Microbiological characteristics of the lower airway in adults with bronchiectasis: a prospective cohort study.

BACKGROUND: Microbial infection and colonization are frequently associated with disease progression and poor clinical outcomes in bronchiectasis. Identification of pathogen spectrum is crucial for precision treatment at exacerbation of bronchiectasis. METHODS: We conducted a prospective cohort study in patients with bronchiectasis exacerbation onset and stable state. Bronchoalveolar lavage fluid (BALF) was collected for conventional microbiological tests (CMTs) and metagenomic Next-Generation Sequencing (mNGS). Bronchiectasis patients were monitored for documenting the time to the next exacerbation during longitudinal follow-up. RESULTS: We recruited 168 eligible participants in the exacerbation cohorts, and 38 bronchiectasis patients at stable state at longitudinal follow-up. 141 bronchiectasis patients at exacerbation onset had definite or probable pathogens via combining CMTs with mNGS reports. We identified that Pseudomonas aeruginosa, non-tuberculous mycobacteria, Haemophilus influenzae, Nocardia spp, and Staphylococcus aureus were the top 5 pathogens with a higher detection rate in our cohorts via combination of CMTs and mNGS analysis. We also observed strong correlations of Pseudomonas aeruginosa, Haemophilus influenzae, non-tuberculous mycobacteria with disease severity, including the disease duration, Bronchiectasis Severity Index, and lung function. Moreover, the adjusted pathogenic index of potential pathogenic microorganism negatively correlated (r = -0.7280, p < 0.001) with the time to the next exacerbation in bronchiectasis. CONCLUSION: We have revealed the pathogenic microbial spectrum in lower airways and the negative correlation of PPM colonization with the time to the next exacerbation in bronchiectasis. These results suggested that pathogens contribute to the progression of bronchiectasis.

Silencing Glypican-1 enhances the antitumor effects of Pictilisib via downregulating PI3K/Akt/ERK signaling in chemo-resistant esophageal adenocarcinoma.

Poorly differentiated esophageal adenocarcinoma (PDEAC) has a dismal prognosis. Glypican-1(GPC-1) is known to be upregulated in several cancer types in contrast to healthy tissues, rendering it as a biomarker. Nevertheless, the potential therapeutic targeting of GPC-1 has not been explored in PDEAC. There is accumulating evidence that GPC-1, via upregulation of PI3K/Akt/ERK signaling, plays a crucial role in the progression and chemoresistance in cancer. Pictilisib, a class I pan PI3K inhibitor, has shown promising antitumor results in clinical trials, however, has not gained widespread success due to acquired drug resistance. This study investigated the role of GPC-1 in chemo-resistant PDEAC and appraises the impact of targeted silencing of GPC-1 on the antitumor effects of Pictilisib in PDEAC cell lines. Immunohistochemistry assays in PDEAC tissue specimens demonstrated a pronounced intensity of staining with GPC-1. Upregulation of GPC-1 was found to be correlated with advanced stage and poor prognosis. In-vitro studies examined the influence of GPC-1 knockdown and Pictilisib, both as individual agents and in combination, on cytotoxicity, cell cycle distribution, apoptosis, and gene expression profiles. Silencing GPC-1 alone showed significantly reduced cell viability, migration, colony formation, epithelial-mesenchymal transition, and stemness in PDEAC cells. Significantly, knockdown of GPC-1 combined with low-dose Pictilisib led to enhancement of cytotoxicity, cell cycle arrest, and apoptosis in ESO-26 and OE-33 cells. In the xenograft mouse model, the combination of Pictilisib and GPC-1 knockdown exhibited synergy. These findings suggest that GPC-1 represents a promising target to augment chemosensitivity in esophageal adenocarcinoma.

Targeting Glypican-1 Reverses Resistance to 5-Fluorouracil in Esophageal Adenocarcinoma Cells.

BACKGROUND/AIM: The primary mode of therapy for individuals with locally advanced esophageal adenocarcinoma (EAC) is neoadjuvant chemotherapy, commonly 5-Fluorouracil (5-FU). However, approximately 30% of these patients develop resistance to therapy. Glypican-1 (GPC-1) has been identified as one of the key drivers of chemoresistance in cancer; however, its role in EAC cells has not been explored. The objective of the present study was to evaluate the role of GPC-1 in chemoresistance to 5-FU in EAC cells. MATERIALS AND METHODS: Cell viability to 5-FU was measured with CCK-8 assay, and GPC-1 expression was validated using western blot. 5-FU resistant cell lines were generated. The effect of lentivirus-mediated GPC-1 knockdown on FLO-1 cell viability, cell cycle, and apoptosis was evaluated. RESULTS: 5-FU resistant EAC cells showed increased GPC-1 expression and knockdown of GPC-1 increased cell death and apoptosis. Importantly, the knockdown of GPC-1 enhanced the antitumor effects of 5-FU in vitro via down-regulating AKT/ERK/ß-catenin signaling. CONCLUSION: Silencing GPC-1 has the potential to augment the efficacy of 5-FU chemotherapy in resistant EAC tumors.

