Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases

<p>Abstract</p> <p>Background</p> <p>Three distinct forms of bovine spongiform encephalopathy (BSE), defined as classical (C-), low (L-) or high (H-) type, have been detected through ongoing active and passive surveillance systems for the disease.</p> <p>The aim of the present study was to compare the ability of two sets of immunohistochemical (IHC) and Western blot (WB) BSE confirmatory protocols to detect C- and atypical (L- and H-type) BSE forms.</p> <p>Obex samples from cases of United States and Italian C-type BSE, a U.S. H-type and an Italian L-type BSE case were tested in parallel using the two IHC sets and WB methods.</p> <p>Results</p> <p>The two IHC techniques proved equivalent in identifying and differentiating between C-type, L-type and H-type BSE. The IHC protocols appeared consistent in the identification of PrP^Scdistribution and deposition patterns in relation to the BSE type examined. Both IHC methods evidenced three distinct PrP^Scphenotypes for each type of BSE: prevailing granular and linear tracts pattern in the C-type; intraglial and intraneuronal deposits in the H-type; plaques in the L-type.</p> <p>Also, the two techniques gave comparable results for PrP^Scstaining intensity on the C- and L-type BSE samples, whereas a higher amount of intraglial and intraneuronal PrP^Scdeposition on the H-type BSE case was revealed by the method based on a stronger demasking step.</p> <p>Both WB methods were consistent in identifying classical and atypical BSE forms and in differentiating the specific PrP^Scmolecular weight and glycoform ratios of each form.</p> <p>Conclusions</p> <p>The study showed that the IHC and WB BSE confirmatory methods were equally able to recognize C-, L- and H-type BSE forms and to discriminate between their different immunohistochemical and molecular phenotypes. Of note is that for the first time one of the two sets of BSE confirmatory protocols proved effective in identifying the L-type BSE form. This finding helps to validate the suitability of the BSE confirmatory tests for BSE surveillance currently in place.</p

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Vaccination of Pigs against Swine Influenza Viruses by Using an NS1-Truncated Modified Live-Virus Vaccine

Swine influenza viruses (SIV) naturally infect pigs and can be transmitted to humans. In the pig, genetic reassortment to create novel influenza subtypes by mixing avian, human, and swine influenza viruses is possible. An SIV vaccine inducing cross-protective immunity between different subtypes and strains circulating in pigs is highly desirable. Previously, we have shown that an H3N2 SIV (A/swine/Texas/4199-2/98 [TX98]) containing a deleted NS1 gene expressing a truncated NS1 protein of 126 amino acids, NS1▴126, was attenuated in swine. In this study, 4-week-old pigs were vaccinated with the TX98 NS1▴126 modified live virus (MLV). Ten days after boosting, pigs were challenged with wild-type homologous H3N2 or heterosubtypic H1N1 SIV and sacrificed 5 days later. The MLV was highly attenuated and completely protected against challenge with the homologous virus. Vaccinated pigs challenged with the heterosubtypic H1N1 virus demonstrated macroscopic lung lesions similar to those of the unvaccinated H1N1 control pigs. Remarkably, vaccinated pigs challenged with the H1N1 SIV had significantly lower microscopic lung lesions and less virus shedding from the respiratory tract than did unvaccinated, H1N1-challenged pigs. All vaccinated pigs developed significant levels of hemagglutination inhibition and enzyme-linked immunosorbent assay titers in serum and mucosal immunoglobulin A antibodies against H3N2 SIV antigens. Vaccinated pigs were seronegative for NS1, indicating the potential use of the TX98 NS1▴126 MLV as a vaccine to differentiate infected from vaccinated animals

Body weight, food and water disappearance, and illness score of mice in 5-day high viral titer inoculation study.

<p>Mice were inoculated intranasally with H1N1 influenza virus (10^7.9 EID₅₀) on day 0 and gavaged with 5% ethanol vehicle control from day -1 to day 4 PI (N=15), or inoculated with virus on day 0 and gavaged with 110 mg/kg <i>H</i>. <i>perforatum</i> extract (N=15), or not inoculated with virus and gavaged with 5% ethanol (N=6). Daily records of body weight (from day -1 to day 5)(A), food (B) and water disappearance (C)(from day -1 to day 4), as well as mouse illness score (D)(from day 1 to day 5) are shown as Mean ± SEM. Significant difference between non-inoculated and inoculated groups is labeled with * (p<0.05), while difference between inoculated vehicle and inoculated <i>H</i>. <i>perforatum</i> group is highlighted with # (p<0.05).</p

RNA transcription profiles the lung of BALB/c mice.

