Rationale: Infection with rhinovirus (RV) triggers exacerbations of asthma and chronic obstructive lung disease. Objectives: We sought to develop a mouse model of RV employing RV1B, a minor group serotype that binds to the low-density lipoprotein receptor. Methods: C57BL/6 mice were inoculated intranasally with RV1B, replication-deficient ultraviolet (UV)-irradiated RV1B, or RV39, a major group virus. Measurements and Main Results: Viral RNA was present in the lungs of RV1B-treated mice, but not in those exposed to UV-irradiated RV1B or RV39. Lung homogenates of RV-treated mice contained infectious RV 4 days after inoculation. RV1B exposure induced neutrophilic and lymphocytic airway inflammation, as well as increased lung expression of KC, macrophage-inflammatory protein-2, and IFN-a and IFNb. RV1B-exposed mice showed airway hyperresponsiveness 1 and 4 days after inoculation. UV-irradiated RV1B induced modest neutrophilic airway inflammation and hyperresponsiveness 1 day after exposure. Both RV1B and UV-irradiated RV1B, but not RV39, increased lung phosphorylation of Akt. Confocal immunofluorescence showed colocalization of RV1B and phospho-Akt in the airway epithelium. Finally, pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 attenuated chemokine production and neutrophil infiltration. Conclusions: We conclude that RV1B induces airway inflammation in vivo. Evidence is presented that viral replication occurs in vivo and is required for maximal responses. On the other hand, viral replication was not required for a subset of RV-induced responses, including neutrophilic inflammation, airway hyperresponsiveness, and Akt phosphorylation. Finally, phosphatidylinositol 3-kinase/Akt signaling is required for maximal RV1B-induced airway neutrophilic inflammation, likely via its essential role in virus internalization.Keywords: asthma; chronic obstructive pulmonary disease; Akt; low-density lipoprotein receptor Viral infections trigger nearly 80% of asthma exacerbations, and rhinovirus (RV) accounts for the majority of virus-induced exacerbations (1, 2). RV also accounts for a substantial percentage of chronic obstructive pulmonary disease (COPD) exacerbations (3, 4). Understanding of RV-induced exacerbations is incomplete, in part because of the absence of an animal model. Rhinovirus RNA has been detected by polymerase chain reaction (PCR) analysis in lower airway cells from volunteers experimentally infected with RV16 (5, 6) and RV capsid protein has been found in airway epithelial cells, albeit sporadically (6). However, RV has not been cultured from the lower airways of immunocompetent subjects, and therefore the extent to which RV infects or replicates in the lower airways of humans remains unclear.RV, a member of the Picornaviridae family of viruses, is responsible for the majority of common colds. The virus is composed of an icosahedral protein capsid and a positive, singlestranded RNA genome. More than 100 serotypes of RV have been identified. These are divided into two groups on the basis o...
Together, these data demonstrate that tracheal aspirate fluid from premature, mechanically ventilated infants contains fibroblasts with cell markers and differentiation potential typically found in mesenchymal stem cells.
The Vectra platform is an advanced multispectral imaging system for biomarker quantitation in tissue microarray (TMA) or intact tissue sections. This is the first study to validate its reliability for quantitating spatially overlapping biomarkers using chromogenic multiplexed immunohistochemistry (IHC) on prostate TMAs. Two TMA cohorts (outcome TMA [oTMA]; progression TMA [pTMA]) were used. The oTMA cohort consists of 462 duplicate cores from 183 prostate cancer (PCa) tissues and 48 benign prostate tissues (BPT) with longer than 5-year outcome information. The pTMA cohort consists of 384 duplicate cores from different disease groups: 43 localized PCa (PCa_local, pT2), 30 aggressive PCa (PCa_aggr, pT3 and 4), 22 metastatic PCa (Met), 25 high-grade prostatic intraepithelial neoplasia (HGPIN), and 48 BPT and 24 benign prostate hyperplasia (BPH). Three oTMAs were stained with AR+ ERG+ E-cadherin (oTMA1), AR+E-cadherin (oTMA2), ERG+E-cadherin (oTMA3), respectively. One pTMA section was stained with E-cadherin and AMACR. TMA slides were then scanned with the Vectra platform. Biomarker expression analysis was performed with Vectra software, Nuance and inForm. IBM SPSS Statistics 19 was used for statistical and correlation analysis. Close concordance was found between the triple and double immunostaining assays used for quantitating spatially overlapping biomarkers AR and ERG using oTMAs (r = 0.897 for AR and r = 0.613 for ERG). Our quantitative results from pTMA cohort showed AMACR expression was significantly increased in HGPIN and PCa compared with benign prostate tissue(P ≤ .001), whereas decreased E-cadherin expression correlated with PCa progression. These results are consistent with previously published data by other groups. Vectra technology is reliable for objective and high-throughput biomarker quantitation and colocalization study using chromogenic multiplexed IHC.
Human papilloma virus (HPV) related oropharyngeal squamous cell carcinoma (OPSCC) has favorable prognosis relative to other head and neck squamous cell carcinomas. Criteria for predicting HPV status based upon p16 staining, including difficult cases with partial staining patterns, have been developed; however, clinical validation of these criteria and the clinical significance of partial p16 staining have not been reported. 81 archival OPSCC cases were initially stained for p16 by immunohistochemistry with clone G175-405. The percentage of p16+ cells and percentage of confluence of p16+ cells were categorized as 25%, 26-75% or >75%. Of all cases, 16 (20%) had partial p16 expression, with 26-75% p16+ cells. Applying previously developed criteria of >75% p16+ cells or >50% positive cells with >25% confluence, 48 (59%) patients were categorized p16+ and demonstrated expected clinical characteristics and superior disease-free survival (DFS) and overall survival (p < 0.001) compared to p16- patients. By themselves, the partial staining patients had intermediate outcomes however, separating the partial staining cases by degree of confluence showed those with >75% confluence had superior DFS (p = 0.042). When the 16 original partial staining cases were re-stained with the alternative anti-p16 E6H4 clone, p16 status remained concordant for all cases, but only 3 of the 16 were interpreted as demonstrating partial staining. This report shows the prevalence of partial p16 staining varies with the antibody utilized and clinically validates the application of a graded evaluation of both the number as well as confluence of positive cells for risk-stratification of patients with OPSCC.
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