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.     

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.     

The effects of antifungal substances on some zoosporic fungi (Kingdom Fungi)

Zoosporic fungi constitute a large group of true fungi which inhabit freshwater, brackish, marine and soil ecosystems. In general, very little is known about the effects of antifungal substances on the growth and survival of most species. This review focuses on experimental research with those isolates which have been studied, especially in some species of Synchytrium, Olpidium, Batrachochytrium, Allomyces, Blastocladiella, Neocallimastix. These genera represent genetically diverse groups. Although the research discussed here is restricted to a small sample, some general conclusions can be reached about zoosporic fungi as a whole. Like many other eukaryotic microorganisms, zoosporic fungi are sensitive to a large number of antibiotics, fungicides, surfactants, bacterial metabolites, metabolic poisons, proteins, heavy metals and other antifungal substances. These include substances commonly released into the environment for the control of plant and animal diseases, for increasing production of domestic animals and in the form of waste products from industry. It is possible that the release of antifungal substances into the environment might cause significant changes in the community structure of zoosporic fungi as well as of other groups of microorganisms which play significant roles in food web dynamics and ecosystem complexity. However, this needs documentation by quantitative studies. For these reasons, extensive research on the effects of antifungal substances is much needed.     

Comorbidities and Crash Involvement among Younger and Older Drivers

Previous studies identified comorbidities as predictors of older driver performance and driving pattern, while the direct impact of comorbidities on road crash risk in elderly drivers is still unknown. The present study is a cross-sectional aimed at investigating the association between levels of comorbidity and crash involvement in adult and elderly drivers. 327 drivers were stratified according to age range in two groups: elderly drivers (age ≥70 years old, referred as older) and adult drivers (age <70 years old, referred as younger). Driving information was obtained through a driving questionnaire. Distance traveled was categorized into low, medium and high on the basis of kilometers driven in a year. CIRS-illness severity (IS) and CIRS-comorbidity indices (CI) in all populations were calculated. Older drivers had a significantly higher crash involvements rate (p = .045) compared with the younger group based on the number of licensed drivers. Dividing comorbidity indices into tertiles among all licensed subjects, the number of current drivers significantly decreased (p<.0001) with increasing level of comorbidity. The number of current drivers among older subjects significantly decreased with increasing comorbidity level (p = .026) while no difference among younger group was found (p = .462). Among younger drivers with increasing comorbidity level, the number of road accidents significantly increased (p = .048) and the logistic regression analysis showed that comorbidity level significantly associated with crash involvement independent of gender and driving exposure. Older subjects with high level of comorbidity are able to self-regulate driving while comorbidity burden represents a significant risk factor for crash involvements among younger drivers.     

New phylogenetic insights into <Emphasis Type="Italic">Saprolegniales</Emphasis> (Oomycota,Straminipila) based upon studies of specimens isolated from Brazil and Argentina

Saprolegniales is a complex and monophyletic order of oomycetes. Their members inhabit terrestrial, freshwater, and marine ecosystems and have a worldwide distribution. In these ecosystems, they are found as saprobes, parasites, or even pathogens of animals and plants of economic importance. In this study, a concatenate phylogeny of the partial LSU and complete ITS rDNA regions is presented, including isolates from Brazil and Argentina, which were sequenced after a detailed morphological analysis. Among the sequenced species, Achlya orion, Leptolegnia eccentrica, Phragmosporangium uniseriatum, and Pythiopsis irregularis are included for the first time in a phylogeny. Our results are in agreement with the recent informal proposals outlined in taxonomic overviews of the Oomycota of G.W. Beakes and collaborators, who placed the family Verrucalvaceae into the Saprolegniales and introduced the family Achlyaceae to group Achlya s.s., Brevilegnia, Dictyuchus, and Thraustotheca. These results also support the transference of Achlya androgyna to Newbya. Leptolegnia appears as paraphyletic, with the separation of L. eccentrica from the other species of this genus. In addition, Phragmosporangium, which is herein sequenced for the first time, clustered as sister to some species of Aphanomyces, including the type species, A. stellatus.     

Controlled synthesis of the DSF cell-cell signal is required for biofilm formation and virulence in Xanthomonas campestris

Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell-cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence.     

Discovery of a novel control element within the 5'-untranslated region of the vascular endothelial growth factor. Regulation of expression using sense oligonucleotides

The regulation of vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis, is controlled primarily through the interactions of control elements located within the 5'- and 3'-untranslated regions, many of which are yet to be described. In this study we examined the 5'-untranslated region of human VEGF for control elements with the aim of regulating expression both in vitro and in vivo using oligonucleotide gene therapy. A potential control element was located, two sense oligonucleotides (S(1) and S(2)) were designed based on its sequence, and a third oligonucleotide (S(3)) was designed as a control and mapped to the 16 base pairs immediately upstream. Retinal cells cultured in the presence of S(1) and S(2) resulted in a 2-fold increase of VEGF protein and a 1.5-fold increase in mRNA 24 h post-transfection whereas S(3) had no significant effect (p > 0.05) compared with controls. Subsequent reporter gene studies confirmed the necessity of this element for up-regulation by S(1). Further in vivo studies showed that S(1) and S(2) mediated an increase in VEGF protein in a rodent ocular model that resulted in angiogenesis. In addition to providing insight into the regulation of the vascular endothelial growth factor, the use of these oligonucleotides to stimulate vascular growth may prove useful for the treatment of ischemic tissues such as those found in the heart following infarct.