The inhibition of YAP Signaling Prevents Chronic Biliary Fibrosis in the Abcb4-/- Model by Modulation of Hepatic Stellate Cell and Bile Duct Epithelium Cell Pathophysiology.

Primary sclerosing cholangitis (PSC) represents a chronic liver disease characterized by poor prognosis and lacking causal treatment options. Yes-associated protein (YAP) functions as a critical mediator of fibrogenesis; however, its therapeutic potential in chronic biliary diseases such as PSC remains unestablished. The objective of this study is to elucidate the possible significance of YAP inhibition in biliary fibrosis by examining the pathophysiology of hepatic stellate cells (HSC) and biliary epithelial cells (BEC). Human liver tissue samples from PSC patients were analyzed to assess the expression of YAP/connective tissue growth factor (CTGF) relative to non-fibrotic control samples. The pathophysiological relevance of YAP/CTGF in HSC and BEC was investigated in primary human HSC (phHSC), LX-2, H69, and TFK-1 cell lines through siRNA or pharmacological inhibition utilizing verteporfin (VP) and metformin (MF). The Abcb4-/- mouse model was employed to evaluate the protective effects of pharmacological YAP inhibition. Hanging droplet and 3D matrigel culture techniques were utilized to investigate YAP expression and activation status of phHSC under various physical conditions. YAP/CTGF upregulation was observed in PSC patients. Silencing YAP/CTGF led to inhibition of phHSC activation and reduced contractility of LX-2 cells, as well as suppression of epithelial-mesenchymal transition (EMT) in H69 cells and proliferation of TFK-1 cells. Pharmacological inhibition of YAP mitigated chronic liver fibrosis in vivo and diminished ductular reaction and EMT. YAP expression in phHSC was effectively modulated by altering extracellular stiffness, highlighting YAP's role as a mechanotransducer. In conclusion, YAP regulates the activation of HSC and EMT in BEC, thereby functioning as a checkpoint of fibrogenesis in chronic cholestasis. Both VP and MF demonstrate effectiveness as YAP inhibitors, capable of inhibiting biliary fibrosis. These findings suggest that VP and MF warrant further investigation as potential therapeutic options for the treatment of PSC.

Insulin Determines Transforming Growth Factor ß Effects on Hepatocyte Nuclear Factor 4α Transcription in Hepatocytes.

Loss of hepatocyte nuclear factor 4α (HNF4α) expression is frequently observed in end-stage liver disease and associated with loss of vital liver functions, thus increasing mortality. Loss of HNF4α expression is mediated by inflammatory cytokines, such as transforming growth factor (TGF)-ß. However, details of how HNF4α is suppressed are largely unknown to date. Herein, TGF-ß did not directly inhibit HNF4α but contributed to its transcriptional regulation by SMAD2/3 recruiting acetyltransferase CREB-binding protein/p300 to the HNF4α promoter. The recruitment of CREB-binding protein/p300 is indispensable for CCAAT/enhancer-binding protein α (C/EBPα) binding, another essential requirement for constitutive HNF4α expression in hepatocytes. Consistent with the in vitro observation, 67 of 98 patients with hepatic HNF4α expressed both phospho-SMAD2 and C/EBPα, whereas 22 patients without HNF4α expression lacked either phospho-SMAD2 or C/EBPα. In contrast to the observed induction of HNF4α, SMAD2/3 inhibited C/EBPα transcription. Long-term TGF-ß incubation resulted in C/EBPα depletion, which abrogated HNF4α expression. Intriguingly, SMAD2/3 inhibitory binding to the C/EBPα promoter was abolished by insulin. Two-thirds of patients without C/EBPα lacked membrane glucose transporter type 2 expression in hepatocytes, indicating insulin resistance. Taken together, these data indicate that hepatic insulin sensitivity is essential for hepatic HNF4α expression in the condition of inflammation.

Concomitant Irradiation to Checkpoint Inhibitor Therapy of Hepatocellular Carcinoma Patients: A Systematic Retrospective, Single-Center Analysis.