Hallmark guided identification and characterization of a novel immune-relevant signature for prognostication of recurrence in stage I-III lung adenocarcinoma.

The high risk of postoperative mortality in lung adenocarcinoma (LUAD) patients is principally driven by cancer recurrence and low response rates to adjuvant treatment. Here, A combined cohort containing 1,026 stage I-III patients was divided into the learning (n = 678) and validation datasets (n = 348). The former was used to establish a 16-mRNA risk signature for recurrence prediction with multiple statistical algorithms, which was verified in the validation set. Univariate and multivariate analyses confirmed it as an independent indicator for both recurrence-free survival (RFS) and overall survival (OS). Distinct molecular characteristics between the two groups including genomic alterations, and hallmark pathways were comprehensively analyzed. Remarkably, the classifier was tightly linked to immune infiltrations, highlighting the critical role of immune surveillance in prolonging survival for LUAD. Moreover, the classifier was a valuable predictor for therapeutic responses in patients, and the low-risk group was more likely to yield clinical benefits from immunotherapy. A transcription factor regulatory protein-protein interaction network (TF-PPI-network) was constructed via weighted gene co-expression network analysis (WGCNA) concerning the hub genes of the signature. The constructed multidimensional nomogram dramatically increased the predictive accuracy. Therefore, our signature provides a forceful basis for individualized LUAD management with promising potential implications.

Physicians' knowledge of invasive fungal disease in China.

BACKGROUND: Invasive fungal disease (IFD) is associated with high morbidity and mortality. Data are lacking regarding physicians' perspectives on the diagnosis and management of IFD in China. OBJECTIVES: To evaluate physicians' perspectives on the diagnosis and management of IFD. METHODS: Based on current guidelines, a questionnaire was designed and administered to 294 physicians working in haematology departments, intensive care units, respiratory departments and infectious diseases departments in 18 hospitals in China. RESULTS: The total score and subsection scores for invasive candidiasis, invasive aspergillosis (IA), cryptococcosis and invasive mucormycosis (IM) were 72.0 ± 12.2 (maximum = 100), 11.1 ± 2.7 (maximum = 19), 43.0 ± 7.8 (maximum = 57), 8.1 ± 2.0 (maximum = 11) and 9.8 ± 2.3 (maximum = 13), respectively. Although the perspectives of the Chinese physicians were in good overall agreement with guideline recommendations, some knowledge gaps were identified. Specific areas in which the physicians' perspectives and guideline recommendations differed included use of the ß-D-glucan test to facilitate the diagnosis of IFD, relative utility of the serum galactomannan test and bronchoalveolar lavage fluid galactomannan test in patients with agranulocytosis, use of imaging in the diagnosis of mucormycosis, risk factors for mucormycosis, indications for initiating antifungal therapy in patients with haematological malignancies, when to start empirical therapy in mechanically ventilated patients, first-line drugs for mucormycosis and treatment courses for IA and IM. CONCLUSION: This study highlights the main areas that could be targeted by training programs to improve the knowledge of physicians treating patients with IFD in China.

Direct and indirect activation of the adenosine triphosphate-sensitive potassium channel to induce spinal cord ischemic metabolic tolerance.

