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.     

The N-glycosylation defect of cwh8Delta yeast cells causes a distinct defect in sphingolipid biosynthesis

CWH8/YGR036c of Saccharomyces cerevisiae has been identifiedas a dolichylpyrophosphate (Dol-PP) phosphatase that removesa phosphate from the Dol-PP generated by the oligosaccharyltransferase(OST), while it adds N-glycans to nascent glycoproteins in theendoplasmic reticulum (ER). Lack of CWH8 was proposed to interruptthe so called dolichol (Dol) cycle by trapping Dol in the formof Dol-PP in the ER lumen. Indeed, cwh8D mutants display a severedeficiency in N-glycosylation. We find that cwh8D mutants havestrongly reduced levels of inositolphosphorylceramide (IPC),whereas its derivative, mannosyl-(inositol-P)₂-ceramide (M(IP)₂C)is not affected. Microsomes of cwh8D contain normal ceramidesynthase and IPC synthesis activities. Within a large panelof mutants affecting Dol dependent pathways such as N- or O-glycosylation,or glycosylphosphatidyl inositol (GPI)-anchoring, only the mutantshaving a deficiency of N-glycan addition show the defect inIPC biosynthesis. By mutating genes required for the additionof N-glycans or by treating cells with tunicamycin (Tm) onecan similarly reduce the steady state level of IPC and exactlyreproduce the phenotype of cwh8D cells. Some potential mechanismsby which the lack of N-glycans could lead to the sphingolipidabnormality were further explored.     

Dragon (repulsive guidance molecule b) inhibits IL-6 expression in macrophages

Despite the strong interest in the NK cell-mediated immunity toward malignant cells and viruses, there is a relative lack of data on the interplay between NK cells and filamentous fungi, especially Aspergillus fumigatus, which is the major cause of invasive aspergillosis. By studying the in vitro interaction between human NK cells and A. fumigatus, we found only germinated morphologies to be highly immunogenic, able to induce a Th1-like response, and capable of upregulating cytokines such as IFN-γ and TNF-α. Moreover, priming NK cells with human rIL-2 and stimulating NK cells by direct NK cell-pathogen contact were essential to induce damage against A. fumigatus. However, the most interesting finding was that NK cells did not mediate anti-Aspergillus cytotoxicity through degranulation of their cytotoxic proteins (perforin, granzymes, granulysine), but via an alternative mechanism involving soluble factor(s). To our knowledge, our study is the first to demonstrate that IFN-γ, released by NK cells, directly damages A. fumigatus, attributing new properties to both human NK cells and IFN-γ and suggesting them as possible therapeutic tools against IA.     

Catheter-related infections

Nosocomial infections are a leading cause of morbidity and mortality among hospitalized patients. These infections have made newspaper headlines recently in many countries, and both patients and their relatives are now perfectly aware of their existence and of the risks which are inherent to any medical activity. However, significant improvements in the knowledge of the pathophysiology and epidemiology of nosocomial infections allow us to prevent them efficiently. Accordingly, they should no longer be considered as an inevitable tribute to pay to the continuous progress of medicine, but as a real challenge in the process of improving the quality of patient care. This is particularly the case for bloodstream infections, of which at least 80% are considered to be catheter associated. This paper reviews the epidemiology and impact of infections associated with the use of intravenous catheters. Principles of therapy are reviewed, as well as major aspects of prevention.     

Efficacy of a New Educational Tool to Improve Handrubbing Technique amongst Healthcare Workers: A Controlled,Before-After Study

Introduction

Hand hygiene is a key component of infection control in healthcare. WHO recommends that healthcare workers perform six specific poses during each hand hygiene action. SureWash (Glanta Ltd, Dublin, Ireland) is a novel device that uses video-measurement technology and immediate feedback to teach this technique. We assessed the impact of self-directed SureWash use on healthcare worker hand hygiene technique and evaluated the device''s diagnostic capacity.

Methods

A controlled before-after study: subjects in Group A were exposed to the SureWash for four weeks followed by Group B for 12 weeks. Each subject''s hand hygiene technique was assessed by blinded observers at baseline (T₀) and following intervention periods (T₁ and T₂). Primary outcome was performance of a complete hand hygiene action, requiring all six poses during an action lasting ≥20 seconds. The number of poses per hand hygiene action (maximum 6) was assessed in a post-hoc analysis. SureWash''s diagnostic capacity compared to human observers was assessed using ROC curve analysis.

Results

Thirty-four and 29 healthcare workers were recruited to groups A and B, respectively. No participants performed a complete action at baseline. At T₁, one Group A participant and no Group B participants performed a complete action. At baseline, the median number of poses performed per action was 2.0 and 1.0 in Groups A and B, respectively (p = 0.12). At T₁, the number of poses per action was greater in Group A (post-intervention) than Group B (control): median 3.8 and 2.0, respectively (p<0.001). In Group A, the number of poses performed twelve weeks post-intervention (median 3.0) remained higher than baseline (p<0.001). The area under the ROC curves for the 6 poses ranged from 0.59 to 0.88.

Discussion

While no impact on complete actions was demonstrated, SureWash significantly increased the number of poses per hand hygiene action and demonstrated good diagnostic capacity.     

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.