Muscarinic receptor-stimulated Ca2+ signaling and inositol lipid metabolism in avian salt gland cells

Activation of muscarinic cholinergic receptors was studied by measuring agonist-stimulated inositol lipid turnover and changes in [Ca2+]i in dissociated salt gland secretory cells. Carbachol stimulation of quin2-loaded cells results in a sustained 4-fold increase in [Ca2+]i, while incorporation of [32P]Pi into phosphatidylinositol (PI) and phosphatidate are similarly increased. [3H]Inositol phosphates, measured in the presence of Li+, increased 13-fold. The stimulated increment in [Ca2+]i required extracellular Ca2+, whereas [3H]inositol phosphate accumulation was independent of external Ca2+. Dose-response curves for carbachol-induced increments in [Ca2+]i, PI labeling, and labeled inositol phosphate release are similar, with EC50 values of 6, 4.5 and 8 [mu]M, respectively. Dissociation constants for atropine vs. the quin2 and phospholipid responses are 0.59 +/- 0.3 nM and 0.48 +/- 0.28 nM, respectively. These cells thus provide a model system for the study of non-exocytotic secretion as a consequence of stimulated inositol lipid turnover.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25973/1/0000039.pd

Impact of human papillomavirus (HPV) 16 and 18 vaccination on prevalent infections and rates of cervical lesions after excisional treatment

BackgroundHuman papillomavirus vaccines prevent human papillomavirus infection and cervical precancers. The impact of vaccinating women with a current infection or after treatment for an human papillomavirus-associated lesion is not fully understood.ObjectivesTo determine whether human papillomavirus-16/18 vaccination influences the outcome of infections present at vaccination and the rate of infection and disease after treatment of lesions.Study DesignWe included 1711 women (18−25 years) with carcinogenic human papillomavirus infection and 311 women of similar age who underwent treatment for cervical precancer and who participated in a community-based trial of the AS04-adjuvanted human papillomavirus-16/18 virus-like particle vaccine. Participants were randomized (human papillomavirus or hepatitis A vaccine) and offered 3 vaccinations over 6 months. Follow-up included annual visits (more frequently if clinically indicated), referral to colposcopy of high-grade and persistent low-grade lesions, treatment by loop electrosurgical excisional procedure when clinically indicated, and cytologic and virologic follow-up after treatment. Among women with human papillomavirus infection at the time of vaccination, we considered type-specific viral clearance, and development of cytologic (squamous intraepithelial lesions) and histologic (cervical intraepithelial neoplasia) lesions. Among treated women, we considered single-time and persistent human papillomavirus infection, squamous intraepithelial lesions, and cervical intraepithelial neoplasia 2 or greater. Outcomes associated with infections absent before treatment also were evaluated. Infection-level analyses were performed and vaccine efficacy estimated.ResultsMedian follow-up was 56.7 months (women with human papillomavirus infection) and 27.3 months (treated women). There was no evidence of vaccine efficacy to increase clearance of human papillomavirus infections or decrease incidence of cytologic/histologic abnormalities associated with human papillomavirus types present at enrollment. Vaccine efficacy for human papillomavirus 16/18 clearance and against human papillomavirus 16/18 progression from infection to cervical intraepithelial neoplasia 2 or greater were −5.4% (95% confidence interval −19,10) and 0.3% (95% confidence interval −69,41), respectively. Among treated women, 34.1% had oncogenic infection and 1.6% had cervical intraepithelial neoplasia 2 or greater detected after treatment, respectively, and of these 69.8% and 20.0% were the result of new infections. We observed no significant effect of vaccination on rates of infection/lesions after treatment. Vaccine efficacy estimates for human papillomavirus 16/18 associated persistent infection and cervical intraepithelial neoplasia 2 or greater after treatment were 34.7% (95% confidence interval −131, 82) and −211% (95% confidence interval −2901, 68), respectively. We observed evidence for a partial and nonsignificant protective effect of vaccination against new infections absent before treatment. For incident human papillomavirus 16/18, human papillomavirus 31/33/45, and oncogenic human papillomavirus infections post-treatment, vaccine efficacy estimates were 57.9% (95% confidence interval −43, 88), 72.9% (95% confidence interval 29, 90), and 36.7% (95% confidence interval 1.5, 59), respectively.ConclusionWe find no evidence for a vaccine effect on the fate of detectable human papillomavirus infections. We show that vaccination does not protect against infections/lesions after treatment. Evaluation of vaccine protection against new infections after treatment and resultant lesions warrants further consideration in future studies

Cross-protection of the Bivalent Human Papillomavirus (HPV) Vaccine Against Variants of Genetically Related High-Risk HPV Infections

BackgroundResults from the Costa Rica Vaccine Trial (CVT) demonstrated partial cross-protection by the bivalent human papillomavirus (HPV) vaccine, which targets HPV-16 and HPV-18, against HPV-31, -33, and -45 infection and an increased incidence of HPV-51 infection.MethodsA study nested within the CVT intention-to-treat cohort was designed to assess high-risk HPV variant lineage-specific vaccine efficacy (VE). The 2 main end points were (1) long-term incident infections persisting for ≥2 years and/or progression to high-grade squamous intraepithelial lesions (ie, cervical intraepithelial neoplasia grade 2/3 [CIN 2/3]) and (2) incident transient infections lasting for <2 years. For efficiency, incident infections due to HPV-16, -18, -31, -33, -35, -45, and -51 resulting in persistent infection and/or CIN 2/3 were matched (ratio, 1:2) to the more-frequent transient viral infections, by HPV type. Variant lineages were determined by sequencing the upstream regulatory region and/or E6 region.ResultsVEs against persistent or transient infections with HPV-16, -18, -33, -35, -45, and -51 did not differ significantly by variant lineage. As the possible exception, VEs against persistent infection and/or CIN 2/3 due to HPV-31 A/B and HPV-31C variants were -7.1% (95% confidence interval [CI], -33.9% to 0%) and 86.4% (95% CI, 65.1%-97.1%), respectively (P = .02 for test of equal VE). No difference in VE was observed by variant among transient HPV-31 infections (P = .68).ConclusionsOverall, sequence variation at the variant level does not appear to explain partial cross-protection by the bivalent HPV vaccine

