Human Respiratory Coronaviruses Detected In Patients with Influenza-Like Illness in Arkansas, USA.

Acute respiratory viruses often result in significant morbidity and mortality. The potential impact of human respiratory coronavirus (CoV) infections was underestimated until the severe acute respiratory syndrome (SARS-CoV) outbreak in 2003, which showed that new, highly pathogenic coronaviruses could be introduced to humans, highlighting the importance of monitoring the circulating coronaviruses. The use of sensitive molecular methods has contributed to the differential diagnosis of viruses circulating in humans. Our study aim was to investigate the molecular epidemiology of human CoV strains circulating in Arkansas, their genetic variability and their association with reported influenza-like symptoms. We analyzed 200 nasal swab samples, collected by the Arkansas Department of Health in 2010, for influenza diagnosis. All samples were from patients showing acute respiratory symptoms while testing negative for influenza. Samples were pre-screened, using a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) multiprobe for coronavirus, and subjected to confirmatory pancoronavirus and/or strain-specific reverse transcriptase (RT)-PCR followed by sequence analysis. Seventy-nine samples (39.5%) were positive by qRT-PCR and 35 samples (17.5%) were confirmed by conventional RT-PCR. Twenty-three of the confirmed samples (59%) were sequenced. The most frequent strain detected was HCoV-OC43-like followed by NL63-like; only one sample was positive for HCoV-229E and one for HCoV-HKU1. Feline-like CoV strains were detected in three samples, representing possible evidence of interspecies transmission or a new human strain. Seventeen percent of the coronavirus positive samples were also positive for other respiratory viruses, such as Respiratory Syncytial Virus (RSV), Parainfluenza 2 and 3, and Rhinovirus. Thus, HCoV-OC43, NL63, HKU1 and new feline-like strains were circulating in Arkansas in 2010. HCoV was the sole respiratory virus detected in 16% of the patients who showed acute respiratory symptoms with negative diagnoses for influenza virus.

Adiponectin translation is increased by the PPARgamma agonists pioglitazone and omega-3 fatty acids.

Adiponectin, made exclusively by adipocytes, is a 30-kDa secretory protein assembled posttranslationally into low-molecular weight, middle-molecular weight, and high-molecular weight homo-oligomers. PPARgamma ligand thiozolidinediones, which are widely used in the treatment of type II diabetes, increase adiponectin levels. PPARgamma also has several putative ligands that include fatty acid derivatives. Overnight treatment of rat adipocytes with pioglitazone, docosahexaenoic acid (DHA), or eicosapentaenoic acid (EPA) triggered a twofold increase in the synthesis and secretion of HMW adiponectin, and this increase was blocked by the addition of PPARgamma inhibitor GW-9662. Inhibition of glycosylation using 2,2'-dipyridyl decreased the synthesis of high-molecular weight adiponectin by pioglitazone, EPA, and DHA, but there was increased secretion of trimeric adiponectin resulting from increased translation. Although pioglitazone, DHA, and EPA increased adiponectin synthesis by more than 60%, there was no increase in total protein synthesis and no corresponding change in adiponectin mRNA expression, indicating the upregulation of translation. We examined the possibility of transacting factors in the cytoplasmic extracts from adipocytes treated with pioglitazone or DHA. In vitro translation of adiponectin mRNA was inhibited by S-100 fraction of control adipocytes and increased by S-100 extracts from adipocytes treated with pioglitazone or DHA. Consistent with this observation, both pioglitazone and DHA treatments increased the association of adiponectin mRNA with the heavier polysome fractions. Together, these data suggest that pioglitazone and the fish oils DHA or EPA are PPARgamma agonists in adipocytes with regard to adiponectin expression, and the predominant mode of adiponectin stimulation is via an increase in translation.

Calcium is involved in formation of high molecular weight adiponectin.

BACKGROUND: Adiponectin, an adipocyte-specific secretory protein, is known to circulate as different isoforms in the blood stream. METHODS: Using sucrose gradients and Western blotting on nondenaturing gels, adiponectin isoforms were examined in human serum, plasma, adipose tissue, and cells. The medium from human adipose tissue and human and mouse adipocytes were also examined for changes in isoform formation upon treatment with EGTA. RESULTS: Comparison of adiponectin complexes revealed distinct differences in distribution of high molecular weight (HMW) forms between human serum and plasma, with an apparent difference in molecular weight. Variation in molecular weight suggested a probable dissociation of the HMW isoforms in the presence of EDTA in the plasma. Examination of human serum samples treated with EDTA or EGTA showed a partial dissociation of the HMW isoform, while the addition of excess calcium, but not magnesium, to human plasma resulted in partial restoration of HMW adiponectin. When human adipose tissue-secreted adiponectin was treated with EGTA, there was a decrease in the HMW isoform by 61% (+/- 1.89%) and a corresponding increase in low molecular weight (LMW) and middle molecular weight (MMW) isoforms, compared to untreated samples. Analysis of mouse and human adipocytes also showed a reduction in HMW isoforms with a corresponding increase in MMW and LMW isoforms upon treatment with EGTA. The Simpson-Golabi-Behmel syndrome (SGBS) human adipocyte cell line, which primarily synthesizes LMW isoforms, produced increasing amounts of HMW adiponectin upon treatment with calcium in a dose-dependent manner. CONCLUSION: These data indicate that calcium promotes the formation of HMW adiponectin, and calcium sequestration decreases HMW adiponectin. Because of the importance of HMW adiponectin in insulin sensitivity, these data demonstrate the importance of assay conditions and sample preparation in the measurement of adiponectin isoforms.

