Soraya Utokaparch

Toll-Like Receptor 4-Mediated Activation of p38 Mitogen-Activated Protein Kinase Is a Determinant of Respiratory Virus Entry and Tropism

ABSTRACT Respiratory viruses exert a heavy toll of morbidity and mortality worldwide. Despite this burden there are few specific treatments available for respiratory virus infections. Since many viruses utilize host cell enzymatic machinery such as protein kinases for replication, we determined whether pharmacological inhibition of kinases could, in principle, be used as a broad antiviral strategy for common human respiratory virus infections. A panel of green fluorescent protein (GFP)-expressing recombinant respiratory viruses, including an isolate of H1N1 influenza virus (H1N1/Weiss/43), was used to represent a broad range of virus families responsible for common respiratory infections (Adenoviridae, Paramyxoviridae, Picornaviridae, and Orthomyxoviridae). Kinase inhibitors were screened in a high-throughput assay that detected virus infection in human airway epithelial cells (1HAEo-) using a fluorescent plate reader. Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling was able to significantly inhibit replication by all viruses tested. Therefore, the pathways involved in virus-mediated p38 and extracellular signal-regulated kinase (ERK) MAPK activation were investigated using bronchial epithelial cells and primary fibroblasts derived from MyD88 knockout mouse lungs. Influenza virus, which activated p38 MAPK to approximately 10-fold-greater levels than did respiratory syncytial virus (RSV) in 1HAEo- cells, was internalized about 8-fold faster and more completely than RSV. We show for the first time that p38 MAPK is a determinant of virus infection that is dependent upon MyD88 expression and Toll-like receptor 4 (TLR4) ligation. Imaging of virus-TLR4 interactions showed significant clustering of TLR4 at the site of virus-cell interaction, triggering phosphorylation of downstream targets of p38 MAPK, suggesting the need for a signaling receptor to activate virus internalization.

The Relationship Between Respiratory Viral Loads and Diagnosis in Children Presenting to a Pediatric Hospital Emergency Department

Background: Respiratory viral infections account for a considerable proportion of pediatric emergency room visits. Illnesses range in severity from mild upper respiratory tract infections to serious lower respiratory tract infections (LRTI). The relationship between viral load and specific viruses to clinical diagnosis made by physicians in this setting is poorly understood. Methods: We applied a real-time, quantitative polymerase chain reaction (qPCR) panel for 13 common respiratory viruses to 195 frozen, archival nasopharyngeal aspirate specimens obtained from symptomatic children ≤24 months of age presenting to the emergency room. Mean total viral load and number of viruses per archival nasopharyngeal aspirate specimen were compared between LRTI (n = 70) and non-LRTI (1 or more of upper respiratory tract infection, fever, or cough) (n = 125), as were yield and concordance of qPCR results to viral culture/direct fluorescence assay (DFA). Results: Children with LRTI had significantly increased total viral load and harbored more viruses than the non-LRTI group. Respiratory syncytial virus-A and -B were significantly associated with LRTI, and parainfluenza virus-1 with non-LRTI. Individual loads of parainfluenza virus-2 and human rhinovirus were increased in LRTI versus non-LRTI. Quantitative PCR yielded more viruses (including coinfections, where a “dominant virus” was typically identified) than viral culture/DFA and documented nucleic acid from pathogens not tested by culture/DFA including human rhinovirus; coronaviruses -OC43, -229E, and -NL63; and metapneumovirus. Conclusions: In symptomatic children presenting to the emergency room, total viral load is related to clinical diagnosis; specific viruses are associated with particular clinical diagnoses, and qPCR has a higher yield than other viral diagnostic methods.

A New Role for the Muscle Repair Protein Dysferlin in Endothelial Cell Adhesion and Angiogenesis

Objective—Ferlins are known to regulate plasma membrane repair in muscle cells and are linked to muscular dystrophy and cardiomyopathy. Recently, using proteomic analysis of caveolae/lipid rafts, we reported that endothelial cells (EC) express myoferlin and that it regulates membrane expression of vascular endothelial growth factor receptor 2 (VEGFR-2). The goal of this study was to document the presence of other ferlins in EC. Methods and Results—EC expressed another ferlin, dysferlin, and that in contrast to myoferlin, it did not regulate VEGFR-2 expression levels or downstream signaling (nitric oxide and Erk1/2 phosphorylation). Instead, loss of dysferlin in subconfluent EC resulted in deficient adhesion followed by growth arrest, an effect not observed in confluent EC. In vivo, dysferlin was also detected in intact and diseased blood vessels of rodent and human origin, and angiogenic challenge of dysferlin-null mice resulted in impaired angiogenic response compared with control mice. Mechanistically, loss of dysferlin in cultured EC caused polyubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31), an adhesion molecule essential for angiogenesis. In addition, adenovirus-mediated gene transfer of PECAM-1 rescued the abnormal adhesion of EC caused by dysferlin gene silencing. Conclusion—Our data describe a novel pathway for PECAM-1 regulation and broaden the functional scope of ferlins in angiogenesis and specialized ferlin-selective protein cargo trafficking in vascular settings.

Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo

Angiogenesis consists in the growth of new blood vessels from pre-existing ones. Although anti-angiogenesis interventions have been shown to have therapeutic properties in human diseases such as cancer, their effect is only partial and the identification of novel modulators of angiogenesis is warranted. Recently, we reported the unexpected proteomic identification in endothelial cells (EC) of Myoferlin, a member of the Ferlin family of transmembrane proteins. Ferlins are well known to regulate the fusion of lipid vesicles at the plasma membrane in muscle cells, and we showed that Myoferlin gene knockdown not only decreases lipid vesicle fusion in EC but also attenuates Vascular Endothelial Growth Factor (VEGF) Receptor-2 (VEGFR-2) expression. Herein, we show that Myoferlin gene silencing in cultured EC also results in attenuated expression of a second tyrosine kinase receptor, Tie-2, which is another well-described angiogenic receptor. Most importantly, we provide evidence that delivery of a low-volume Myoferlin siRNA preparation in mouse tissues results in attenuated angiogenesis and edema formation. This provides the first evidence that acute Myoferlin knockdown has anti-angiogenic effects and validates Myoferlin as an anti-angiogenesis target. Furthermore, this supports the unexpected but increasingly accepted concept that proper tyrosine kinase receptors expression at the plasma membrane requires Myoferlin.

Respiratory viral detection and small airway inflammation in lung tissue of patients with stable, mild COPD.

Abstract Background: Viral respiratory tract infections are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). In lung tissue specimens from patients with stable, mild COPD and from control smokers without airflow obstruction, we determined the prevalence and load of nucleic acid from common respiratory viruses and concomitant inflammation of small airways measuring less than 2-mm in diameter. Methods: Frozen lung tissue obtained from patients with stable, mild COPD (n = 20) and control subjects (n = 20) underwent real-time quantitative PCR (qPCR) for 13 respiratory viruses, and quantitative histology for inflammation of small airways. The two groups were compared for viral prevalence and load, and airway inflammation. The relationship between viral load and airway inflammatory cells was also analyzed. Results: Viral nucleic acid were detected in lung tissue of 18/40 (45.0%) of the individuals studied and included seven co-infections that were characterized by a “dominant virus” contributing to most of the total measured viral load. Lung tissue of COPD patients had a significantly higher prevalence of viral nucleic acid (particularly influenza A virus), and increased inflammation of small airways by macrophages and neutrophils versus controls. In qPCR-positive individuals, linear regression analysis showed a direct correlation between viral load and airway neutrophils, and between influenza A virus load and airway macrophages. Conclusion: The lung tissue of patients with stable, mild COPD has a higher prevalence and load of respiratory viruses versus non-obstructed control subjects, and increased inflammation of small airways. Respiratory viruses may represent potential targets in COPD patient management.

Loss of GD1-positive Lactobacillus correlates with inflammation in human lungs with COPD

Objectives The present study assesses the relationship between contents of GD1 (glycerol dehydratase)-positive Lactobacillus, presence of Lactobacillus and the inflammatory response measured in host lung tissue in mild to moderate chronic obstructive pulmonary disease (COPD). We hypothesise that there will be a loss of GD1 producing Lactobacillus with increasing severity of COPD and that GD1 has anti-inflammatory properties. Setting Secondary care, 1 participating centre in Vancouver, British Columbia, Canada. Participants 74 individuals who donated non-cancerous portions of their lungs or lobes removed as treatment for lung cancer (normal lung function controls (n=28), persons with mild (GOLD 1) (n=21) and moderate (GOLD 2) COPD (n=25)). Outcome measures Primary outcome measure was GD1 positivity within each group and whether or not this impacted quantitative histological measures of lung inflammation. Secondary outcome measures included Lactobacillus presence and quantification, and quantitative histological measurements of inflammation and remodelling in early COPD. Results Total bacterial count (p>0.05) and prevalence of Lactobacillus (p>0.05) did not differ between groups. However, the GD1 gene was detected more frequently in the controls (14%) than in either mild (5%) or moderate (0%) COPD (p<0.05) samples. Macrophage and neutrophil volume fractions (0.012±0.005 (mean±SD) vs 0.026±0.017 and 0.005±0.002 vs 0.015±0.014, respectively) in peripheral lung tissue were reduced in samples positive for the GD1 gene (p<0.0035). Conclusions A reduction in GD1 positivity is associated with an increased tissue immune inflammatory response in early stage COPD. There is potential for Lactobacillus to be used as a possible therapeutic, however, validation of these results need to be completed before an anti-inflammatory role of Lactobacillus in COPD can be confirmed.

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.