OBJECTIVES: The mitochondrial adenosine triphosphate-sensitive potassium channel is central to pharmacologically induced tolerance to spinal cord injury. We hypothesized that both direct and nitric oxide-dependent indirect activation of the adenosine triphosphate-sensitive potassium channel contribute to the induction of ischemic metabolic tolerance. METHODS: Spinal cord injury was induced in adult male C57BL/6 mice through 7 minutes of thoracic aortic crossclamping. Pretreatment consisted of intraperitoneal injection 3 consecutive days before injury. Experimental groups were sham (no pretreatment or ischemia, n = 10), spinal cord injury control (pretreatment with normal saline, n = 27), Nicorandil 1.0 mg/kg (direct and indirect adenosine triphosphate-sensitive potassium channel opener, n = 20), Nicorandil 1 mg/kg + carboxy-PTIO 1 mg/kg (nitric oxide scavenger, n = 21), carboxy-PTIO (n = 12), diazoxide 5 mg/kg (selective direct adenosine triphosphate-sensitive potassium channel opener, n = 25), and DZ 5 mg/kg+ carboxy-PTIO 1 mg/kg, carboxy-PTIO (n = 23). Limb motor function was assessed using the Basso Mouse Score (0-9) at 12-hour intervals for 48 hours after ischemia. RESULTS: Motor function was significantly preserved at all time points after ischemia in the Nicorandil pretreatment group compared with ischemic control. The addition of carboxy-PTIO partially attenuated Nicorandil's motor-preserving effect. Motor function in the Nicorandil + carboxy-PTIO group was significantly preserved compared with the spinal cord injury control group (P < .001), but worse than in the Nicorandil group (P = .078). Motor preservation in the diazoxide group was similar to the Nicorandil + carboxy-PTIO group. There was no significant difference between the diazoxide and diazoxide + carboxy-PTIO groups. CONCLUSIONS: Both direct and nitric oxide-dependent indirect activation of the mitochondrial adenosine triphosphate-sensitive potassium channel play an important role in pharmacologically induced motor function preservation.

Glypican 1 promotes proliferation and migration in esophagogastric adenocarcinoma via activating AKT/GSK/ß-catenin pathway.

Background: Glypican 1 (GPC1) is a heparan sulphate proteoglycan cell membrane protein. It is implicated in driving cancers of the breast, brain, pancreas, and prostate; however, its role in esophagogastric cancer (EGAC) remains unexplored. The aim of the study was to investigate and elucidate the molecular mechanistic of GPC1 in human EGAC. Methods: Thirty tissue and 120 microarray sections of EGAC were evaluated with Anti-GPC1 immunohistochemistry. Loss and gain of GPC1 function were performed using lentivirus transfection in EGAC cell lines. Mechanistically, AKT/GSK/ß-catenin pathway was evaluated using AKT inhibitor MK-2206 and Wnt/ß-catenin stimulant LiCl. Results: GPC1 overexpression was found in 102 cases (68%). Overexpression of GPC1 correlated with lymph node metastasis, poor differentiation and decreased overall survival. Lentivirus mediated GPC1 knockdown resulted in decreased cell proliferation, migration, invasion, and colony formation. Knockdown caused G0/G1 cell cycle arrest, increased apoptosis, and reduced epithelial mesenchymal transition (EMT). GPC1 mediated its effects by activation of AKT/GSK/ß-catenin pathway. Conclusions: This is the first descriptive study to decipher the role of GPC1 in EGAC. Our results suggest that GPC1 regulates cell proliferation and growth and may serve as an attractive oncotarget in EGAC.

Simvastatin Inhibits Histologic Changes Associated with Gastroduodenal Reflux in a Murine Model.

BACKGROUND: Observational studies demonstrate a protective effect of statins on the development and progression of esophageal adenocarcinoma. The role of statins in the prevention of reflux-induced esophageal changes remains unknown. AIMS: Using a mixed gastroduodenal reflux mouse model, we hypothesized that oral administration of simvastatin would attenuate reflux-induced mucosal changes of the distal esophagus. METHODS: Human Barrett's (CPB) and esophageal adenocarcinoma (FLO1 and OE19) cells were treated with simvastatin. Cell proliferation and apoptosis were evaluated using the MTS proliferation and annexin V apoptosis assays, respectively. A reflux mouse model was generated by performing a side-to-side anastomosis between the gastroesophageal junction and first portion of the duodenum (duodeno-gastroesophageal anastomosis, DGEA). DGEA mice were fed a standard or simvastatin-containing diet following surgery. Mice were euthanized 6 weeks post-operatively. RESULTS: Simvastatin significantly decreased proliferation and increased apoptosis in all cell lines. Compared to control animals, mice undergoing DGEA who were fed a standard diet demonstrated a fourfold increase in mucosal thickness and significant increase in proliferating cells (p < 0.0001). DGEA mice fed a simvastatin-containing diet had an attenuated response to reflux, with a significant reduction in mucosal hyperplasia and proliferation (p < 0.0001). DGEA mice fed a simvastatin-containing diet demonstrated significant upregulation of procaspase-3 (p = 0.009) and cleaved caspase-3 (p = 0.034) in the distal esophagus. CONCLUSIONS: We demonstrate for the first time a reduction in reflux-induced histologic changes of the distal esophagus following oral administration of simvastatin in vivo. These findings identify simvastatin as a potential preventative agent to inhibit the development and progression of reflux-induced esophageal injury.

