Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Clinical Utility of Waist Circumference in Predicting All‐cause Mortality in a Preventive Cardiology Clinic Population: A PreCIS Database Study

Although obesity is a risk factor for mortality, it is unclear whether waist circumference (WC) is a better predictor of mortality than BMI in a clinical setting of patients at high risk for coronary artery disease (CAD). Thus, we compared the association between WC and BMI with all‐cause mortality in relation to traditional CAD risk factors in a high‐risk cohort. Study population included 5,453 consecutive new patients seen between 1996 and 2005 for management of CAD risk factors in a preventive cardiology clinic. Mortality was determined from the Social Security Death Index. There were 359 deaths over a median follow‐up of 5.2 years. Mortality was greater in high (>102 cm in men and >88 cm in women) vs. normal WC in both genders (P < 0.01). The unadjusted Cox proportional hazard ratio (HR) for continuous WC (per cm) was 1.02 (P < 0.001) in both genders and remained significant after adjustment for CAD risk factors (HR = 1.01 in men, HR = 1.03 in women, both P < 0.05). BMI did not associate statistically with mortality. WC associated with diabetes mellitus (DM) and CAD prevalence (P < 0.001). BMI associated only with DM (P < 0.001) and this association disappeared when WC was added to the model. We conclude that WC is an independent predictor of all‐cause mortality in a preventive cardiology population. These data affirm the clinical importance of WC measurements for mortality, DM, and CAD risk prediction and suggest that obesity‐specific interventions targeting WC in addition to traditional risk factor management may favorably impact these outcomes.     

Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p

We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to contact the ligand directly. Instead, these mutations alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor-agonist interactions involve at least two sites, of which only one is specific for the activated conformation of the receptor.     

Nutrition and somatomedin--XII. Fractionation of somatomedins and somatomedin inhibitors in normal and diabetic rats

Bioassayable somatomedins and somatomedin inhibitors were examined after chromatographic separation, using serum from normal rats (enriched in somatomedins) and diabetic rats (enriched in somatomedin inhibitors). At neutral pH, gel filtration on Sephacryl S-300 revealed somatomedins at mol. wt approximately 140,000 (presumably carrier-bound) and inhibitors at mol. wts approximately 250,000, approximately 24,000 and approximately 1,000. At acid pH, gel filtration on Sephadex G-50 revealed somatomedins at mol. wt approximately 8,000 (presumably carrier-free) and a single inhibitor at mol. wt approximately 21,000. Ion exchange chromatography revealed that the inhibitor(s) may be more acidic than the somatomedins, but only low quantities of somatomedins were recovered. Sephadex G-50 fractionation was applied to pathophysiologic models in rats: 3 days of fasting were associated with a 62% fall in somatomedins and a 159% rise in inhibitors; 2 days of diabetes were associated with a 60% fall in somatomedins and a 344% rise in inhibitors. Since chromatography on Sephadex G-50 at pH 2.4 appears to provide adequate separation of somatomedins and somatomedin inhibitors with good estimated recovery of biological activity, this simple approach may be a probe useful in examining the regulation of somatomedins and somatomedin inhibitors in vivo.     

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

Extra precision glide docking,free energy calculation and molecular dynamics studies of 1,2-diarylethane derivatives as potent urease inhibitors

For the latter half of the twentieth century, most medical professionals considered bacterial infection to be a primary cause of gastrointestinal ulcers in human beings. In 1994, the World Health Organization (WHO) recognized Helicobacter pylori, the bacterium most closely linked to ulcer development, as a type I carcinogen. Biological research has shown that there is a positive correlation between the number of species in the Helicobacter genus and the number of medical conditions associated with Helicobacter infection, both of which are increasing rapidly. N-Benzylaniline derivatives, frequently used in industrial manufacturing, are being considered as a strong candidate for ongoing drug modeling in search of novel therapies. The basic goal behind this study was to determine the potency of experimentally proved data, and to determine favorable substituents to enhance potency, and thereafter to support this finding through theoretical modification of the existing base skeleton by addition of suitable substituents. Ligands were investigated thoroughly by paying attention to the urease-inhibitory properties present in the selected series. Initially, docking was performed on ligands with protein to produce efficient docking poses. Molecular dynamics (MD) simulations were also performed to precisely understand the interactions between ligands and proteins. Thereafter, MM-GBSA was used in order to validate the methods and results. Good interaction was observed with amino acids Arg338, Ala169, Asp223, His322, and Asn168. This study also revealed that the electron rich hydroxyl group (–OH) substituent plays an important role during bond formation. In addition, various hydrogen bonds, ionic bonds, and pi–pi stacking bonds make significant contributions towards urease inhibition. Therefore, further research utilizing electron-rich moieties may lead to novel and efficacious urease inhibitors.     

KNL1 Binding to PP1 and Microtubules Is Mutually Exclusive

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