A microRNA processing defect in smokers' macrophages is linked to SUMOylation of the endonuclease DICER.

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

Inositol-requiring enzyme 1 inhibits respiratory syncytial virus replication.

Despite being a major health problem, respiratory syncytial virus (RSV) infections remain without specific therapy. Identification of novel host cellular responses that play a role in the pathogenesis of RSV infection is needed for therapeutic development. The endoplasmic reticulum (ER) stress response is an evolutionarily conserved cellular signaling cascade that has been implicated in multiple biological phenomena, including the pathogenesis of some viral infections. In this study, we investigate the role of the ER stress response in RSV infection using an in vitro A549 cell culture model. We found that RSV infection induces a non-canonical ER stress response with preferential activation of the inositol-requiring enzyme 1 (IRE1) and activated transcription factor 6 (ATF6) pathways with no concomitant significant activation of the protein kinase R-like ER kinase (PERK) pathway. Furthermore, we discovered that IRE1 has an inhibitory effect on RSV replication. Our data characterize, for the first time, the nature of the ER stress response in the setting of RSV infection and identify the IRE1 stress pathway as a novel cellular anti-RSV defense mechanism.

Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway.

Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.

Thyrotoxicosis associated Wernicke's encephalopathy.

BACKGROUND: Wernicke's encephalopathy is a rare disorder resulting from nutrition deficiency in thiamine (vitamin B1). It is most often associated with chronic alcohol abuse. It can accompany other disorders such as hyperemesis gravidarum and hyperthyroidism. CASE: We present a case of severe Wernicke's encephalopathy that developed in a male non-alcoholic inpatient that was precipitated by poor nutritional intake and development of thyrotoxicosis. It is likely that the hypermetabolic state from thyrotoxicosis contributed to the development of Wernicke's encephalopathy, which was extremely debilitating. CONCLUSION: Wernicke's encephalopathy is a severe, life-threatening illness that can be a consequence of hyperthyroidism in addition to alcohol abuse and can be easily prevented with appropriate supplementation of thiamine.

Serotyping of hepatitis C virus in hemodialysis patients: comparison with a standardized genotyping assay.

The aims of this study were to investigate the prevalence of hepatitis C virus (HCV) genotypes and serotypes in anti-HCV-positive hemodialysis patients and determine the concordance between genotyping and serotyping methods. Sixty-two hemodialysis patients were included in this study. HCV RNA was determined using polymerase chain reaction assay and genotypes using a line probe assay. HCV serotyping was performed with competitive enzyme-linked immunosorbent assay. Genotype 4 (52 patients) was the most predominant genotype, followed by type 1 (10 patients). The most prevalent HCV serotype was type 4 (41 patients), followed by serotype 1 (6 patients). We detected multiple serotypes in 4 patients and untypeable strains in 11. The overall sensitivity of serotyping assay was 82% for the study patients. According to the genotyping results, the sensitivity of serotyping was 60% and 86.5% for HCV types 1 and 4, respectively. There was a 100% concordance between results of serotyping and genotyping in the identification of HCV type 1 and 91% concordance in HCV type 4. Serological typing method may be of great value in microbiology laboratories that require a simple assay for identification of HCV genotypes, although the sensitivity of this assay may be limited by the immunocompetence of infected hemodialysis patients.

Cigarette smoking decreases global microRNA expression in human alveolar macrophages.

Human alveolar macrophages are critical components of the innate immune system. Cigarette smoking-induced changes in alveolar macrophage gene expression are linked to reduced resistance to pulmonary infections and to the development of emphysema/COPD. We hypothesized that microRNAs (miRNAs) could control, in part, the unique messenger RNA (mRNA) expression profiles found in alveolar macrophages of cigarette smokers. Activation of macrophages with different stimuli in vitro leads to a diverse range of M1 (inflammatory) and M2 (anti-inflammatory) polarized phenotypes that are thought to mimic activated macrophages in distinct tissue environments. Microarray mRNA data indicated that smoking promoted an "inverse" M1 mRNA expression program, defined by decreased expression of M1-induced transcripts and increased expression of M1-repressed transcripts with few changes in M2-regulated transcripts. RT-PCR arrays identified altered expression of many miRNAs in alveolar macrophages of smokers and a decrease in global miRNA abundance. Stratification of human subjects suggested that the magnitude of the global decrease in miRNA abundance was associated with smoking history. We found that many of the miRNAs with reduced expression in alveolar macrophages of smokers were predicted to target mRNAs upregulated in alveolar macrophages of smokers. For example, miR-452 is predicted to target the transcript encoding MMP12, an important effector of smoking-related diseases. Experimental antagonism of miR-452 in differentiated monocytic cells resulted in increased expression of MMP12. The comprehensive mRNA and miRNA expression profiles described here provide insight into gene expression regulation that may underlie the adverse effects cigarette smoking has on alveolar macrophages.

Coordinated DNA methylation and gene expression changes in smoker alveolar macrophages: specific effects on VEGF receptor 1 expression.

