constitutive catecholamine release, whereas an NPY-immunoneutralizing antibody markedly attenuated the secretion. Taken together, these data suggest that NPY originating from the adrenal medulla locally enhances the secretion of catecholamines, presumably by acting via the putative y3 receptor.

Risk Factors for Anal Cancer in Persons Infected With HIV: A Nested Case-Control Study in the Swiss HIV Cohort Study.

Although persons infected with human immunodeficiency virus (HIV), particularly men who have sex with men, are at excess risk for anal cancer, it has been difficult to disentangle the influences of anal exposure to human papillomavirus (HPV) infection, immunodeficiency, and combined antiretroviral therapy. A case-control study that included 59 anal cancer cases and 295 individually matched controls was nested in the Swiss HIV Cohort Study (1988-2011). In a subset of 41 cases and 114 controls, HPV antibodies were tested. A majority of anal cancer cases (73%) were men who have sex with men. Current smoking was significantly associated with anal cancer (odds ratio (OR) = 2.59, 95% confidence interval (CI): 1.25, 5.34), as were antibodies against L1 (OR = 4.52, 95% CI: 2.00, 10.20) and E6 (OR = â^?, 95% CI: 4.64, â^?) of HPV16, as well as low CD4+ cell counts, whether measured at nadir (OR per 100-cell/μL decrease = 1.53, 95% CI: 1.18, 2.00) or at cancer diagnosis (OR per 100-cell/μL decrease = 1.24, 95% CI: 1.08, 1.42). However, the influence of CD4+ cell counts appeared to be strongest 6-7 years prior to anal cancer diagnosis (OR for <200 vs. â0/00¥500 cells/μL = 14.0, 95% CI: 3.85, 50.9). Smoking cessation and avoidance of even moderate levels of immunosuppression appear to be important in reducing long-term anal cancer risks.

Establishment of HEV RNA reverse transcriptase PCR assays for the diagnosis of acute and chronic hepatitis E

Background and aim: H epatitis E v irus (HEV) infection has emerged as a c ause o f travel-related a nd autochthonous a cute hepatitis as well as chronic hepatitis in immunosuppressed patients. While t ravel-related cases a re c aused primarily b y infections w ith HEV of g enotype 1 ( HEV-1), autochthonous c ases a nd chronic cases a re d ue t o genotype 3 (HEV-3), which is s hared between humans and diverse animal species. The aim of this study was to establish HEV RNA detection assays f or q uantitative v iral load testing and genotyping. Methods: V iral RNA was p urified from plasma or s erum a nd converted to cDNA prior to (1) multiplex real-time PCR for HEV RNA quantification and (2) multiplex PCR coupled to DNA sequencing for HEV genotype determination. Real-time PCR was d esigned to match a ll known HEV genotypes available i n Genbank while PCR was designed using conserved primers flanking a variable region of the HEV RNA. Results: In a validation panel, the newly developed assays allowed for the reliable detection and genotyping of HEV-1 or HEV-3. Cases of t ravel-related and a utochthonous a cute h epatitis E a s well a s chronic hepatitis E i n immunosuppressed patients have b een identified using t hese a ssays a nd will be p resented in detail. Anti- HEV antibodies were n egative i n three well-characterized patients with chronic hepatitis E after organ transplantation. Conclusions: We developed and validated a quantitative HEV RNA detection assay that c an now be o ffered on a r outine basis (www.chuv.ch/imul/imu-collaborations-viral_hepatitis). Genotyping can also be offered on selected cases. HEV RNA detection is key in diagnosing chronic hepatitis E i n immunosuppressed patients with unexplained transaminase elevations, as serology can be negative in these patients.

Secretory IgA-mediated neutralization of Shigella flexneri prevents intestinal tissue destruction by down-regulating inflammatory circuits.

Shigella, a Gram-negative invasive enteropathogenic bacterium responsible for bacillary dysentery, causes the rupture, invasion, and inflammatory destruction of the human colonic mucosa. We explored the mechanisms of protection mediated by Shigella LPS-specific secretory IgA (SIgA), the major mucosal Ab induced upon natural infection. Bacteria, SIgA, or SIgA-S. flexneri immune complexes were administered into rabbit ligated intestinal loops containing a Peyer's patch. After 8 h, localizations of bacteria, SIgA, and SIgA-S. flexneri immune complexes were examined by immunohistochemistry and confocal microscopy imaging. We found that anti-Shigella LPS SIgA, mainly via immune exclusion, prevented Shigella-induced inflammation responsible for the destruction of the intestinal barrier. Besides this luminal trapping, a small proportion of SIgA-S. flexneri immune complexes were shown to enter the rabbit Peyer's patch and were internalized by dendritic cells of the subepithelial dome region. Local inflammatory status was analyzed by quantitative RT-PCR using newly designed primers for rabbit pro- and anti-inflammatory mediator genes. In Peyer's patches exposed to immune complexes, limited up-regulation of the expression of proinflammatory genes, including TNF-alpha, IL-6, Cox-2, and IFN-gamma, was observed, consistent with preserved morphology. In contrast, in Peyer's patches exposed to Shigella alone, high expression of the same mediators was measured, indicating that neutralizing SIgA dampens the proinflammatory properties of Shigella. These results show that in the form of immune complexes, SIgA guarantees both immune exclusion and neutralization of translocated bacteria, thus preserving the intestinal barrier integrity by preventing bacterial-induced inflammation. These findings add to the multiple facets of the noninflammatory properties of SIgA.