Thiazolidinediones and the risk of lung, prostate, and colon cancer in patients with diabetes.

PURPOSE: Peroxisome proliferator-activated receptor gamma (PPARgamma) mediates cell cycle arrest and adipocyte differentiation; has tumor suppressor activity in liposarcoma, lung, and prostate cancers; and suppresses colonic polyp formation in adenomatous polyposis coli (APC)min/+ mice. To assess the influence of thiazolidinediones (TZDs), which are PPAR ligands used to treat diabetes mellitus, a retrospective analysis of a database from 10 Veteran Affairs medical centers was conducted. PATIENTS AND METHODS: Data on male patients 40 years and older diagnosed to have diabetes mellitus between 1997 and 2003 were obtained from the Veterans Integrated Services Network 16 (VISN 16) data warehouse. Subsequent diagnoses of colorectal, lung, and prostate cancer and use of TZD, other antidiabetic agents, and insulin were identified. Cox regression with time-dependent covariates was used to estimate the association between TZD use and cancer risk. Relative risks were adjusted for confounders (age, race/ethnicity, body mass index, use of insulin, and other oral antidiabetic agents). RESULTS: Of 87,678 individuals, 1,137 had colorectal cancer, 3,246 had prostate cancer, and 1,371 had lung cancer. We observed a 33% reduction in lung cancer risk among TZD users compared with nonusers after adjusting for confounder interactions (relative risk, 0.67; 95% CI, 0.51 to 0.87). The risk reduction for colorectal and prostate cancers did not reach statistical significance. CONCLUSION: TZD use was associated with reduced risk of lung cancer. Further studies are warranted to confirm our findings.

Pioglitazone increases secretion of high-molecular-weight adiponectin from adipocytes.

Adiponectin is an adipocyte-derived serum protein that plays important roles in energy homeostasis, obesity, and insulin sensitivity. Using sucrose gradients and Western blotting of nondenaturing gels, we examined the adiponectin isoforms secreted from human adipose tissue, human and mouse adipocytes, and cell lines in response to pioglitazone added in vitro. The predominant form secreted from adipose tissue in vitro was the high-molecular-weight (HMW) isoform, with small amounts of low-molecular-weight (LMW) forms present. The addition of pioglitazone (1-3 micromM) in vitro increased the secretion of the HMW isoform, with no significant effect on the other isoforms. Human adipose tissue was also examined for changes in adiponectin mRNA levels upon pioglitazone treatment. No difference was detected, suggesting that the effect of pioglitazone is not at the transcriptional level but, rather, at a posttranscriptional phase of the secretory pathway. Additional experiments were conducted to determine whether adiponectin expression was mechanistically similar in other adipose cells. Examination of primary human adipocytes revealed an increase in intracellular HMW isoform with a decline in LMW forms following pioglitazone treatment, with a corresponding increase in the secreted HMW form. Similar results were observed with primary mouse adipocytes, 3T3-F422A cells, and SGBS human adipocyte cells, although differences in the distribution of HMW and LMW isoforms were apparent between cell types. Although there are differences in isoforms between species, in all cases pioglitazone served to increase the secretion of the HMW form of adiponectin.

Role of an arbovirus nonstructural protein in cellular pathogenesis and virus release.

The insect-borne Bluetongue virus (BTV) is considered the prototypic Orbivirus, a member of the Reovirus family. One of the hallmarks of Orbivirus infection is the production of large numbers of intracellular tubular structures of unknown function. For BTV these structures are formed as the polymerization product of a single 64-kDa nonstructural protein, NS1, encoded by the viral double-stranded RNA genome segment 6. Although the NS1 protein is the most abundant viral protein synthesized in infected cells, its function has yet to be determined. One possibility is that NS1 tubules may be involved in the translocation of newly formed viral particles to the plasma membrane, and NS1-specific monoclonal antibodies have been shown to react with viral particles leaving infected cells. In the present study we generated a mammalian cell line that expresses a recombinant single-chain antibody fragment (scFv) derived from an NS1-specific monoclonal antibody (10B1) and analyzed the effect that this intracellular antibody has on BTV replication. Normally, BTV infection of mammalian cells in culture results in a severe cytopathic effect within 24 to 48 h postinfection manifested by cell rounding, apoptosis, and lytic release of virions into the culture medium. However, infection of scFv-expressing cells results in a marked reduction in the stability of NS1 and formation of NS1 tubules, a decrease in cytopathic effect, an increased release of infectious virus into the culture medium, and budding of virions from the plasma membrane. These results suggest that NS1 tubules play a direct role in the cellular pathogenesis and morphogenesis of BTV.