INTRODUCTION: Immunotherapy has been established as the standard treatment option for patients with advanced hepatocellular carcinoma (aHCC). Despite the increased efficacy, disease progression occurs in a relevant proportion of patients even after an objective response. Combination concepts with locoregional therapy are currently under investigation for hepatic disease but are also in discussion for the control of distant metastasis. Radiotherapy is a highly effective treatment modality for local tumor control. It is also thought to increase the efficacy of checkpoint inhibition and sensitize distant lesions to the effects of immunotherapy, but may potentially increase adverse effects. In our center, few patients with aHCC treated with immune checkpoint inhibitors (ICIs) received concomitant radiotherapy for symptom or disease control. The aim of this study was to retrospectively analyze adverse effects and efficacy of concomitant radiotherapy in patients with aHCC treated with checkpoint inhibition. METHODS: To this aim, patients who received a combination of ICI and radiotherapy in our institution were retrospectively considered for analysis. The predefined inclusion criterion was radiotherapy after initiated checkpoint inhibition and continuation of ICI therapy for at least 8 weeks. Adverse effects and efficacy measurements were performed according to local standards. RESULTS: The database search of 2016-2021 revealed six consecutive patients fulfilling the predefined criteria for concomitant ICI and radiotherapy. Three patients received high-dose-rate brachytherapy (15 Gy) to treat progredient hepatic lesions. Two patients received stereotactic body radiotherapy (SBRT) (25-30 Gy) for symptom control, and 1 patient received brachytherapy and SBRT to treat metastases. No severe adverse events were reported in the period (<6 months) after concomitant radiotherapy. In 5 out of 6 cases, long-term tumor control could be achieved by this therapeutic combination. CONCLUSION: A good efficacy of concomitant radiotherapy and checkpoint inhibition has been achieved with no safety concerns. Further investigations should evaluate the safety, appropriate clinical context, and efficacy of this promising approach.

Changing treatment landscape associated with improved survival in advanced hepatocellular carcinoma: a nationwide, population-based study.

BACKGROUND AND AIMS: The treatment of hepatocellular carcinoma (HCC) is undergoing a historic transformation with the approval of several new systemic therapies in the last few years. This study aimed to examine the impact of this changing landscape on survival and costs in a Western nationwide, real-world cohort. METHODS: A nationwide representative claims database (InGef) was screened for HCC cases between 2015 and 2020. Survival in an era with only sorafenib (period A, January 2015 to July 2018) and after approval of lenvatinib and other systemic treatments (period B, August 2018 to December 2020) was analysed. Health care costs were assessed. RESULTS: We identified 2876 individuals with HCC in the study period. The proportion of patients receiving systemic therapy increased significantly over time, from 11.8% in 2015 to 15.1% in 2020 (p < 0.0001). The median overall survival in period B was 6.5 months (95% confidence interval [CI]: 4.9-8.9) and in period A was 5.3 months (95% CI: 4.5-6.3; p = 0.046). In period B, the median overall survival with lenvatinib was 9.7 months (95% CI: 6.3-18.4) versus 4.8 months with sorafenib (95% CI: 4.0-7.1, p = 0.008). Costs for prescription drugs per patient increased from €6150 in 2015 to €9049 in 2020 (p < 0.0001), and costs for outpatient care per patient increased from €1646 to €2149 (p = 0.0240). CONCLUSION: The approval of new systemic therapies resulted in a survival benefit in patients with HCC. The magnitude of the effect is modest and associated with a moderate increase in health costs.

Thyroidal Angiotensin-Converting Enzyme 2 Protein Expression and Thyroid Function Tests in Patients with COVID-19: Results from a Retrospective Case Series and a Prospective Cohort Study.

