Endothelial dysfunction and cardiovascular risk in post-COVID-19 patients after 6- and 12-months SARS-CoV-2 infection.

INTRODUCTION: SARS-CoV-2 infection causes severe endothelial damage, an essential step for cardiovascular complications. Endothelial-colony forming cells (ECFCs) act as a biomarker of vascular damage but their role in SARS-CoV-2 remain unclear. The aim of this study was to assess whether the number of ECFCs and angiogenic biomarkers remained altered after 6 and 12-months post-infection and whether this imbalance correlated with the presence of long-COVID syndrome and other biological parameters measured. METHODS: Seventy-two patients were recruited at different time-points after overcoming COVID-19 and thirty-one healthy controls. All subjects were matched for age, gender, BMI, and comorbidities. ECFCs were obtained from peripheral blood and cultured with specific conditions. RESULTS: The results confirm the presence of a long-term sequela in post-COVID-19 patients, with an abnormal increase in ECFC production compared to controls (82.8% vs. 48.4%, P < 0.01) that is maintained up to 6-months (87.0% vs. 48.4%, P < 0.01) and 12-months post-infection (85.0% vs. 48.4%, P < 0.01). Interestingly, post-COVID-19 patients showed a significant downregulation of angiogenesis-related proteins compared to controls indicating a clear endothelial injury. Troponin, NT-proBNP and ferritin levels, markers of cardiovascular risk and inflammation, remained elevated up to 12-months post-infection. Patients with lower numbers of ECFC exhibited higher levels of inflammatory markers, such as ferritin, suggesting that ECFCs may play a protective role. Additionally, long-COVID syndrome was associated with higher ferritin levels and with female gender. CONCLUSIONS: These findings highlight the presence of vascular sequela that last up to 6- and 12-months post-infection and point out the need for preventive measures and patient follow-up.

Long-term respiratory consequences of COVID-19 related pneumonia: a cohort study.

BACKGROUND: Our aims were to describe respiratory sequelae up to 12 months after discharge in COVID-19 patients with severe pneumonia requiring non-invasive respiratory support therapies. METHODS: This study was undertaken at University Hospital Doctor Josep Trueta (Girona, Spain) between March 2020 and June 2020. Three months after discharge, we evaluated their dyspnoea and performed Saint George's respiratory questionnaire, pulmonary function tests, blood test, 6-min walking test, and a high-resolution CT (HRCT). At the six and 12-month follow-up, we repeated all tests except for pulmonary function, 6-min walking test, and HRCT, which were performed only if abnormal findings had been previously detected. RESULTS: Out of the 94 patients recruited, 73% were male, the median age was 62.9 years old, and most were non-smokers (58%). When comparing data three and 12 months after discharge, the percentage of patients presenting dyspnoea ≥ 2 decreased (19% vs 7%), the quality-of-life total score improved (22.8% vs 18.9%; p = 0.019), there were less abnormal results in the pulmonary function tests (47% vs 23%), the 6-min walking test distance was enhanced (368.3 m vs 390.7 m, p = 0.020), ground glass opacities findings waned (51.6% vs 11.5%), and traction bronchiectasis increased (5.6% vs 15.9%). Only age showed significant differences between patients with and without pulmonary fibrotic-like changes. CONCLUSION: Most patients improved their clinical condition, pulmonary function, exercise capacity and quality of life one year after discharge. Nonetheless, pulmonary fibrotic-like changes were observed during the follow-ups.

TL-532, a novel specific Toll-like receptor 3 agonist rationally designed for targeting cancers: discovery process and biological characterization.