Expression of Adhesion Molecules in a Gastroduodenal Reflux Murine Model.

BACKGROUND: Various adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), have been shown to play a role in inflammation as well as contribute to tumor progression and prognosis. We hypothesized that gastroduodenal reflux upregulates ICAM-1 and VCAM-1 expression in the distal esophagus, serving as possible early markers of pathologic esophageal disease. METHODS: Normal human esophageal epithelial cells (HET1A), Barrett cells (CPB), and esophageal adenocarcinoma cells (FLO1 and OE33) were treated with deoxycholic acid at increasing concentrations for 24 hours. Adhesion molecule expression was assessed using immunoblotting. A surgical mouse reflux model was generated by performing a side-to-side anastomosis between the gastroesophageal junction and the first portion of the duodenum (duodenum-gastroesophageal anastomosis). Esophageal sections were evaluated using hematoxylin and eosin staining, immunohistochemistry, and immunofluorescence. RESULTS: Deoxycholic acid induced a significant increase in ICAM-1 and VCAM-1 expression in HET1A, CPB, FLO1, and OE33 cells. Animals undergoing duodenum-gastroesophageal anastomosis demonstrated a significant increase in mucosal hyperplasia (P < .0001) and cellular proliferation (P < .0001) compared with control animals. Immunofluorescence and Western blot analysis of the lower esophagus demonstrated significant upregulation of ICAM-1 (P = .005), with no change in VCAM-1 expression (P = .82). CONCLUSIONS: Our results reveal that ICAM-1 and VCAM-1 are upregulated in response to in vitro reflux treatment of normal esophageal epithelial cells. However, our investigation using a mouse reflux model found ICAM-1 is noticeably upregulated without a concomitant increase in VCAM-1. These findings identify ICAM-1, but not VCAM-1, as a potential player in early esophageal disease developing from chronic reflux exposure.

Optimizing Nicorandil for Spinal Cord Protection in a Murine Model of Complex Aortic Intervention.

There are currently no clinically utilized pharmacological agents for the induction of metabolic tolerance to spinal cord ischemia-reperfusion injury in the setting of complex aortic intervention. Nicorandil, a nitric oxide donor and ATP-sensitive potassium (KATP) channel opener, has shown promise in neuroprotection. However, the optimized clinical application of the drug and its mechanism of neuroprotection remains unclear. We hypothesized that 3-days pretreatment would confer the most effective neuroprotection, mediated by mitochondrial KATP channel activation. Spinal cord injury was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Time course: mice received 0.1 mg/kg nicorandil for 10 min, 4 hours, and 3 consecutive days prior to ischemia compared with control. Dose challenge: mice received 3-days nicorandil pretreatment comparing 0.1 mg/kg, 1.0 mg/kg, 5.0 mg/kg, and saline administration. Mitochondrial KATP channel blocker 5-hydroxy-decanoate (5HD) was co-administered to elucidate mechanism. Limb motor function was evaluated, and viable anterior horn neurons quantified. Nicorandil pretreatment at 4 hours and 3 days before ischemia demonstrated significant motor function preservation; administration 10 minutes before ischemia showed no neuroprotection. All nicorandil doses showed significant motor function preservation. Three days administration of Nicorandil 1.0 mg/kg was most potent. Neuroprotection was completely abolished by 5HD co-administration. Histological analysis showed significant neuron preservation with nicorandil pretreatment, which was attenuated by 5HD co-administration. Three days administration of Nicorandil 1.0 mg/kg showed near-total motor function preservation in a murine spinal cord ischemia-reperfusion model, mediated by the mitochondrial KATP channel.

Earlier diagnosis improves COVID-19 prognosis: a nationwide retrospective cohort analysis.