Sequence-Based Hepatitis B Virus Antiviral Resistance Testing in Switzerland

BACKGROUND: A growing number of patients with chronic hepatitis B is being treated for extended periods with nucleoside and/or nucleotide analogs. In this context, antiviral resistance represents an increasingly common and complex issue. METHODS: Mutations in the hepatitis B virus (HBV) reverse transcriptase (rt) gene and viral genotypes were determined by direct sequencing of PCR products and alignment with reference sequences deposited in GenBank. RESULTS: Plasma samples from 60 patients with chronic hepatitis B were analyzed since March 2009. The predominant mutation pattern identified in patients with virological breakthrough was rtM204V/I ± different compensatory mutations, conferring resistance to L-nucleosides (lamivudine, telbivudine, emtricitabine) and predisposing to entecavir resistance (n = 18). Complex mutation patterns with a potential for multidrug resistance were identified in 2 patients. Selection of a fully entecavir resistant strain was observed in a patient exposed to lamivudine alone. Novel mutations were identified in 1 patient. Wild-type HBV was identified in 9 patients with suspected virological breakthrough, raising concerns about treatment adherence. No preexisting resistance mutations were identified in treatment-naïve patients (n = 13). Viral genome amplification and sequencing failed in 16 patients, of which only 2 had a documented HBV DNA > 1000 IU/ml. HBV genotypes were D in 28, A in 6, B in 4, C in 3 and E in 3 patients. Results will be updated in August 2010 and therapeutic implications discussed. CONCLUSIONS: With expanding treatment options and a growing number of patients exposed to nucleoside and/or nucleotide analogs, sequence-based HBV antiviral resistance testing is expected to become a cornerstone in the management of chronic hepatitis B.

Skeletal muscle mitochondria in the elderly: effects of physical fitness and exercise training.

CONTEXT: Sarcopenia is thought to be associated with mitochondrial (Mito) loss. It is unclear whether the decrease in Mito content is consequent to aging per se or to decreased physical activity. OBJECTIVES: The objective of the study was to examine the influence of fitness on Mito content and function and to assess whether exercise could improve Mito function in older adults. DESIGN AND SUBJECTS: Three distinct studies were conducted: 1) a cross-sectional observation comparing Mito content and fitness in a large heterogeneous cohort of older adults; 2) a case-control study comparing chronically endurance-trained older adults and sedentary (S) subjects matched for age and gender; and 3) a 4-month exercise intervention in S. SETTING: The study was conducted at a university-based clinical research center. OUTCOMES: Mito volume density (MitoVd) was assessed by electron microscopy from vastus lateralis biopsies, electron transport chain proteins by Western blotting, mRNAs for transcription factors involved in M biogenesis by quantitative RT-PCR, and in vivo oxidative capacity (ATPmax) by (31)P-magnetice resonance spectroscopy. Peak oxygen uptake was measured by graded exercise test. RESULTS: Peak oxygen uptake was strongly correlated with MitoVd in 80 60- to 80-year-old adults. Comparison of chronically endurance-trained older adults vs S revealed differences in MitoVd, ATPmax, and some electron transport chain protein complexes. Finally, exercise intervention confirmed that S subjects are able to recover MitoVd, ATPmax, and specific transcription factors. CONCLUSIONS: These data suggest the following: 1) aging per se is not the primary culprit leading to Mito dysfunction; 2) an aerobic exercise program, even at an older age, can ameliorate the loss in skeletal muscle Mito content and may prevent aging muscle comorbidities; and 3) the improvement of Mito function is all about content.

Nineteen additional unpredicted transcripts from human chromosome 21.

The identification of all human chromosome 21 (HC21) genes is a necessary step in understanding the molecular pathogenesis of trisomy 21 (Down syndrome). The first analysis of the sequence of 21q included 127 previously characterized genes and predicted an additional 98 novel anonymous genes. Recently we evaluated the quality of this annotation by characterizing a set of HC21 open reading frames (C21orfs) identified by mapping spliced expressed sequence tags (ESTs) and predicted genes (PREDs), identified only in silico. This study underscored the limitations of in silico-only gene prediction, as many PREDs were incorrectly predicted. To refine the HC21 annotation, we have developed a reliable algorithm to extract and stringently map sequences that contain bona fide 3' transcript ends to the genome. We then created a specific 21q graphical display allowing an integrated view of the data that incorporates new ESTs as well as features such as CpG islands, repeats, and gene predictions. Using these tools we identified 27 new putative genes. To validate these, we sequenced previously cloned cDNAs and carried out RT-PCR, 5'- and 3'-RACE procedures, and comparative mapping. These approaches substantiated 19 new transcripts, thus increasing the HC21 gene count by 9.5%. These transcripts were likely not previously identified because they are small and encode small proteins. We also identified four transcriptional units that are spliced but contain no obvious open reading frame. The HC21 data presented here further emphasize that current gene prediction algorithms miss a substantial number of transcripts that nevertheless can be identified using a combination of experimental approaches and multiple refined algorithms.