Toll-like receptor 3 (TLR3) is an innate immune receptor that recognizes double-stranded RNA (dsRNA) and induces inflammation in immune and normal cells to initiate anti-microbial responses. TLR3 acts also as a death receptor only in cancer cells but not in their normal counterparts, making it an attractive target for cancer therapies. To date, all of the TLR3-activating dsRNAs used at preclinical or clinical stages have major drawbacks such as structural heterogeneity, toxicity, and lack of specificity and/or efficacy. We conducted the discovery process of a new family of TLR3 agonists that are chemically manufactured on solid-phase support and perfectly defined in terms of sequence and size. A stepwise discovery process was performed leading to the identification of TL-532, a 70 base pair dsRNA that is potent without transfection reagent and is highly specific for TLR3 without activating other innate nucleic sensors such as RIG-I/MDA5, TLR7, TLR8, and TLR9. TL-532 induces inflammation in murine RAW264.7 myeloid macrophages, in human NCI-H292 lung cancer cells, and it promotes immunogenic apoptosis in tumor cells in vitro and ex vivo without toxicity towards normal primary cells. In conclusion, we identified a novel TLR3 agonist called TL-532 that has promising anticancer properties.

A Chemically Defined TLR3 Agonist with Anticancer Activity.

Toll-like receptor 3 (TLR3) agonists such as polyinosinic:polycytidylic acid (poly(I:C)) have immunostimulatory effects that can be taken advantage of to induce anticancer immune responses in preclinical models. In addition, poly(I:C) has been introduced into clinical trials to demonstrate its efficacy as an adjuvant and to enhance the immunogenicity of locally injected tumors, thus reverting resistance to PD-L1 blockade in melanoma patients. Here, we report the pharmacokinetic, pharmacodynamic, mechanistic and toxicological profile of a novel TLR3 agonist, TL-532, a chemically synthesized double-stranded RNA that is composed by blocks of poly(I:C) and poly(A:U) (polyadenylic - polyuridylic acid). In preclinical models, we show that TL-532 is bioavailable after parenteral injection, has an acceptable toxicological profile, and stimulates the production of multiple chemokines and interleukins that constitute pharmacodynamic markers of its immunostimulatory action. When given at a high dose, TL-532 monotherapy reduced the growth of bladder cancers growing on mice. In addition, in immunodeficient mice lacking formylpeptide receptor-1 (FPR1), TL-532 was able to restore the response of orthotopic subcutaneous fibrosarcoma to immunogenic chemotherapy. Altogether, these findings may encourage further development of TL-532 as an immunotherapeutic anticancer agent.

Toll-like receptor 3 downregulation is an escape mechanism from apoptosis during hepatocarcinogenesis.

BACKGROUND & AIMS: Low levels of toll-like receptor 3 (TLR3) in patients with hepatocellular carcinoma (HCC) are associated with poor prognosis, primarily owing to the loss of inflammatory signaling and subsequent lack of immune cell recruitment to the liver. Herein, we explore the role of TLR3-triggered apoptosis in HCC cells. METHODS: Quantitative reverse transcription PCR, western blotting, immunohistochemistry and comparative genomic hybridization were used to analyze human and mouse HCC cell lines, as well as surgically resected primary human HCCs, and to study the impact of TLR3 expression on patient outcomes. Functional analyses were performed in HCC cells, following the restoration of TLR3 by lentiviral transduction. The role of TLR3-triggered apoptosis in HCC was analyzed in vivo in a transgenic mouse model of HCC. RESULTS: Lower expression of TLR3 in tumor compared to non-tumor matched tissue was observed at both mRNA and protein levels in primary HCC, and was predictive of shorter recurrence-free survival after surgical resection in both univariate (hazard ratio [HR] 1.79; 95% CI 1.04-3.06; p = 0.03) and multivariate analyses (HR 1.73; CI 1.01-2.97; p = 0.04). Immunohistochemistry confirmed frequent downregulation of TLR3 in human and mouse primary HCC cells. None of the 6 human HCC cell lines analyzed expressed TLR3, and ectopic expression of TLR3 following lentiviral transduction not only restored the inflammatory response but also sensitized cells to TLR3-triggered apoptosis. Lastly, in the transgenic mouse model of HCC, absence of TLR3 expression was accompanied by a lower rate of preneoplastic hepatocyte apoptosis and accelerated hepatocarcinogenesis without altering the tumor immune infiltrate. CONCLUSION: Downregulation of TLR3 protects transforming hepatocytes from direct TLR3-triggered apoptosis, thereby contributing to hepatocarcinogenesis and poor patient outcome. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a heterogeneous disease associated with a poor prognosis. In patients with HCC, TLR3 downregulation is associated with reduced survival. Herein, we show that the absence of TLR3 is associated with a lower rate of apoptosis, and subsequently more rapid hepatocarcinogenesis, without any change to the immune infiltrate in the liver. Therefore, the poor prognosis associated with low TLR3 expression in HCC is likely linked to tumors ability to escape apoptosis. TLR3 may become a promising therapeutic target in TLR3-positive HCC.