BACKGROUND: Risk of adverse outcomes in COVID-19 patients by stratifying by the time from symptom onset to confirmed diagnosis status is still uncertain. METHODS: We included 1,590 hospitalized COVID-19 patients confirmed by real-time RT-PCR assay or high-throughput sequencing of pharyngeal and nasal swab specimens from 575 hospitals across China between 11 December 2019 and 31 January 2020. Times from symptom onset to confirmed diagnosis, from symptom onset to first medical visit and from first medical visit to confirmed diagnosis were described and turned into binary variables by the maximally selected rank statistics method. Then, survival analysis, including a log-rank test, Cox regression, and conditional inference tree (CTREE) was conducted, regarding whether patients progressed to a severe disease level during the observational period (assessed as severe pneumonia according to the Chinese Expert Consensus on Clinical Practice for Emergency Severe Pneumonia, admission to an intensive care unit, administration of invasive ventilation, or death) as the prognosis outcome, the dependent variable. Independent factors included whether the time from symptom onset to confirmed diagnosis was longer than 5 days (the exposure) and other demographic and clinical factors as multivariate adjustments. The clinical characteristics of the patients with different times from symptom onset to confirmed diagnosis were also compared. RESULTS: The medians of the times from symptom onset to confirmed diagnosis, from symptom onset to first medical visit, and from first medical visit to confirmed diagnosis were 6, 3, and 2 days. After adjusting for age, sex, smoking status, and comorbidity status, age [hazard ratio (HR): 1.03; 95% CI: 1.01-1.04], comorbidity (HR: 1.84; 95% CI: 1.23-2.73), and a duration from symptom onset to confirmed diagnosis of >5 days (HR: 1.69; 95% CI: 1.10-2.60) were independent predictors of COVID-19 prognosis, which echoed the CTREE models, with significant nodes such as time from symptom onset to confirmed diagnosis, age, and comorbidities. Males, older patients with symptoms such as dry cough/productive cough/shortness of breath, and prior COPD were observed more often in the patients who procrastinated before initiating the first medical consultation. CONCLUSIONS: A longer time from symptom onset to confirmed diagnosis yielded a worse COVID-19 prognosis.

Reactive Oxygen Species Mediate Nicorandil-induced Metabolic Tolerance to Spinal Cord Injury.

BACKGROUND: Spinal cord injury remains a devastating complication of thoracoabdominal aortic surgery. We previously demonstrated that pretreatment with nicorandil preserved motor function in a murine spinal cord injury model through mitochondrial adenosine triphosphate-sensitive potassium channel activation. We hypothesized that the neuroprotective effect of nicorandil is mediated by downstream generation of reactive oxygen species. METHODS: Spinal cord injury was induced by 7 minutes of thoracic aortic cross-clamping in adult male C57BL/6 mice. Five groups were evaluated: ischemic control (n = 19); nicorandil 1.0 mg/kg (n = 17); nicorandil 1.0 mg/kg plus N acetyl L-cysteine (NAC [reactive oxygen species scavenger, n = 18)]) 150 mg/kg; NAC 150 mg/kg (n = 13); and sham (n = 10). Limb motor function and the number of viable neurons within the anterior horn of the spinal cord were evaluated. RESULTS: Mice in the sham group showed no functional deficits after surgery. Compared with ischemic control, motor function was significantly preserved in the nicorandil pretreatment group at every timepoint after ischemia. In the nicorandil plus NAC group, the motor-preserving effect of nicorandil was completely abolished (P < .001). Viable neuron quantification showed significant neuron preservation in the nicorandil group (29.± 2.6) compared with the ischemic control group (18.5 ± 2.1, P = .024) and nicorandil plus NAC group (14 ± 8.3, P = .001); no significant difference was observed between the ischemic control group and nicorandil plus NAC group (P = 0.768). CONCLUSIONS: Reactive oxygen species generation plays a key role in the nicorandil-induced metabolic tolerance to spinal cord injury. Manipulation of mitochondrial adenosine triphosphate-sensitive potassium channels may lead to improvement in preventing spinal cord injury after thoracoabdominal aortic interventions.

Definition and Risks of Cytokine Release Syndrome in 11 Critically Ill COVID-19 Patients With Pneumonia: Analysis of Disease Characteristics.

Corona virus disease 2019 (COVID-19) patients with severe immune abnormalities are at risk of cytokine release syndrome (CRS). The definition, prevention, and treatment of symptoms of CRS in critically ill patients with COVID-19 are important problems. We report a single-center case series of 11 COVID-19 patients with acute respiratory distress syndrome from The First Affiliated Hospital of Guangzhou Medical University in China from 26 January 2020 to 18 February 2020. The termination date of follow-up was 19 February 2020. Eight patients were determined to have characteristics of CRS, including pulmonary inflammation, fever, and dysfunction of nonpulmonary organs. An increase in interleukin-6 in peripheral blood was the highest risk factor and an early indicator of CRS in COVID-19.

Risk Factors of Fatal Outcome in Hospitalized Subjects With Coronavirus Disease 2019 From a Nationwide Analysis in China.