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Differences in dispersion of influenza virus lipids and proteins during fusion

Digitally enhanced low-light-level fluorescence video microscopy and immunochemical staining were used to examine influenza virus envelope lipid and protein redistribution during pH-induced fusion. Video microscopy was performed using viruses labeled with either the lipid analogue octadecylrhodamine B (R18) or fluorescein isothiocyanate (FITC) covalently linked to envelope proteins. Viruses were bound to human red blood cells, and the pattern and intensity of fluorescence were monitored for 30 min while cell-virus complexes were perfused with pH 7.4 or 4.8 media at temperatures either above or below 20°C. R18 showed complete redistribution and dequenching by 30 min at all incubation temperatures, confirming reports that viral fusion occurs at subphysiological temperatures. FITC-labeled protein showed spatial redistribution at 28°C but no change at low temperature. Electron microscopy observations of immunochemical staining of viral proteins confirmed both that protein redistribution at 37°C was slower than R18 and the failure of movement within 30 min at 16°C. Video microscopy monitoring of RNA staining by acridine orange of virus-cell complexes showed redistribution to the RBCs at all temperatures but only after low pH-induced fusion. The results are consistent with differential dispersion of viral components into the RBC and the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation

Early Activation of MAP Kinases by Influenza A Virus X-31 in Murine Macrophage Cell Lines

<div><p>Early molecular responses to Influenza A (FLUA) virus strain A/X-31 H3N2 in macrophages were explored using J774.A1 and RAW 264.7 murine cell lines. NF-kappa B (NFκB) was reported to be central to FLUA host-response in other cell types. Our data showed that FLUA activation of the classical NFκB dependent pathway in these macrophages was minimal. Regulator proteins, IkappaB-alpha and –beta (IκBα, IκBβ), showed limited degradation peaking at 2 h post FLUA exposure and p65 was not observed to translocate from the cytoplasm to the nucleus. Additionally, the non-canonical NFκB pathway was not activated in response to FLUA. The cells did display early increases in TNFα and other inflammatory cytokine and chemokine production. Mitogen activated phosphokinase (MAPK) signaling pathways are also reported to control production of inflammatory cytokines in response to FLUA. The activation of the MAPKs, cJun kinases 1 and 2 (JNK 1/2), extracellular regulated kinases 1 and 2 (ERK 1/2), and p38 were investigated in both cell lines between 0.25 and 3 h post-infection. Each of these kinases showed increased phosphorylation post FLUA exposure. JNK phosphorylation occurred early while p38 phosphorylation appeared later. Phosphorylation of ERK 1/2 occurred earlier in J774.A1 cells compared to RAW 264.7 cells. Inhibition of MAPK activation resulted in decreased production of most FLUA responsive cytokines and chemokines in these cells. The results suggest that in these monocytic cells the MAPK pathways are important in the early response to FLUA.</p></div

Effects of MAPK inhibitors on FLUA-induced CK/CHK from RAW 264.7 cells at short time points.

<p>Supernatants from cells treated with the indicated inhibitors as described in Methods then exposed to FLUA were collected 3 or 6 h after exposure, along with those from mock infected controls. They were assayed for selected CK/CHK by multiplexed fluid phased antibody based analysis. Cytokine concentrations were calculated based on known concentration standards run at the same time. Data shown are the mean ± SEM, n = 3 independent experiments. FO–FLUA only; JEI–both JNK and ERK Inhibition; JI–JNK inhibition; EI–ERK inhibition.</p

Effects of MAPK inhibitors on FLUA-induced CK/CHK from cell lines at 24 h.

<p>Supernatants from cells pre-treated with the indicated inhibitors as described in Methods then exposed to FLUA were collected 24 h after exposure, along with those from mock infected controls. They were assayed for selected CK/CHK by multiplexed fluid phased antibody based analysis. Cytokine concentrations were calculated based on known concentration standards run at the same time. Data shown are the mean ± SEM, n = 3 independent experiments. FO–FLUA only; JEI–both JNK and ERK Inhibition; JI–JNK inhibition; EI–ERK inhibition.</p

NFkB is not activated by FLUA at early time-points.

<p><b>A.</b> In J774.A1 cells IκBα levels were reduced at 2 h while IκBβ levels showed a gradual decline across the time points (but approximated control levels at 2 h). In RAW 264.7 cells, IκBα stayed near or above control levels while IκBβ levels showed a mild decline. Representative Western blots of cell lysates collected at the indicated time points post exposure to X-31 FLUA. Control and LPS treated (1 µg/ml for 15 minutes) cells were mock infected. <b>B.</b> Densitometry graphs from the Western blots. Data are normalized to actin, background is subtracted and protein levels are expressed as percentage of the mock infected control. Means are shown ± SEM, n = 3 to 6 independent experiments depending on time point. <b>C.</b> Nuclear localization of NFκB subunit p65 is not increased by X-31 infection of J774.A1 cells. Cells were infected or mock infected for the indicated periods, fixed, permeabilized and stained with anti-p65 antibody. LPS cells received 1 µg/ml LPS 30 minutes before fixation. LPS did increase NFκB nuclear localization as indicated by immunofluorescence. LPS activation was not blocked by prior X-31 exposure. 60X scale fields are shown with 255X digitally zoomed insets.</p