Release of c-FLIP brake selectively sensitizes human cancer cells to TLR3-mediated apoptosis.

Toll-like receptor 3 (TLR3) mediates innate immune responses by sensing viral dsRNA, but also induces apoptosis selectively in cancer cells. Our analysis by immunohistochemistry revealed that TLR3 is frequently overexpressed in 130 non-small cell lung cancer (NSCLC) patients' samples compared with normal bronchial epithelium (P < 0.0001, Mann-Whitney test), supporting the therapeutic potential of TLR3 ligand for this type of cancer. However, a proportion of TLR3-expressing cancer cell lines, including NSCLC, remain resistant to TLR3-mediated apoptosis, and the underlying mechanism of resistance remains unclear. We here investigated the molecular basis conferring resistance to non-transformed vs. transformed cells against TLR3-mediated cell death. In non-transformed epithelial cells cellular FLICE-like inhibitory protein (c-FLIP) and cellular Inhibitor of APoptosis (cIAPs) ubiquitin ligases exerted an efficient double brake on apoptosis signaling. In contrast, releasing only one of these two brakes was sufficient to overcome the resistance of 8/8 cancer cell lines tested. Remarkably, the release of the c-FLIP, but not cIAPs, brake only results in the sensitization of all human cancer cells to TLR3-mediated apoptosis. Taking advantage of the difference between transformed and non-transformed cells, we developed a rational strategy by combining the chemotherapeutic agent paclitaxel, which decreases c-FLIP expression, with TLR3 ligand. This combination was highly synergistic for triggering apoptosis in cancer cells but not in non-transformed cells. In vivo, the combination of paclitaxel with dsRNA delayed tumor growth and prolonged survival in a mouse xenograft lung tumor model. In conclusion, combining the release of the c-FLIP brake with TLR3 ligand synergizes to selectively kill cancer cells, and could represent an efficient and safe therapy against TLR3-expressing cancers such as NSCLC.

Characterization of Pattern Recognition Receptor Expression and Functionality in Liver Primary Cells and Derived Cell Lines.

Different liver cell types are endowed with immunological properties, including cell-intrinsic innate immune functions that are important to initially control pathogen infections. However, a full landscape of expression and functionality of the innate immune signaling pathways in the major human liver cells is still missing. In order to comparatively characterize these pathways, we purified primary human hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells (LSEC), and Kupffer cells (KC) from human liver resections. We assessed mRNA and protein expression level of the major innate immune sensors, as well as checkpoint-inhibitor ligands in the purified cells, and found Toll-like receptors (TLR), RIG-I-like receptors, as well as several DNA cytosolic sensors to be expressed in the liver microenvironment. Amongst the cells tested, KC were shown to be most broadly active upon stimulation with PRR ligands emphasizing their predominant role in innate immune sensing the liver microenvironment. By KC immortalization, we generated a cell line that retained higher innate immune functionality as compared to THP1 cells, which are routinely used to study monocyte/macrophages functions. Our findings and the establishment of the KC line will help to understand immune mechanisms behind antiviral effects of TLR agonists or checkpoint inhibitors, which are in current preclinical or clinical development.

Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes.

