Merkel cell polyomavirus (MCPyV) in the context of Immunosuppression. Genetic analysis of noncoding control region (NCCR) variability among a HIV-1-positive population

Background: Since limited data are available about the prevalence of Merkel cell polyomavirus (MCPyV) and the genetic variability of its noncoding control region (NCCR) in the context of immunosuppression, this study aimed to investigate the distribution of MCPyV in anatomical sites other than the skin and the behavior of NCCR among an HIV-1-positive population. Methods: Urine, plasma, and rectal swabs specimens from a cohort of 66 HIV-1-positive patients were collected and subjected to quantitative real-time polymerase chain reaction (qPCR) for MCPyV DNA detection. MCPyV-positive samples were amplified by nested PCR targeting the NCCR, and NCCRs alignment was carried out to evaluate the occurrence of mutations and to identify putative binding sites for cellular factors. Results: MCPyV DNA was detected in 10/66 urine, in 7/66 plasma, and in 23/66 rectal samples, with a median value of 5 × 102 copies/mL, 1.5 × 102 copies/mL, and 2.3 × 103 copies/mL, respectively. NCCR sequence analysis revealed a high degree of homology with the MCC350 reference strain in urine, whereas transitions, transversions, and single or double deletions were observed in plasma and rectal swabs. In these latter samples, representative GTT and GTTGA insertions were also observed. Search for putative binding sites of cellular transcription factors showed that in several strains, deletions, insertions, or single base substitutions altered the NCCR canonical configuration. Conclusions: Sequencing analysis revealed the presence of numerous mutations in the NCCR, including insertions and deletions. Whether these mutations may have an impact on the pathogenic features of the virus remains to be determined. qPCR measured on average a low viral load in the specimens analyzed, with the exception of those with the GTTGA insertion

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions

Cocaine-induced renal infarction: report of a case and review of the literature

BACKGROUND: Cocaine abuse has been known to have detrimental effects on the cardiovascular system. Its toxicity has been associated with myocardial ischemia, cerebrovascular accidents and mesenteric ischemia. The pathophysiology of cocaine-related renal injury is multifactorial and involves renal hemodynamic changes, alterations in glomerular matrix synthesis, degradation and oxidative stress, and possibly induction of renal atherogenesis. Renal infarction as a result of cocaine exposure, however, is rarely reported in the literature. CASE PRESENTATION: A 48 year-old male presented with a four-day history of severe right flank pain following cocaine use. On presentation, he was tachycardic, febrile and had severe right costovertebral angle tenderness. He had significant proteinuria, leukocytosis and elevated serum creatinine and lactate dehydrogenase. Radiographic imaging studies as well as other screening tests for thromboembolic events, hypercoagulability states, collagen vascular diseases and lipid disorders were suggestive of Cocaine-Induced Renal Infarction (CIRI) by exclusion. CONCLUSION: In a patient with a history of cocaine abuse presenting with fevers and flank pain suggestive of urinary tract infection or nephrolithiasis, cocaine-induced renal infarction must be considered in the differential diagnosis. In this article, we discuss the prior reported cases of CIRI and thoroughly review the literature available on this disorder. This is important for several reasons. First, it will allow us to discuss and elaborate on the mechanism of renal injury caused by cocaine. In addition, this review will demonstrate the importance of considering the diagnosis of CIRI in a patient with documented cocaine use and an atypical presentation of acute renal injury. Finally, we will emphasize the need for a consensus on optimal treatment of this disease, for which therapy is not yet standardized

APP Processing Induced by Herpes Simplex Virus Type 1 (HSV-1) Yields Several APP Fragments in Human and Rat Neuronal Cells

Lifelong latent infections of the trigeminal ganglion by the neurotropic herpes simplex virus type 1 (HSV-1) are characterized by periodic reactivation. During these episodes, newly produced virions may also reach the central nervous system (CNS), causing productive but generally asymptomatic infections. Epidemiological and experimental findings suggest that HSV-1 might contribute to the pathogenesis of Alzheimer's disease (AD). This multifactorial neurodegenerative disorder is related to an overproduction of amyloid beta (Aβ) and other neurotoxic peptides, which occurs during amyloidogenic endoproteolytic processing of the transmembrane amyloid precursor protein (APP). The aim of our study was to identify the effects of productive HSV-1 infection on APP processing in neuronal cells. We found that infection of SH-SY5Y human neuroblastoma cells and rat cortical neurons is followed by multiple cleavages of APP, which result in the intra- and/or extra-cellular accumulation of various neurotoxic species. These include: i) APP fragments (APP-Fs) of 35 and 45 kDa (APP-F35 and APP-F45) that comprise portions of Aβ; ii) N-terminal APP-Fs that are secreted; iii) intracellular C-terminal APP-Fs; and iv) Aβ1-40 and Aβ1-42. Western blot analysis of infected-cell lysates treated with formic acid suggests that APP-F35 may be an Aβ oligomer. The multiple cleavages of APP that occur in infected cells are produced in part by known components of the amyloidogenic APP processing pathway, i.e., host-cell β-secretase, γ-secretase, and caspase-3-like enzymes. These findings demonstrate that HSV-1 infection of neuronal cells can generate multiple APP fragments with well-documented neurotoxic potentials. It is tempting to speculate that intra- and extracellular accumulation of these species in the CNS resulting from repeated HSV-1 reactivation could, in the presence of other risk factors, play a co-factorial role in the development of AD

