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Rationale: Patients with chronic obstructive pulmonary disease (COPD) are susceptible to respiratory viral infections that cause exacerbations. The mechanisms underlying this susceptibility are not understood. Effectors of the adaptive immune response-CD8(+) T cells that clear viral infections-are present in increased numbers in the lungs of patients with COPD, but they fail to protect against infection and may contribute to the immunopathology of the disease., Objectives: CD8(+) function and signaling through the programmed cell death protein (PD)-1 exhaustion pathway were investigated as a potential key mechanism of viral exacerbation of the COPD lung., Methods: Tissue from control subjects and patients with COPD undergoing lung resection was infected with live influenza virus ex vivo. Viral infection and expression of lung cell markers were analyzed using flow cytometry., Measurements and Main Results: The proportion of lung CD8(+) T cells expressing PD-1 was greater in COPD (mean, 16.2%) than in controls (4.4%, P = 0.029). Only epithelial cells and macrophages were infected with influenza, and there was no difference in the proportion of infected cells between controls and COPD. Infection up-regulated T-cell PD-1 expression in control and COPD samples. Concurrently, influenza significantly up-regulated the marker of cytotoxic degranulation (CD107a) on CD8(+) T cells (P = 0.03) from control subjects but not on those from patients with COPD. Virus-induced expression of the ligand PD-L1 was decreased on COPD macrophages (P = 0.04) with a corresponding increase in IFN-γ release from infected COPD explants compared with controls (P = 0.04)., Conclusions: This study has established a signal of cytotoxic immune dysfunction and aberrant immune regulation in the COPD lung that may explain both the susceptibility to viral infection and the excessive inflammation associated with exacerbations.

Unlabelled: Equine hepacivirus (EHCV; nonprimate hepacivirus) is a hepatotropic member of the Flaviviridae family that infects horses. Although EHCV is the closest known relative to hepatitis C virus (HCV), its complete replication kinetics in vivo have not been described, and direct evidence that it causes hepatitis has been lacking. In this study, we detected EHCV in 2 horses that developed post-transfusion hepatitis. Plasma and serum from these horses were used to experimentally transmit EHCV to 4 young adult Arabian horses, two 1-month-old foals (1 Arabian and 1 Arabian-pony cross), and 2 foals (1 Arabian and 1 Arabian-pony cross) with severe combined immunodeficiency (SCID). Our results demonstrated that EHCV had infection kinetics similar to HCV and that infection was associated with acute and chronic liver disease as measured by elevations of liver-specific enzymes and/or by histopathology. Although most of these animals were coinfected with equine pegivirus (EPgV), also a flavivirus, EPgV viral loads were much lower and often undetectable in both liver and blood. Three additional young adult Arabian-pony crosses and 1 SCID foal were then inoculated with plasma containing only EHCV, and evidence of mild hepatocellular damage was observed. The different levels of liver-specific enzyme elevation, hepatic inflammation, and duration of viremia observed during EHCV infection suggested that the magnitude and course of liver disease was mediated by the virus inoculum and/or by host factors, including breed, age, and adaptive immune status., Conclusion: This work documents the complete infection kinetics and liver pathology associated with acute and chronic EHCV infection in horses and further justifies it as a large animal model for HCV., (© 2015 by the American Association for the Study of Liver Diseases.)

Background: The number and complexity of clinical laboratory tests is rapidly expanding, presenting primary care physicians with challenges in accurately, efficiently, and safely ordering and interpreting diagnostic tests. The objective of this study was to identify challenges primary care physicians face related to diagnostic laboratory testing and solutions they believe are helpful and available to them., Methods: In this study, sponsored by the Centers for Disease Control and Prevention, a random sample of general internal medicine and family medicine physicians from the American Medical Association Masterfile were surveyed in 2011., Results: 1768 physicians (5.6%) responded to the survey. Physicians reported ordering diagnostic laboratory tests for an average of 31.4% of patient encounters per week. They reported uncertainty about ordering tests in 14.7% and uncertainty in interpreting results in 8.3% of these diagnostic encounters. The most common problematic challenges in ordering tests were related to the cost to patients and insurance coverage restrictions. Other challenges included different names for the same test, tests not available except as part of a test panel, and different tests included in panels with the same names. The most common problematic challenges in interpreting and using test results were not receiving the results and confusing report formats. Respondents endorsed a variety of information technology and decision support solutions to improve test selection and results interpretation, but these solutions were not widely available at the time of the survey. Physicians infrequently sought assistance or consultation from laboratory professionals but valued these consultations when they occurred., Conclusions: Primary care physicians routinely experience uncertainty and challenges in ordering and interpreting diagnostic laboratory tests. With more than 500 million primary care patient visits per year, the level of uncertainty reported in this study potentially affects 23 million patients per year and raises significant concerns about the safe and efficient use of laboratory testing resources. Improvement in information technology and clinical decision support systems and quick access to laboratory consultations may reduce physicians' uncertainty and mitigate these challenges.