Background: Infection with SARS-CoV-2 has initially been known as a respiratory disease but in the course of the pandemic the understanding has emerged that severity is owing to fatal inflammatory responses apart from lung injury. In this context, endocrine disorders such as thyroiditis as well as pituitary dysfunction in addition to nonthyroidal illness syndrome have been described. Furthermore, angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 cell receptor, has been detected in most endocrine tissues, including the thyroid gland. Objective: To evaluate histopathologic changes and compare thyroidal ACE2 protein expression in thyroid tissue from patients who died from severe COVID-19 with thyroid tissue from patients without SARS-CoV-2 infection in a retrospective case series. Furthermore, to assess and compare alterations in thyroid function tests (TFTs) between patients with or without SARS-CoV-2 infection as well as association of TFTs with the severity of the disease in a prospective cohort study. Methods: Thyroid tissue of deceased COVID-19 patients (n = 23) was analyzed for histopathology and ACE2 expression by immunohistochemical staining. A total of 153 patients with confirmed SARS-CoV-2 were evaluated regarding TFTs and divided into a severe (intubation, intensive care treatment) and an intermediate group. Results: Thyroidal ACE2 expression was detected in 87% of the deceased COVID-19 patients. Normal thyroid tissue from patients without SARS-CoV-2 infection showed no ACE2 protein expression. Half of the severely ill COVID-19 patients had low free triiodothyronine (fT3) levels. Combination of low fT3 and thyrotropin (TSH) was associated significantly with deadly disease. Conclusion: The high percentage of positive ACE2 immunostaining in deceased patients compared with normal thyroid tissue of patients without SARS-CoV-2 infection suggests involvement of the thyroid in COVID-19, although further research will have to show the pathogenic role of thyroidal ACE2 in COVID-19. Abnormal fT3 and a TSH of ≤0.5 mU/L were associated with a fatal outcome in our severely ill SARS-CoV-2 patient cohort. Therefore, assessment of TFTs is crucial in the treatment of severely ill COVID-19 patients. Trial Registration: COVID-19 Registry of the LMU University Hospital Munich (CORKUM), WHO trial ID DRKS00021225.

Ipilimumab and nivolumab in advanced hepatocellular carcinoma after failure of prior immune checkpoint inhibitor-based combination therapies: a multicenter retrospective study.

INTRODUCTION: Immune checkpoint inhibitor (ICI)-based regimens are transforming the landscape of hepatocellular carcinoma (HCC) treatment. We describe the effect of combined ipilimumab and nivolumab in patients with advanced HCC after the failure of prior ICI-based combination treatments. METHODS: The clinical course of patients with advanced HCC who received combined ipilimumab and nivolumab after prior ICI-based combination therapies was assessed. Progression-free survival (PFS), overall response rate (ORR) and disease control rate (DCR) per RECIST v1.1 and mRECIST, overall survival (OS), and safety were analyzed. RESULTS: Of 109 patients treated with atezolizumab and bevacizumab or other ICI-based combination treatments, ten patients received subsequent therapy with ipilimumab and nivolumab. The majority of patients had Barcelona Clinic Liver Cancer (BCLC) Stage C (80%) HCC and a preserved liver function as defined by Child-Pugh A (80%). At a median follow-up of 15.3 months, ORR for ipilimumab and nivolumab was 30% with a DCR of 40%. Median PFS was 2.9 months and the median OS was 7.4 months. CONCLUSION: This retrospective study demonstrates that combined ipilimumab and nivolumab can be effective and tolerable after prior ICI-based combination therapies and provides a rationale for the prospective clinical evaluation of this treatment sequencing.

FOXA2 prevents hyperbilirubinaemia in acute liver failure by maintaining apical MRP2 expression.

OBJECTIVE: Multidrug resistance protein 2 (MRP2) is a bottleneck in bilirubin excretion. Its loss is sufficient to induce hyperbilirubinaemia, a prevailing characteristic of acute liver failure (ALF) that is closely associated with clinical outcome. This study scrutinises the transcriptional regulation of MRP2 under different pathophysiological conditions. DESIGN: Hepatic MRP2, farnesoid X receptor (FXR) and Forkhead box A2 (FOXA2) expression and clinicopathologic associations were examined by immunohistochemistry in 14 patients with cirrhosis and 22 patients with ALF. MRP2 regulatory mechanisms were investigated in primary hepatocytes, Fxr -/- mice and lipopolysaccharide (LPS)-treated mice. RESULTS: Physiologically, homeostatic MRP2 transcription is mediated by the nuclear receptor FXR/retinoid X receptor complex. Fxr-/- mice lack apical MRP2 expression and rapidly progress into hyperbilirubinaemia. In patients with ALF, hepatic FXR expression is undetectable, however, patients without infection maintain apical MRP2 expression and do not suffer from hyperbilirubinaemia. These patients express FOXA2 in hepatocytes. FOXA2 upregulates MRP2 transcription through binding to its promoter. Physiologically, nuclear FOXA2 translocation is inhibited by insulin. In ALF, high levels of glucagon and tumour necrosis factor α induce FOXA2 expression and nuclear translocation in hepatocytes. Impressively, ALF patients with sepsis express low levels of FOXA2, lose MRP2 expression and develop severe hyperbilirubinaemia. In this case, LPS inhibits FXR expression, induces FOXA2 nuclear exclusion and thus abrogates the compensatory MRP2 upregulation. In both Fxr -/- and LPS-treated mice, ectopic FOXA2 expression restored apical MRP2 expression and normalised serum bilirubin levels. CONCLUSION: FOXA2 replaces FXR to maintain MRP2 expression in ALF without sepsis. Ectopic FOXA2 expression to maintain MRP2 represents a potential strategy to prevent hyperbilirubinaemia in septic ALF.

TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status.

Chemotherapy, the standard treatment for pancreatic ductal adenocarcinoma (PDAC), has only a modest effect on the outcome of patients with late-stage disease. Investigations of the genetic features of PDAC have demonstrated a frequent occurrence of mutations in genes involved in homologous recombination (HR), especially in the breast cancer susceptibility gene 2 (BRCA2). Olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, is approved as a maintenance treatment for patients with advanced PDAC with germline BRCA1/2 mutations following a platinum-containing first-line regimen. Limitations to the use of PARP inhibitors are represented by the relatively small proportion of patients with mutations in BRCA1/2 genes and the modest capability of these substances of inducing objective response. We have previously shown that pancreatic cancer with BRCA2 mutations exhibits a remarkably enhanced sensitivity towards tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) receptor-stimulating agents. We thus aimed to investigate the effect of combined treatment with PARP inhibitors and TRAIL receptor-stimulating agents in pancreatic cancer and its dependency on the BRCA2 gene status. The respective effects of TRAIL-targeting agents and the PARP inhibitor olaparib or of their combination were assessed in pancreatic cancer cell lines and patient-derived organoids. In addition, BRCA2-knockout and -complementation models were investigated. The effects of these agents on apoptosis, DNA damage, cell cycle, and receptor surface expression were assessed by immunofluorescence, Western blot, and flow cytometry. PARP inhibition and TRAIL synergized to cause cell death in pancreatic cancer cell lines and PDAC organoids. This effect proved independent of BRCA2 gene status in three independent models. Olaparib and TRAIL in combination caused a detectable increase in DNA damage and a concentration-dependent cell cycle arrest in the G2/M and S cell cycle phases. Olaparib also significantly increased the proportion of membrane-bound death receptor 5. Our results provide a preclinical rationale for the combination of PARP inhibitors and TRAIL receptor agonists for the treatment of pancreatic cancer and suggest that the use of PARP inhibitors could be extended to patients without BRCA2 mutations if used in combination with TRAIL agonists.

Hepatic perfusion as a new predictor of prognosis and mortality in critical care patients with acute-on-chronic liver failure.

Background and aims: Liver diseases are frequent causes of morbidity and mortality worldwide. Liver diseases can lead to cirrhosis, with the risk of acute-on-chronic liver failure (ACLF). For the detection of changes in hepatic hemodynamics, Doppler ultrasonography is a well-established method. We investigated hepatic hemodynamics via serial Doppler ultrasonography to determine the predictive value of changes in hepatic perfusion for the outcome in patients with severe liver diseases compared to established prognostic models such as the MELD (Model for End-Stage Liver Disease) or CLIF-C (Chronic Liver Failure-Consortium) ACLF score. Methods: In this prospective cohort study, hepatic perfusion was quantified at baseline before the initiation of treatment and every third day by means of serial measurements of the hepatic artery resistance index (HARI) and the maximum portal vein velocity (PVv) using Doppler ultrasonography in 50 consecutive patients with severe liver diseases admitted to a medical intensive care unit (MICU). The recorded hemodynamic parameters were compared to the MELD score, and the CLIF-C ACLF score to analyze their utility for the prediction of the outcome of patients with severe liver diseases, liver cirrhosis, and ACLF. Results: The changes (delta) obtained by serial measurements of the MELD score, HARI, and PVv were analyzed through scatter plots. Bivariate correlation analysis yielded a new positive linear correlation between the delta-HARI and the delta-MELD score (r = 0.469; p < 0.001). In addition, our data revealed a new negative linear correlation between delta-PVv and the delta-MELD score (r = -0.279, p = 0.001). The leading cause of MICU mortality was acute-on-chronic liver failure (ACLF). A subgroup analysis of patients with liver cirrhosis revealed a positive linear correlation between the delta-HARI and the delta-CLIF-C-ACLF score (r = 0.252, p = 0.005). Of clinical relevance, non-survivors of ACLF exhibited a significantly higher mean value for the delta-HARI (0.010 vs. -0.005; p = 0.015) and a lower mean value for the delta-PVv (-0.7 vs. 1.9 cm/s; p = 0.037) in comparison to survivors of ACLF. Conclusion: This study shows the prognostic value of the assessment of hepatic perfusion in critical care patients with severe liver diseases by bedside Doppler ultrasound examination and its utility as an accurate predictor of the outcome in patients with ACLF. Increasing HARI and a decreasing PVv are predictors of an adverse outcome. Delta-HARI and delta-PVv are new biomarkers of prognosis and ACLF-related mortality in patients with liver diseases. Delta-HARI and delta-PVv may be helpful in guiding clinical decision-making, especially in catecholamine and fluid management.