Caveolin-1 enhances resveratrol-mediated cytotoxicity and transport in a hepatocellular carcinoma model

<p>Abstract</p> <p>Background</p> <p>Resveratrol (RES), an estrogen analog, is considered as a potential cancer chemo-preventive agent. However, it remains unclear how RES is transported into cells. In this study, we observed that Caveolin-1(CAV1) expression can increase the cytotoxic and pro-apoptotic activity of RES in a dose- and time-dependent manner both <it>in vitro </it>and <it>in vivo </it>in a Hepatocellular Carcinoma animal model.</p> <p>Methods</p> <p>High performance liquid chromatography (HPLC) demonstrated that RES intra-cellular concentration is increased about 2-fold in cells stably expressing CAV1 or CAVM1 (a scaffolding domain (81-101AA)-defective CAV1 mutant) compared to the untransduced human Hepatoblastoma cell line (HepG2) or after transduction with the green fluorescent protein (GFP) control vector. The increased intra-cellular transport of RES was abolished in cells stably expressing CAVM2 (a cholesterol shuttle domain (143-156AA)-defective CAV1 mutant) or CAVRNAi. In order to further characterize CAV1-dependent RES transport, we synthesized RES-dansyl chloride derivatives as fluorescent probes to visualize the transport process, which demonstrated a distribution consistent with that of CAV1 in HepG2 cells.</p> <p>Results</p> <p>In addition, RES endocytosis was not mediated by estrogen receptor (ER) α and β, as suggested by lack of competitive inhibition by estrogen or Tamoxifen. Pathway analysis showed that RES can up-regulate the expression of endogenous CAV1; this activates further the MAPK pathway and caspase-3 expression.</p> <p>Discussion</p> <p>This study provides novel insights about the role played by CAV1 in modulating cellular sensitivity to RES through enhancement of its internalization and trafficking.</p

A Canadian Critical Care Trials Group project in collaboration with the international forum for acute care trialists - Collaborative H1N1 Adjuvant Treatment pilot trial (CHAT): study protocol and design of a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Swine origin influenza A/H1N1 infection (H1N1) emerged in early 2009 and rapidly spread to humans. For most infected individuals, symptoms were mild and self-limited; however, a small number developed a more severe clinical syndrome characterized by profound respiratory failure with hospital mortality ranging from 10 to 30%. While supportive care and neuraminidase inhibitors are the main treatment for influenza, data from observational and interventional studies suggest that the course of influenza can be favorably influenced by agents not classically considered as influenza treatments. Multiple observational studies have suggested that HMGCoA reductase inhibitors (statins) can exert a class effect in attenuating inflammation. The Collaborative H1N1 Adjuvant Treatment (CHAT) Pilot Trial sought to investigate the feasibility of conducting a trial during a global pandemic in critically ill patients with H1N1 with the goal of informing the design of a larger trial powered to determine impact of statins on important outcomes.</p> <p>Methods/Design</p> <p>A multi-national, pilot randomized controlled trial (RCT) of once daily enteral rosuvastatin versus matched placebo administered for 14 days for the treatment of critically ill patients with suspected, probable or confirmed H1N1 infection. We propose to randomize 80 critically ill adults with a moderate to high index of suspicion for H1N1 infection who require mechanical ventilation and have received antiviral therapy for ≤ 72 hours. Site investigators, research coordinators and clinical pharmacists will be blinded to treatment assignment. Only research pharmacy staff will be aware of treatment assignment. We propose several approaches to informed consent including a priori consent from the substitute decision maker (SDM), waived and deferred consent. The primary outcome of the CHAT trial is the proportion of eligible patients enrolled in the study. Secondary outcomes will evaluate adherence to medication administration regimens, the proportion of primary and secondary endpoints collected, the number of patients receiving open-label statins, consent withdrawals and the effect of approved consent models on recruitment rates.</p> <p>Discussion</p> <p>Several aspects of study design including the need to include central randomization, preserve allocation concealment, ensure study blinding compare to a matched placebo and the use novel consent models pose challenges to investigators conducting pandemic research. Moreover, study implementation requires that trial design be pragmatic and initiated in a short time period amidst uncertainty regarding the scope and duration of the pandemic.</p> <p>Trial Registration Number</p> <p><a href="http://www.controlled-trials.com/ISRCTN45190901">ISRCTN45190901</a></p

Antiviral activity and possible mechanisms of action of pentagalloylglucose (PGG) against influenza A virus.

Influenza A virus (IAV) infection is a major public health threat leading to significant morbidity and mortality. The emergence of drug-resistant virus strains highlights the urgent need to develop novel antiviral drugs with alternative modes of action. Pentagalloylglucose (PGG), a naturally occurring polyphenolic compound, possesses a broad spectrum of biological activities. In this study, we found that PGG has anti-influenza-virus activity, and investigated its possible mechanism(s) of action in vitro. Both pre-incubation of virus prior to infection and post-exposure of infected cells with PGG significantly inhibited virus yields. Influenza-virus-induced hemagglutination of chicken red blood cells was inhibited by PGG treatment, suggesting that PGG can inhibit IAV infection by interacting with the viral hemagglutinin. PGG did not affect viral protein synthesis or nuclear transport of viral nucleoprotein (NP) but greatly reduced plasma membrane accumulation of NP protein at the late stage of the replication cycle. Furthermore, PGG significantly reduced virus budding and progeny virus release from infected cells. This study revealed for the first time that PGG can inhibit IAV replication with a dual mode of action and offers new insights into its underlying mechanisms of antiviral action