Current therapies for chronic hepatitis B virus (HBV) infections are effective at decreasing the viral load in serum, but do not lead to viral eradication. Recent studies highlighted the therapeutic or "adjuvant" potential of immune-modulators. Our aim was to explore the direct anti-HBV effect of Toll-Like-Receptors (TLR) agonists in hepatocytes. HBV-infected primary human hepatocytes (PHH) or differentiated HepaRG cells (dHepaRG) were treated with various TLR agonists. Amongst all TLR ligands tested, Pam3CSK4 (TLR1/2-ligand) and poly(I:C)-(HMW) (TLR3/MDA5-ligand) were the best at reducing all HBV parameters. No or little viral rebound was observed after treatment arrest, implying a long-lasting effect on cccDNA. We also tested Riboxxol that features improved TLR3 specificity compared to poly(I:C)-(HMW). This agonist demonstrated a potent antiviral effect in HBV-infected PHH. Whereas, poly(I:C)-(HMW) and Pam3CSK4 mainly induced the expression of classical genes from the interferon or NF-κB pathway respectively, Riboxxol had a mixed phenotype. Moreover, TLR2 and TLR3 ligands can activate hepatocytes and immune cells, as demonstrated by antiviral cytokines produced by stimulated hepatocytes and peripheral blood mononuclear cells. In conclusion, our data highlight the potential of innate immunity activation in the direct control of HBV replication in hepatocytes, and support the development of TLR-based antiviral strategies.

Interaction between Toll-Like Receptor 9-CpG Oligodeoxynucleotides and Hepatitis B Virus Virions Leads to Entry Inhibition in Hepatocytes and Reduction of Alpha Interferon Production by Plasmacytoid Dendritic Cells.

We previously reported that Toll-like receptor 9 (TLR9)-CpG oligonucleotides could inhibit the establishment of hepatitis B virus (HBV) infections in hepatocytes. Our aim was to uncover the underlying mechanisms of this inhibition. HepaRG cells, RPMI-B lymphoblastoma cells, and primary plasmacytoid dendritic cells (pDCs) exposed to HBV and TLR9 ligands/agonists in various configurations were used. We observed an inhibition of HBV infection upon TLR9 stimulations only when agonist was applied during inoculation. This inhibition was independent of interleukin-6 (IL-6)/interferon-inducible protein 10 (IP-10) production as well as of TLR9 expression in hepatocytes. We further demonstrated an entry inhibition mechanism by showing a noncovalent binding of TLR9 agonist to HBV particles. Besides inhibiting HBV entry into hepatocytes, this biophysical interaction between HBV virions and TLR9 agonist was responsible for a reduction of alpha interferon (IFN-α) expression by pDCs. Interestingly, subviral particles composed of only HBsAg were able to genuinely inhibit the TLR9 pathway, without titrating TLR9 ligands. To conclude, our data suggest that synthetic TLR9-CpG oligonucleotides can strongly inhibit HBV entry by "coating" HBV virions and thereby preventing their interaction with cellular receptor. This titration effect of TLR9 agonist is also artifactually responsible for the inhibition of TLR9 engagement in pDCs, whereas a genuine inhibition of this innate pathway was confirmed with HBsAg subviral particles.

Toll-like receptor 7 agonist GS-9620 induces prolonged inhibition of HBV via a type I interferon-dependent mechanism.

BACKGROUND & AIMS: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. METHODS: Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. RESULTS: GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. CONCLUSIONS: Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. LAY SUMMARY: GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.

Expression and functionality of Toll- and RIG-like receptors in HepaRG cells.

BACKGROUND & AIMS: HepaRG cells are considered as the best surrogate model to primary human hepatocyte (PHH) culture to investigate host-pathogen interactions. Yet their innate immune functions remain unknown. In this study, we explored the expression and functionality of Toll-like (TLR) and retinoic acid-inducible gene-1 (RIG-I)-like receptors (RLR) in these cells. METHODS: Gene and protein expression levels of TLR-1 to 9 and RLR in HepaRG were mainly compared to PHH, by RT-qPCR, FACS, and Western blotting. Their functionality was assessed, by measuring the induction of toll/rig-like themselves and several target innate gene expressions, as well as the secretion of IL-6, IP-10, and type I interferon (IFN), upon agonist stimulation. Their functionality was also shown by measuring the antiviral activity of some TLR/RLR agonists against hepatitis B virus (HBV) infection. RESULTS: The basal gene and protein expression profile of TLR/RLR in HepaRG cells was similar to PHH. Most receptors, except for TLR-7 and 9, were expressed as proteins and functionally active as shown by the induction of some innate genes, as well as by the secretion of IL-6 and IP-10, upon agonist stimulation. The highest levels of IL-6 and IP-10 secretion were obtained by TLR-2 and TLR-3 agonist stimulation respectively. The highest preventive anti-HBV activity was obtained following TLR-2, TLR-4 or RIG-I/MDA-5 stimulations, which correlated with their high capacity to produce both cytokines. CONCLUSIONS: Our results indicate that HepaRG cells express a similar pattern of functional TLR/RLR as compared to PHH, thus qualifying HepaRG cells as a surrogate model to study pathogen interactions within a hepatocyte innate system.