Comparative Response of HCC Cells to TKIs: Modified in vitro Testing and Descriptive Expression Analysis.

Introduction: Although the treatment paradigm for hepatocellular carcinoma (HCC) has recently shifted in favour of checkpoint inhibitor (CPI)-based treatment options, the tyrosine kinase inhibitors (TKI) currently approved for the treatment of HCC are expected to remain the cornerstone of HCC treatment alone or in combination with CPIs. Despite considerable research efforts, no biomarker capable of predicting the response to specific TKIs has been validated. Thus, personalized approaches to HCC may aid in determining optimal treatment lines for 2nd and 3rd lines. To identify new biomarkers, we examined differential sensitivity and investigated potential transcriptomic predictors. Methods: To this aim, the sensitivity of nine HCC cell lines to sorafenib, lenvatinib, regorafenib, and cabozantinib was evaluated by a prolonged treatment scheme to determine their respective growth rate inhibition concentrations (GR50). Subgroups discriminated by GR50 values underwent differential expression and gene set enrichment analysis (GSEA). Results: The nine cell lines showed broadly different sensitivities to different TKIs. GR50 values of sorafenib and regorafenib clustered closer in all cell lines, whereas treatments with lenvatinib and cabozantinib showed diversified GR50 values. GSEA showed the activation of specific pathways in sensitive vs non-sensitive cell lines. A signature consisting of 14 biomarkers (GAGE12H, GJB6, PTCHD3, PRH1-PRR4, C6orf222, HBB, C17orf99, GOLGA6A, CRYAA, CCL23, RP11-347C12.3, RP11-514O12.4, FAM180B, and TMPRSS4) discriminates the cell lines' response into three distinct treatment profiles: 1) equally sensible to sorafenib, regorafenib and cabozantinib, 2) sensible to lenvatinib, and 3) more sensible to regorafenib than sorafenib. Conclusion: We observed diverse responses to either of the four TKIs. Subgroup analysis of TKI effectiveness showed distinct transcriptomic profiles and signaling pathways associated with responsiveness. This prompts more extensive studies to explore and validate pharmacogenomic and transcriptomic strategies for a personalized treatment approach, particularly after the failure of CPI treatment.

A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.

BACKGROUND AND AIMS: It remains unknown how patients with liver failure maintain essential albumin levels. Here, we delineate a hierarchical transcription regulatory network that ensures albumin expression under different disease conditions. APPROACH AND RESULTS: We examined albumin levels in liver tissues and serum in 157 patients, including 84 with HCC, 38 decompensated cirrhosis, and 35 acute liver failure. Even in patients with liver failure, the average serum albumin concentrations were 30.55 g/L. In healthy subjects and patients with chronic liver diseases, albumin was expressed in hepatocytes. In patients with massive hepatocyte loss, albumin was expressed in liver progenitor cells (LPCs). The albumin gene (ALB) core promoter possesses a TATA box and nucleosome-free area, which allows constitutive RNA polymerase II binding and transcription initiation. Chromatin immunoprecipitation assays revealed that hepatocyte nuclear factor 4 alpha (HNF4α), CCAAT/enhancer-binding protein alpha (C/EBPα), and forkhead box A2 (FOXA2) bound to the ALB enhancer. Knockdown of either of these factors reduced albumin expression in hepatocytes. FOXA2 acts as a pioneer factor to support HNF4α and C/EBPα. In hepatocytes lacking HNF4α and C/EBPα expression, FOXA2 synergized with retinoic acid receptor (RAR) to maintain albumin transcription. RAR nuclear translocation was induced by retinoic acids released by activated HSCs. In patients with massive hepatocyte loss, LPCs expressed HNF4α and FOXA2. RNA sequencing and quantitative PCR analyses revealed that lack of HNF4α and C/EBPα in hepatocytes increased hedgehog ligand biosynthesis. Hedgehog up-regulates FOXA2 expression through glioblastoma family zinc finger 2 binding to the FOXA2 promoter in both hepatocytes and LPCs. CONCLUSIONS: A hierarchical regulatory network formed by master and pioneer transcription factors ensures essential albumin expression in various pathophysiological conditions.