<p>Expression levels of SOCS3 relative to GAPDH in lungs collected at the end of the 5-day high viral titer inoculation study (10^7.9 EID₅₀, N=15 for inoculated groups and N=6 for non-inoculated group) and 10-day low viral tier inoculation study (10^5.1 EID₅₀, N=13 for each group) were measured and shown as log relative transcription level (Mean ± SEM). Values at the same time point are differentiated by individual over bar labels when significant differences were found (p<0.05). ND=not determined (no non-inoculated group in the 10 day study).</p

Mouse BAL cell population in 5-day high viral titer inoculation study.

<p>Mice were intranasally inoculated with H1N1 (10^7.9 EID₅₀) influenza virus on day 0 and gavaged with 5% ethanol vehicle control from day -1 to day 4 PI (N=15), or inoculated with virus on day 0 and gavaged with 110 mg/kg <i>H</i>. <i>perforatum</i> extract (N=15), or not inoculated with virus and gavaged with 5% ethanol (N=6). BAL cells were subject to flow-cytometry. Total cell counts (A), and percentage of BAL cells being neutrophils (CD11b⁺ GR1⁺ high SSC)(B), cytotoxic T cells (CD8b⁺ low SSC)(C), induced macrophages (CD11b⁺ GR1^- high auto-fluorescent)(D), resident alveolar macrophages (CD11b^- GR1^- high auto-fluorescent)(E) and inflammatory monocytes (CD11b⁺ GR1^int low SSC)(F) are shown as Mean ± SEM for each treatment group. Values without same over bar label are statistically different from each other (p<0.05, a > b > c).</p

Lung index and viral titer of mice.

<p>Mice were inoculated intranasally with 10^5.0 and 10^7.9 EID₅₀ of H1N1 influenza virus respectively on day 0 and gavaged with 5% ethanol vehicle control from day -1 to day 6 (N=12) or day 5 PI (N=15), or inoculated with virus on day 0 and gavaged with 110 mg/kg <i>H</i>. <i>perforatum</i> extract in 5% ethanol (N=12 for low viral titer study and N=15 for high viral titer study), or not inoculated with virus and gavaged with 5% ethanol (N=6 for 6-day study and N=5 for 5-day study). At the end of the study, the mouse lungs were weighed and used to calculate lung index (A and C) by histopathology, shown as Mean ± SEM. Lung viral titer (B and D) was measured using qRT-PCR, with log values for relative viral titer shown as Mean ± SEM. Values without the same label are different from each other statistically (p<0.05, a > b > c).</p

Lung lesion score and lung viral titer in 10-day long term low viral titer inoculation study.

<p>Mice were inoculated intranasally with H1N1 influenza virus (10^5.1 EID50) on day 0 and gavaged with 5% ethanol vehicle control (N=13), or 110 mg/kg <i>H</i>. <i>perforatum</i> extract (N=13) from day 5 to day 9 PI. Microscopic lung lesion scores are shown in box plots. Lung viral titer was measured using qRT-PCR and shown as relative titer in log scale (Mean ± SEM). Values without same over bar label are statistically different from each other (p<0.05).</p

Schematic diagram for the proposed underlying mechanism.

<p>SOCS3 was a negative regulator that undermines innate immunity against influenza virus infection. <i>H. perforatum</i> extract elevated SOCS3, and thus impaired the required anti-viral immune defense system, resulting in uncontrolled infection and sustained inflammation.</p

Cytokines released by A549 human bronchial epithelial cells.

<p>DMSO vehicle control or 30 µg/mL <i>H</i>. <i>perforatum</i> extract in DMSO were applied to cells, with or without H1N1 influenza virus inoculation. Cytokine levels for IL-6 (A), TNF-α (B), IP-10 (C), and MCP-1 (D) after 24 hrs of treatment are shown as Mean ± SEM (N=3). Significant difference between vehicle and <i>H</i>. <i>perforatum</i> extract treatments are noted with * (p<0.05).</p