Efficacy of continuous positive airway pressure treatment on 5-year survival in patients with ischaemic stroke and obstructive sleep apnea: a randomized controlled trial.

The main purpose of the present analysis is to assess the influence of introducing early nasal continuous positive airway pressure (nCPAP) treatment on cardiovascular recurrences and mortality in patients with a first-ever ischaemic stroke and moderate-severe obstructive sleep apnea (OSA) with an apnea-hypopnea index (AHI) ≥20 events h(-1) during a 5-year follow-up. Patients received conventional treatment for stroke and were assigned randomly to the nCPAP group (n = 71) or the control group (n = 69). Cardiovascular events and mortality were registered for all patients. Survival and cardiovascular event-free survival analysis were performed after 5-year follow-up using the Kaplan-Meier test. Patients in the nCPAP group had significantly higher cardiovascular survival than the control group (100 versus 89.9%, log-rank test 5.887; P = 0.015) However, and also despite a positive tendency, there were no significant differences in the cardiovascular event-free survival at 68 months between the nCPAP and control groups (89.5 versus 75.4%, log-rank test 3.565; P = 0.059). Early nCPAP therapy has a positive effect on long-term survival in ischaemic stroke patients and moderate-severe OSA.

Cleaved/associated TLR3 represents the primary form of the signaling receptor.

TLR3 belongs to the family of intracellular TLRs that recognize nucleic acids. Endolysosomal localization and cleavage of intracellular TLRs play pivotal roles in signaling and represent fail-safe mechanisms to prevent self-nucleic acid recognition. Indeed, cleavage by cathepsins is required for native TLR3 to signal in response to dsRNA. Using novel Abs generated against TLR3, we show that the conserved loop exposed in LRR12 is the single cleavage site that lies between the two dsRNA binding sites required for TLR3 dimerization and signaling. Accordingly, we found that the cleavage does not dissociate the C- and N-terminal fragments, but it generates a very stable "cleaved/associated" TLR3 present in endolysosomes that recognizes dsRNA and signals. Moreover, comparison of wild-type, noncleavable, and C-terminal-only mutants of TLR3 demonstrates that efficient signaling requires cleavage of the LRR12 loop but not dissociation of the fragments. Thus, the proteolytic cleavage of TLR3 appears to fulfill function(s) other than separating the two fragments to generate a functional receptor.

Factors associated with hospital admission for exacerbation of chronic obstructive pulmonary disease.

INTRODUCTION: Exacerbations of Chronic Obstructive Pulmonary Disease (COPD) that require hospital admission have a major impact on the progression of disease and generate high health costs. METHOD: A multi-center, cross-sectional, observational, study was conducted with the aim to identify factors associated with hospital admission in patients with COPD. We obtained data of socio-demographic and anthropometric characteristics, quality of life, respiratory symptoms, anxiety and depression, physical activity and pulmonary function tests. We analyzed their association with hospital admission with a multivariate analysis using a logistic regression model. RESULTS: We analyzed 127 patients, 50 (39%) of whom had been hospitalized. 93.7% were men, mean age 67 years (SD=9) and a FEV1 of 41.9% (SD=15.3). In the first model obtained, the baseline SpO(2), the BODE index and emergency room (ER) visits were associated with hospital admission and the area under the ROC curve (AUC) was 0.809. In a second model we included only variables readily available (without the 6 minutes walking test) and only the SpO(2) and previous visits to the ER were significant with an AUC ROC 0.783. CONCLUSIONS: hospital admission for exacerbation of COPD is associated with poor SpO(2), higher BODE index score and a greater number of visits to the ER. In case you do not have the 6 minutes walking test, the other two variables offer a similar discriminative ability.