Follistatin-controlled activin-HNF4α-coagulation factor axis in liver progenitor cells determines outcome of acute liver failure.

BACKGROUND AND AIMS: In patients with acute liver failure (ALF) who suffer from massive hepatocyte loss, liver progenitor cells (LPCs) take over key hepatocyte functions, which ultimately determines survival. This study investigated how the expression of hepatocyte nuclear factor 4α (HNF4α), its regulators, and targets in LPCs determines clinical outcome of patients with ALF. APPROACH AND RESULTS: Clinicopathological associations were scrutinized in 19 patients with ALF (9 recovered and 10 receiving liver transplantation). Regulatory mechanisms between follistatin, activin, HNF4α, and coagulation factor expression in LPC were investigated in vitro and in metronidazole-treated zebrafish. A prospective clinical study followed up 186 patients with cirrhosis for 80 months to observe the relevance of follistatin levels in prevalence and mortality of acute-on-chronic liver failure. Recovered patients with ALF robustly express HNF4α in either LPCs or remaining hepatocytes. As in hepatocytes, HNF4α controls the expression of coagulation factors by binding to their promoters in LPC. HNF4α expression in LPCs requires the forkhead box protein H1-Sma and Mad homolog 2/3/4 transcription factor complex, which is promoted by the TGF-ß superfamily member activin. Activin signaling in LPCs is negatively regulated by follistatin, a hepatocyte-derived hormone controlled by insulin and glucagon. In contrast to patients requiring liver transplantation, recovered patients demonstrate a normal activin/follistatin ratio, robust abundance of the activin effectors phosphorylated Sma and Mad homolog 2 and HNF4α in LPCs, leading to significantly improved coagulation function. A follow-up study indicated that serum follistatin levels could predict the incidence and mortality of acute-on-chronic liver failure. CONCLUSIONS: These results highlight a crucial role of the follistatin-controlled activin-HNF4α-coagulation axis in determining the clinical outcome of massive hepatocyte loss-induced ALF. The effects of insulin and glucagon on follistatin suggest a key role of the systemic metabolic state in ALF.

Pulmonary function impairment of asymptomatic and persistently symptomatic patients 4 months after COVID-19 according to disease severity.

OBJECTIVE: Evaluation of pulmonary function impairment after COVID-19 in persistently symptomatic and asymptomatic patients of all disease severities and characterisation of risk factors. METHODS: Patients with confirmed SARS-CoV-2 infection underwent prospective follow-up with pulmonary function testing and blood gas analysis during steady-state cycle exercise 4 months after acute illness. Pulmonary function impairment (PFI) was defined as reduction below 80% predicted of DLCOcSB, TLC, FVC, or FEV1. Clinical data were analyzed to identify risk factors for impaired pulmonary function. RESULTS: 76 patients were included, hereof 35 outpatients with mild disease and 41 patients hospitalized due to COVID-19. Sixteen patients had critical disease requiring mechanical ventilation, 25 patients had moderate-severe disease. After 4 months, 44 patients reported persisting respiratory symptoms. Significant PFI was prevalent in 40 patients (52.6%) occurring among all disease severities. The most common cause for PFI was reduced DLCOcSB (n = 39, 51.3%), followed by reduced TLC and FVC. The severity of PFI was significantly associated with mechanical ventilation (p < 0.001). Further risk factors for DLCO impairment were COPD (p < 0.001), SARS-CoV-2 antibody-Titer (p = 0.014) and in hospitalized patients CT score. A decrease of paO2 > 3 mmHg during cycle exercise occurred in 1/5 of patients after mild disease course. CONCLUSION: We characterized pulmonary function impairment in asymptomatic and persistently symptomatic patients of different severity groups of COVID-19 and identified further risk factors associated with persistently decreased pulmonary function. Remarkably, gas exchange abnormalities were revealed upon cycle exercise in some patients with mild disease courses and no preexisting pulmonary condition.