BACKGROUND: The novel coronavirus disease 2019 (COVID-19) has become a global health emergency. The cumulative number of new confirmed cases and deaths are still increasing out of China. Independent predicted factors associated with fatal outcomes remain uncertain. RESEARCH QUESTION: The goal of the current study was to investigate the potential risk factors associated with fatal outcomes from COVID-19 through a multivariate Cox regression analysis and a nomogram model. STUDY DESIGN AND METHODS: A retrospective cohort of 1,590 hospitalized patients with COVID-19 throughout China was established. The prognostic effects of variables, including clinical features and laboratory findings, were analyzed by using Kaplan-Meier methods and a Cox proportional hazards model. A prognostic nomogram was formulated to predict the survival of patients with COVID-19. RESULTS: In this nationwide cohort, nonsurvivors included a higher incidence of elderly people and subjects with coexisting chronic illness, dyspnea, and laboratory abnormalities on admission compared with survivors. Multivariate Cox regression analysis showed that age ≥ 75 years (hazard ratio [HR], 7.86; 95% CI, 2.44-25.35), age between 65 and 74 years (HR, 3.43; 95% CI, 1.24-9.5), coronary heart disease (HR, 4.28; 95% CI, 1.14-16.13), cerebrovascular disease (HR, 3.1; 95% CI, 1.07-8.94), dyspnea (HR, 3.96; 95% CI, 1.42-11), procalcitonin level > 0.5 ng/mL (HR, 8.72; 95% CI, 3.42-22.28), and aspartate aminotransferase level > 40 U/L (HR, 2.2; 95% CI, 1.1-6.73) were independent risk factors associated with fatal outcome. A nomogram was established based on the results of multivariate analysis. The internal bootstrap resampling approach suggested the nomogram has sufficient discriminatory power with a C-index of 0.91 (95% CI, 0.85-0.97). The calibration plots also showed good consistency between the prediction and the observation. INTERPRETATION: The proposed nomogram accurately predicted clinical outcomes of patients with COVID-19 based on individual characteristics. Earlier identification, more intensive surveillance, and appropriate therapy should be considered in patients at high risk.

Comorbidity and its impact on 1590 patients with COVID-19 in China: a nationwide analysis.

BACKGROUND: The coronavirus disease 2019 (COVID-19) outbreak is evolving rapidly worldwide. OBJECTIVE: To evaluate the risk of serious adverse outcomes in patients with COVID-19 by stratifying the comorbidity status. METHODS: We analysed data from 1590 laboratory confirmed hospitalised patients from 575 hospitals in 31 provinces/autonomous regions/provincial municipalities across mainland China between 11 December 2019 and 31 January 2020. We analysed the composite end-points, which consisted of admission to an intensive care unit, invasive ventilation or death. The risk of reaching the composite end-points was compared according to the presence and number of comorbidities. RESULTS: The mean age was 48.9 years and 686 (42.7%) patients were female. Severe cases accounted for 16.0% of the study population. 131 (8.2%) patients reached the composite end-points. 399 (25.1%) reported having at least one comorbidity. The most prevalent comorbidity was hypertension (16.9%), followed by diabetes (8.2%). 130 (8.2%) patients reported having two or more comorbidities. After adjusting for age and smoking status, COPD (HR (95% CI) 2.681 (1.424-5.048)), diabetes (1.59 (1.03-2.45)), hypertension (1.58 (1.07-2.32)) and malignancy (3.50 (1.60-7.64)) were risk factors of reaching the composite end-points. The hazard ratio (95% CI) was 1.79 (1.16-2.77) among patients with at least one comorbidity and 2.59 (1.61-4.17) among patients with two or more comorbidities. CONCLUSION: Among laboratory confirmed cases of COVID-19, patients with any comorbidity yielded poorer clinical outcomes than those without. A greater number of comorbidities also correlated with poorer clinical outcomes.

Early and late changes in natural killer cells in response to ledipasvir/sofosbuvir treatment.