A 2-year-old, spayed female, German short-haired pointer was presented with a 1-day history of non-ambulatory paraplegia with absent deep pain perception. A computed tomography scan revealed an irregular eighth thoracic vertebral body and an extradural compressive lesion. Decompression was performed and abnormal tissues were submitted for analysis. Findings were consistent with a Cryptococcus gattii infection.

Protective immunity against influenza virus infection is mediated by neutralizing antibodies, but the precise role of T cells in human influenza immunity is uncertain. We conducted influenza infection studies in healthy volunteers with no detectable antibodies to the challenge viruses H3N2 or H1N1. We mapped T cell responses to influenza before and during infection. We found a large increase in influenza-specific T cell responses by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. Preexisting CD4+, but not CD8+, T cells responding to influenza internal proteins were associated with lower virus shedding and less severe illness. These CD4+ cells also responded to pandemic H1N1 (A/CA/07/2009) peptides and showed evidence of cytotoxic activity. These cells are an important statistical correlate of homotypic and heterotypic response and may limit severity of influenza infection by new strains in the absence of specific antibody responses. Our results provide information that may aid the design of future vaccines against emerging influenza strains.

Rationale: Respiratory syncytial virus (RSV) is the leading cause of childhood lower respiratory infection, yet viable therapies are lacking. Two major challenges have stalled antiviral development: ethical difficulties in performing pediatric proof-of-concept studies and the prevailing concept that the disease is immune-mediated rather than being driven by viral load., Objectives: The development of a human experimental wild-type RSV infection model to address these challenges., Methods: Healthy volunteers (n = 35), in five cohorts, received increasing quantities (3.0-5.4 log plaque-forming units/person) of wild-type RSV-A intranasally., Measurements and Main Results: Overall, 77% of volunteers consistently shed virus. Infection rate, viral loads, disease severity, and safety were similar between cohorts and were unrelated to quantity of RSV received. Symptoms began near the time of initial viral detection, peaked in severity near when viral load peaked, and subsided as viral loads (measured by real-time polymerase chain reaction) slowly declined. Viral loads correlated significantly with intranasal proinflammatory cytokine concentrations (IL-6 and IL-8). Increased viral load correlated consistently with increases in multiple different disease measurements (symptoms, physical examination, and amount of nasal mucus)., Conclusions: Viral load appears to drive disease manifestations in humans with RSV infection. The observed parallel viral and disease kinetics support a potential clinical benefit of RSV antivirals. This reproducible model facilitates the development of future RSV therapeutics.

Rationale: Frequent chronic obstructive pulmonary disease (COPD) exacerbations are a major cause of hospital admission and mortality and are associated with increased airway inflammation. Macrolides have airway antiinflammatory actions and may reduce the incidence of COPD exacerbations., Objectives: To determine whether regular therapy with macrolides reduces exacerbation frequency., Methods: We performed a randomized, double-blind, placebo-controlled study of erythromycin administered at 250 mg twice daily to patients with COPD over 12 months, with primary outcome variable being the number of moderate and/or severe exacerbations (treated with systemic steroids, treated with antibiotics, or hospitalized)., Measurements and Main Results: We randomized 109 outpatients: 69 (63%) males, 52 (48%) current smokers, mean (SD) age 67.2 (8.6) years, FEV1 1.32 (0.53) L, FEV1% predicted 50 (18)%. Thirty-eight (35%) of the patients had three or more exacerbations in the year before recruitment, with no differences between treatment groups. There were a total of 206 moderate to severe exacerbations: 125 occurred in the placebo arm. Ten in the placebo group and nine in the macrolide group withdrew. Generalized linear modeling showed that the rate ratio for exacerbations for the macrolide-treated patients compared with placebo-treated patients was 0.648 (95% confidence interval: 0.489, 0.859; P = 0.003) and that these patients had shorter duration exacerbations compared with placebo. There were no differences between the macrolide and placebo arms in terms of stable FEV1, sputum IL-6, IL-8, myeloperoxidase, bacterial flora, serum C-reactive protein, or serum IL-6 or in changes in these parameters from baseline to first exacerbation over the 1-year study period., Conclusions: Macrolide therapy was associated with a significant reduction in exacerbations compared with placebo and may be useful in decreasing the excessive disease burden in this important patient population. Clinical trial registered with www.clinicaltrials.gov (NCT 00147667).

Chronic obstructive pulmonary disease (COPD) is not only an established major cause of mortality and morbidity but is increasing in worldwide prevalence despite current medical interventions. The natural history of COPD is punctuated by periods of acute symptomatic, physiologic, and functional deterioration or exacerbations. These events are responsible for considerable additional morbidity and mortality and impact on patients' long-term health status. Despite advances in understanding disease mechanisms and in treatment, exacerbations continue to be the major cause of COPD-associated hospitalization, and provision for their management incurs considerable health care costs. Although pharmacologic therapies may improve clinical outcomes, these benefits must be optimized by prompt diagnosis and delivery. This will require improved understanding of this complex disease by physicians and patients alike.

The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Weaned plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls., (Copyright 2003 Annals of Botany Company)