Polyomavirus exerts detrimental effects on renal function in patients after lung transplantation.

INTRODUCTION: Chronic kidney disease (CKD) is associated with significant morbidity and mortality after lung transplantation (LTX). Calcineurin inhibitor (CNI) nephrotoxicity is the leading cause of CKD. After kidney transplantation, polyomavirus-associated nephropathy (PyVAN) is a well-recognized problem. This study aims to evaluate the role of polyomavirus in patients after LTX. METHODS: From January 2017 to January 2020, all lung transplant recipients who performed follow-up visits in our center were included in the study and retrospectively assessed. We measured renal function (creatinine levels before and after transplantation), JCPyV, and BKPyV load by polymerase chain reaction (PCR) in serum and urine samples after transplantation. RESULTS: In total, 104 consecutive patients (59 males, 56.7%) with a mean age of 49.6 ± 11.1 years were identified. JCPyV was found in urine of 36 patients (34.6%) and serum of 3 patients (2.9%). BKPyV was found in urine of 40 patients (38.5%) and serum of 4 patients (3.8%), respectively. Urine evidence for JCPyV (p < 0.001, coefficient: +21.44) and BKPyV (p < 0.001, coefficient: +29.65) correlated highly with further kidney function decline. CONCLUSION: Kidney function deterioration is associated with JCPyV and BKPyV viruria in patients after LTX. This might indicate a role of PyVAN in lung transplant recipients.

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 followed by community and nosocomial spread, Germany, February to May 2020.

BackgroundIn the SARS-CoV-2 pandemic, viral genomes are available at unprecedented speed, but spatio-temporal bias in genome sequence sampling precludes phylogeographical inference without additional contextual data.AimWe applied genomic epidemiology to trace SARS-CoV-2 spread on an international, national and local level, to illustrate how transmission chains can be resolved to the level of a single event and single person using integrated sequence data and spatio-temporal metadata.MethodsWe investigated 289 COVID-19 cases at a university hospital in Munich, Germany, between 29 February and 27 May 2020. Using the ARTIC protocol, we obtained near full-length viral genomes from 174 SARS-CoV-2-positive respiratory samples. Phylogenetic analyses using the Auspice software were employed in combination with anamnestic reporting of travel history, interpersonal interactions and perceived high-risk exposures among patients and healthcare workers to characterise cluster outbreaks and establish likely scenarios and timelines of transmission.ResultsWe identified multiple independent introductions in the Munich Metropolitan Region during the first weeks of the first pandemic wave, mainly by travellers returning from popular skiing areas in the Alps. In these early weeks, the rate of presumable hospital-acquired infections among patients and in particular healthcare workers was high (9.6% and 54%, respectively) and we illustrated how transmission chains can be dissected at high resolution combining virus sequences and spatio-temporal networks of human interactions.ConclusionsEarly spread of SARS-CoV-2 in Europe was catalysed by superspreading events and regional hotspots during the winter holiday season. Genomic epidemiology can be employed to trace viral spread and inform effective containment strategies.

Dynamics of SARS-CoV-2 shedding in the respiratory tract depends on the severity of disease in COVID-19 patients.

A fraction of COVID-19 patients progress to a severe disease manifestation with respiratory failure and the necessity of mechanical ventilation. Identifying patients at risk is critical for optimised care and early therapeutic interventions. We investigated the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shedding relative to disease severity.We analysed nasopharyngeal and tracheal shedding of SARS-CoV-2 in 92 patients with diagnosed COVID-19. Upon admission, standardised nasopharyngeal swab or sputum samples were collected. If patients were mechanically ventilated, endotracheal aspirate samples were additionally obtained. Viral shedding was quantified by real-time PCR detection of SARS-CoV-2 RNA.45% (41 out of 92) of COVID-19 patients had a severe disease course with the need for mechanical ventilation (severe group). At week 1, the initial viral shedding determined from nasopharyngeal swabs showed no significant difference between nonsevere and severe cases. At week 2, a difference could be observed as the viral shedding remained elevated in severely ill patients. A time-course of C-reactive protein, interleukin-6 and procalcitonin revealed an even more protracted inflammatory response following the delayed drop of virus shedding load in severely ill patients. A significant proportion (47.8%) of patients showed evidence of prolonged viral shedding (>17 days), which was associated with severe disease courses (73.2%).We report that viral shedding does not differ significantly between severe and nonsevere COVID-19 cases upon admission to the hospital. Elevated SARS-CoV-2 shedding in the second week of hospitalisation, a systemic inflammatory reaction peaking between the second and third week, and prolonged viral shedding are associated with a more severe disease course.

Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma.

OBJECTIVES: Hepatocellular carcinoma (HCC) is a leading cancer-related cause of death. Unfortunately, recurrence is common even after curative treatment of early-stage patients, and no adjuvant treatment has yet been established. Aberrant expression of OLFM4 in human cancers has been reported; yet, its specific function during tumor development remains poorly understood, and its role in HCC is unknown. The purpose of this study is to examine the prognostic significance of OLFM4 and its functional relevance in determining recurrence in patients with early-stage HCC. METHODS: Immunohistochemical staining to assess expression, cellular distribution, and prognostic significance of OLFM4 was performed in a tissue microarray comprising 157 HCC tissues and matched nontumor tissues. In addition, expression of OLFM4-coding mRNA was assessed in a separate patients' cohort. The findings were validated by in vitro functional studies using siRNA directed against OLFM4 to assess its effect on cell motility and proliferation. RESULTS: The fraction of HCC samples exhibiting positive OLFM4 staining was higher in comparison with that observed in hepatocytes from matched nontumor tissue (61% vs 39%). However, cytoplasmic-only staining for OLFM4 was associated with vascular invasion (P = 0.048), MMP-7 expression (P = 0.002), and poorer survival (P = 0.008). A multivariate analysis confirmed the independent significance of OLFM4 in determining patients' outcome (5-year survival [58.3% vs 17.3%; HR: 2.135 {95% confidence interval: 1.135-4.015}; P = 0.019]). Correspondingly, inhibition of OLFM4 by siRNA modulated the expression of MMP-7 and E-cadherin, causing inhibition of cell proliferation, motility, and migration. DISCUSSION: To the best of our knowledge, we provide the first report on the prognostic significance of OLFM4 in HCC and identify its mechanistic role as crucial mediator of MMP family protein and E-Cadherin in determining cell invasion and metastasis formation.

EpiCO (epirubicin, cyclophosphamide and vincristine) as treatment for extrapulmonary high-grade neuroendocrine neoplasms.

High-grade neuroendocrine neoplasms (NEN) comprise a rare entity. Due to the lack of randomized controlled trials, therapy recommendations were mainly extrapolated from its pulmonary analogue, small cell lung cancer and mostly validated in small retrospective case series. The multicentric Nordic NEC Study of gastro-entero-pancreatic (GEP) and cancer of unknown primary (CUP) high-grade neuroendocrine neoplasms showed a significant disease control upon treatment with etoposide and platinum-based chemotherapies 1. Such a combination with etoposide and a platinum (CE) compound is currently considered standard first-line treatment for high-grade GEP/CUP NEN. High-grade mixed-neuroendocrine-non-neuroendocrine neoplasms (MiNEN) formerly termed mixed adeno-neuroendocrine carcinomas (MANEC) also have a poor prognosis and are generally treated like other high-grade NEN. The CE protocol has significant activity in high-grade NEN and MiNEN, but the response is short-lived in most cases with response rates around 50-60 %. Second-line treatment alternatives are not established so far. The need for additional treatment options is evident.Combination chemotherapy with doxorubicin, cyclophosphamide and vincristine (CAV) showed efficacy in small cell lung carcinoma (SCLC) and was considered standard first-line therapy before the era of etoposide and platinum combinations. Due to a better toxicity profile, doxorubicin was replaced by epirubicin, resulting in the combination of epirubicin, cyclophosphamide and vincristine (abbreviated as EpiCO or CEV).In analogy to SCLC, selected patients with high-grade NEN were treated with the EpiCO regimen in second line (or in one patient first line) at our center. In this report we present the retrospective series of 5 cases with metastatic high-grade GEP/CUP NEN/MiNEN who received chemotherapy according to this protocol.