Chronic hepatitis C virus (HCV) infection is characterized by dysregulated natural killer (NK) cell responses. NKs play a critical role in achieving sustained responses to interferon (IFN)-α-based therapy. Rapid sustained HCV-RNA clearance is now achieved with direct-acting antivirals (DAAs). Studies of patients receiving first-wave DAAs suggest NK functional restoration. Here, we investigate the effect of mainstream DAA treatment on NKs. We collected a prospective cohort of male HCV genotype 1-infected patients treated with ledipasvir/sofosbuvir (n = 22). Peripheral blood was obtained at treatment start, week 2 (W2), W4, W8, and W12 of treatment and 12 weeks posttreatment. Flow cytometry was used to characterize NK responses to therapy. Mean baseline viral load was 1.75 million IU/mL. All subjects rapidly cleared virus and remained HCV RNA-negative posttreatment. No change was seen in total NK levels; however, the frequency of immature NKs (clusters of differentiation [CD]56bright) decreased by W2 and was maintained throughout the study. Phenotypic changes were evident by W2/W4, coincident with rapid viral clearance. At W2, T-cell immunoglobulin and mucin-domain containing-3 and CD161 were significantly increased, returning to pretreatment levels by W12. Some changes were not evident until late (W12 or posttreatment). Down-regulation of several activation markers, including NKp30 and tumor necrosis factor-related apoptosis-inducing ligand, was observed at W12 and sustained posttreatment. No difference was observed in IFN-γ production or cytokine-mediated killing of NK-sensitive cell line K562 posttreatment compared to pretreatment. Conclusion: Our phenotype data suggest transient activation followed by dampening of NK cell activity to pretreatment levels. The NK response to ledipasvir/sofosbuvir is not universal in a homogeneous patient cohort. More studies are needed to elucidate the roles of NK cells in IFN-free regimens, which will have implications for protection from re-infection and fibrosis progression. (Hepatology Communications 2018;2:364-375).

Cigarette Smoke-Induced Hypermethylation of the GCLC Gene Is Associated With COPD.

BACKGROUND: Cigarette smoking is a major environmental contributor to COPD, but understanding its epigenetic regulation of oxidative genes involved in the pathogenesis of COPD remains elusive. METHODS: We analyzed DNA methylation on glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferase M1 (GSTM1), glutathione S-transferase P1 (GSTP1), and superoxide dismutase 3 (SOD3) promoters in clinical samples from patients with COPD (current-smoker [CS-COPD]; ex-smoker [ES-COPD]) and subjects with normal pulmonary function (current-smoker [CS-NS]; ex-smoker [ES-NS]; never-smoker [NC]). Expression of GCLC messenger RNA (mRNA) and glutathione (GSH) synthesis in these clinical samples and human bronchial epithelial (BEAS-2B) cells stimulated by cigarette-smoke extract (CSE) was evaluated. GCLC mRNA and protein levels were measured to determine effects of demethylation and deacetylation agents on CSE-treated BEAS-2B cells. RESULTS: The DNA methylation level of the GCLC promoter was significantly increased in CS-COPD, CS-NS, and ES-COPD groups compared with ES-NS and NC groups. However, there were no significant differences in DNA methylation values of GSTM1, GSTP1, and SOD3 promoters among these groups. Expression of GCLC mRNA was downregulated in the lungs, and GSH levels decreased in plasma as a consequence of hypermethylation of the GCLC promoter. Similarly, CSE-treated BEAS-2B cells had hypermethylation of the GCLC gene, mRNA downregulation, and a decreased intracellular GSH level. GCLC expression in CSE-treated BEAS-2B cells was restored by the methylation inhibitor, 5-aza-2'-deoxycytidine, but not by the deacetylation agent, trichostatin A. CONCLUSIONS: Cigarette smoke-induced hypermethylation of the GCLC promoter is related to the initiation and progression of COPD. Our finding may provide a new strategy for COPD intervention by developing demethylation agents targeting GCLC hypermethylation.

HCV-infected cells and differentiation increase monocyte immunoregulatory galectin-9 production.

The lectin galectin-9 may help establish and maintain chronic hepatitis C virus infection. Galectin-9 is elevated in the liver and sera of hepatitis C virus patients, induces apoptosis of hepatitis C virus-specific T cells, and increases inhibitory regulatory T cells. Kupffer cells stain strongly for galectin-9 protein in hepatitis C virus patients. In the current study, we determined stimuli that induce galectin-9 production by monocytes and macrophages in hepatitis C virus infection. With the use of real-time PCR and flow cytometry, we analyzed galectin-9 mRNA and protein from human monocytes cocultured with hepatitis C virus-infected cells or noninfectious hepatitis C virus subgenomic replicon cells. We focused on finding the stimuli for galectin-9 production. Additionally, we measured galectin-9 during monocyte-to-macrophage maturation. Finally, we examined galectin-9 in peripheral monocytes from hepatitis C virus patients using flow cytometry. Galectin-9 mRNA increased 8-fold when primary monocytes were exposed to hepatitis C virus--infected cells. Maximum induction required proximity or contact and did not require IFN-γ or hepatitis C virus virions. Coculture of monocytes with subgenomic replicon cells increased galectin-9 5-fold, and purified exosomes from infected cells stimulated galectin-9 production. Stimulation of monocyte TLR3, -7, and -8 increased galectin-9 production. Differentiation of monocytes to macrophages increased galectin-9, and nonclassic monocytes from hepatitis C virus patients had the highest levels of galectin-9. Hepatitis C virus-infected cells stimulated monocytes to produce galectin-9 in close proximity, possibly, in part, as a result of exosomes and endosomal TLRs. Differentiation of monocytes to macrophages increased galectin-9. Nonclassic monocytes from hepatitis C virus patients express the highest galectin-9 levels, suggesting they may contribute to elevated galectin-9 and adaptive immune inhibition in hepatitis C virus infection.