Cigarette smoking is implicated in numerous diseases, including emphysema and lung cancer. The clinical expression of lung disease in smokers is not well explained by currently defined variations in gene expression or simple differences in smoking exposure. Alveolar macrophages play a critical role in the inflammation and remodeling of the lung parenchyma in smoking-related lung disease. Significant gene expression changes in alveolar macrophages from smokers have been identified. However, the mechanism for these changes remains unknown. One potential mechanism for smoking-altered gene expression is via changes in cytosine methylation in DNA regions proximal to gene-coding sequences. In this study, alveolar macrophage DNA from heavy smokers and never smokers was isolated and methylation status at 25,000 loci determined. We found differential methylation in genes from immune-system and inflammatory pathways. Analysis of matching gene expression data demonstrated a parallel enrichment for changes in immune-system and inflammatory pathways. A significant number of genes with smoking-altered mRNA expression had inverse changes in methylation status. One gene highlighted by this data was the FLT1, and further studies found particular up-regulation of a splice variant encoding a soluble inhibitory form of the receptor. In conclusion, chronic cigarette smoke exposure altered DNA methylation in specific gene promoter regions in human alveolar macrophages.

Respiratory syncytial virus synergizes with Th2 cytokines to induce optimal levels of TARC/CCL17.

Respiratory syncytial virus (RSV) is a ubiquitous virus that preferentially infects airway epithelial cells, causing asthma exacerbations and severe disease in immunocompromised hosts. Acute RSV infection induces inflammation in the lung. Thymus- and activation-regulated chemokine (TARC) recruits Th2 cells to sites of inflammation. We found that acute RSV infection of BALB/c mice increased TARC production in the lung. Immunization of BALB/c mice with individual RSV proteins can lead to the development of Th1- or Th2-biased T cell responses in the lung after RSV infection. We primed animals with a recombinant vaccinia virus expressing either the RSV fusion (F) protein or the RSV attachment (G) protein, inducing Th1- and Th2-biased pulmonary memory T cell responses, respectively. After RSV infection, TARC production significantly increased in the vaccinia virus G-primed animals only. These data suggest a positive feedback loop for TARC production between RSV infection and Th2 cytokines. RSV-infected lung epithelial cells cultured with IL-4 or IL-13 demonstrated a marked increase in the production of TARC. The synergistic effect of RSV and IL-4/IL-13 on TARC production reflected differential induction of NF kappa B and STAT6 by the two stimuli (both are in the TARC promoter). These findings demonstrate that RSV induces a chemokine TARC that has the potential to recruit Th2 cells to the lung.

One year study of bacterial and fungal nosocomial infections among patients in pediatric intensive care unit (PICU) in Alexandria.

UNLABELLED: A 1-year prospective and observational study included all admissions (n=216) until 48 h after discharge to Alexandria PICU between first of May 2003 and end of April 2004. Cultures for bacteria and fungi and antibiotic sensitivity tests (19 antibiotic using Bauer-Kirby disc diffusion method) were obtained (blood, stool, urine and cerebrospinal fluid, if needed) and repeated on suspicion of NIs. All cannulae, endotracheal tube (ET) aspirates and tips, nasogastric tubes and different catheters were cultured. All PICU health care workers (HCWs) were subjected to throat and under-finger nails cultures as well as inanimate objects, both on bimonthly basis. The referral place (ward or emergency), PRISM III score, length of stay (LOS) and fate were recorded. Amongst those patients whose age ranged from 1 to 23 months, 23 per cent had NIs with infection rates of 40/1000 days. Significantly high rates of mortality, LOS and PRISM III score were encountered among patients with NIs (52 per cent vs 30 per cent; 9.4+/-4.8 vs 5.4+/-2.2 days; 14.4+/-7 vs 11.8+/-6 respectively). The descending order of frequency of NIs was blood stream infection (BSI) (47 per cent), urinary tract infection (UTI) (28 per cent), ventilator-associated pneumonia (VAP) (16 per cent) and meningitis (9 per cent). Gr-ve bacilli accounted for 76.7 per cent; Gr+ve cocci 13.3 per cent (with satisfactory sensitivity to cefepime, imipenem and meropenem) and Candida albicans 10 per cent of all NIs. The rate of NIs/1000 device days were: 18.7 per cent for BSI, 10.9 per cent for VAP and 25.5 per cent for UTI. Vulnerable age groups were >6 m for VAP and <6 m for meningitis. Multiple logistic regression analysis identified LOS, PRISM III score and referral from wards a predictors of NI acquisition (odd ratio and 95 per cent confidence interval: 1.537, 1.423-1.659; 1.073, 1.041-1.105 and 0.269, 0.178-0.406 respectively). Bimonthly cultures for HCWs isolated coagulase-ve Staphylococci, while inanimate objects isolated diphtheroids and Candida albicans. CONCLUSION: NIs rate was high (23 per cent) mainly due to severity of condition on admission as shown by high PRISM III score value, the high PRISM III score, LOS and referral from wards were predictors of acquisition of NIs and there is a high incidence of Candida albicans infection (10 per cent of NIs).