Hepatitis C viral infection is associated with activated cytolytic natural killer cells expressing high levels of T cell immunoglobulin- and mucin-domain-containing molecule-3.

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is an inhibitory receptor implicated in T cell exhaustion characteristic of chronic viral infection. Limited data exist on NK cell Tim-3 expression and functional consequences. In chronic hepatitis C virus (HCV)-infected subjects, we found increased Tim-3 on NKs, which was associated with an activated phenotype. The high level of Tim-3 was not reversed by successful IFN-alpha-based antiviral therapy. Tim-3(high) NK cells up-regulated TRAIL in response to IFN-alpha to a greater extent and demonstrated greater lymphokine-activated killing activity, viral control, and degranulation but similar cytokine production than their Tim-3(low) counterparts. Our results suggest that Tim-3 on NKs is associated with activation of this innate lymphocyte population that is polarized towards cytotoxicity in chronic HCV. These findings reveal roles for Tim-3 in the regulation of NKs that might represent targets for treatment of chronic viral infections.

Transmitted/founder hepatitis C viruses induce cell-type- and genotype-specific differences in innate signaling within the liver.

UNLABELLED: Hepatitis C virus (HCV) infection leads to persistence in the majority of cases despite triggering complex innate immune responses within the liver. Although hepatocytes are the preferred site for HCV replication, nonparenchymal cells (NPCs) can also contribute to antiviral immunity. Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference have identified full-length transmitted/founder (T/F) viruses. Here, we tested the effect of HCV T/F viral RNA (vRNA) on innate immune signaling within hepatocytes and NPCs, including the HepG2 and Huh 7.5.1 cell lines, a human liver endothelial cell line (TMNK-1), a plasmacytoid dendritic cell line (GEN2.2), and a monocytic cell line (THP-1). Transfection with hepatitis C T/F vRNA induced robust transcriptional upregulation of type I and III interferons (IFNs) within HepG2 and TMNK-1 cells. Both the THP-1 and GEN2.2 lines demonstrated higher type I and III IFN transcription with genotype 3a compared to genotype 1a or 1b. Supernatants from HCV T/F vRNA-transfected TMNK-1 cells demonstrated superior viral control. Primary human hepatocytes (PHH) transfected with genotype 3a induced canonical pathways that included chemokine and IFN genes, as well as overrepresentation of RIG-I (DDX58), STAT1, and a Toll-like receptor 3 (TLR3) network. Full-length molecular clones of HCV induce broad IFN responses within hepatocytes and NPCs, highlighting that signals imparted by the various cell types within the liver may lead to divergent outcomes of infection. In particular, the finding that HCV genotypes differentially induce antiviral responses in NPCs and PHH might account for relevant clinical-epidemiological observations (higher clearance but greater necroinflammation in persistence with genotype 3). IMPORTANCE: Hepatitis C virus (HCV) has become a major worldwide problem, and it is now the most common viral infection for which there is no vaccine. HCV infection often leads to persistence of the virus and is a leading cause of chronic hepatitis, liver cancer, and cirrhosis. There are multiple genotypes of the virus, and patients infected with different viral genotypes respond to traditional therapy differently. However, the immune response to the virus within the liver has not been fully elucidated. Here, we determined the responses to different genotypes of HCV in cell types of the liver. We found that the immune response varied according to both cell type and HCV genotype, leading to a more pronounced induction of inflammatory pathways after exposure to certain genotypes. Therefore, inflammatory pathways that are being robustly activated by certain HCV genotypes could lead to more severe damage to the liver, inducing diverse outcomes and responses to therapy.