SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.

PPARG activation promotes the proliferation of colorectal cancer cell lines and enhances the antiproliferative effect of 5-fluorouracil.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARG) is a member of the nuclear receptor family. It is involved in the regulation of adipogenesis, lipid metabolism, insulin sensitivity, vascular homeostasis and inflammation. In addition, PPARG agonists, known as thiazolidinediones, are well established in the treatment of type 2 diabetes mellitus. PPARGs role in cancer is a matter of debate, as pro- and anti-tumour properties have been described in various tumour entities. Currently, the specific role of PPARG in patients with colorectal cancer (CRC) is not fully understood. MATERIAL AND METHODS: The prognostic impact of PPARG expression was investigated by immunohistochemistry in a case-control study using a matched pair selection of CRC tumours (n = 246) with either distant metastases to the liver (n = 82), lung (n = 82) or without distant metastases (n = 82). Its effect on proliferation as well as the sensitivity to the chemotherapeutic drug 5-fluorouracil (5-FU) was examined after activation, inhibition, and transient gene knockdown of PPARG in the CRC cell lines SW403 and HT29. RESULTS: High PPARG expression was significantly associated with pulmonary metastasis (p = 0.019). Patients without distant metastases had a significantly longer overall survival with low PPARG expression in their tumours compared to patients with high PPARG expression (p = 0.045). In the pulmonary metastasis cohort instead, a trend towards longer survival was observed for patients with high PPARG expression in their tumour (p = 0.059). Activation of PPARG by pioglitazone and rosiglitazone resulted in a significant dose-dependent increase in proliferation of CRC cell lines. Inhibition of PPARG by its specific inhibitor GW9662 and siRNA-mediated knockdown of PPARG significantly decreased proliferation. Activating PPARG significantly increased the CRC cell lines sensitivity to 5-FU while its inhibition decreased it. CONCLUSION: The prognostic effect of PPARG expression depends on the metastasis localization in advanced CRC patients. Activation of PPARG increased malignancy associated traits such as proliferation in CRC cell lines but also increases sensitivity towards the chemotherapeutic agent 5-FU. Based on this finding, a combination therapy of PPARG agonists and 5-FU-based chemotherapy constitutes a promising strategy which should be further investigated.

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.

Endoscopic Vacuum Therapy for Left-Sided Colorectal Anastomotic Leak Without Fecal Diversion.

BACKGROUND: Endoscopic vacuum therapy for the treatment of rectal anastomotic leak has been shown to be effective and safe. The majority of patients are treated after fecal diversion to avoid further septic complications. OBJECTIVE: To report the effectiveness of endoscopic vacuum therapy for rectal anastomotic leak without diversion compared to secondary stoma creation. DESIGN: Retrospective cohort analysis. SETTINGS: University hospital, single-center. PATIENTS: Patients undergoing sigmoid or rectal resection without fecal diversion during primary surgery who were treated with endoscopic vacuum therapy for clinically relevant anastomotic leak. MAIN OUTCOME MEASURES: Treatment success (sepsis control, granulation and closure of the leak cavity, and no subsequent interventional or surgical procedure required); treatment duration; complications associated with endoscopic vacuum therapy; outpatient treatment; and restoration of intestinal continuity in diverted patients. RESULTS: Fifty-seven patients were included. In 20 patients (35%), endoscopic vacuum therapy was initiated without secondary diversion since the leak was extraperitoneal, and the sponge could be placed into the leak cavity with an adequate seal toward the lumen. In 18 patients (90%), this approach was successful. None of these patients required subsequent diversion in the further course of their disease. In two patients, secondary diversion was necessary due to treatment failure. Balloon dilatation for luminal stenosis was required in two patients. When comparing patient and treatment characteristics of patients with and without a stoma, including treatment success and duration, no significant differences were found. Restoration of intestinal continuity was achieved in 69% of diverted patients. LIMITATIONS: Unrandomized, retrospective study design; confounding factors of treatment assignment; low patient numbers and short follow-up of diverted patients; and low statistical power. CONCLUSION: In this single-institution study, endoscopic vacuum therapy for rectal anastomotic leak was successful in 90% of patients without diversion with regard to sepsis control, granulation of the leak cavity, avoidance of surgery, and long-term stoma-free survival. See Video Abstract at http://links.lww.com/DCR/B737.TERAPIA ENDOSCÓPICA POR ASPIRACIÓN AL VACÍO EN CASOS DE FUGA ANASTOMÓTICA RECTO-CÓLICA IZQUIERDA SIN OSTOMÍA DE PROTECCIÓNANTECEDENTES:Se ha demostrado que la terapia endoscópica por aspiración al vacío en casos de fuga anastomótica recto-cólica izquierda en el tratamiento de la fuga anastomótica rectal es eficaz y segura. La mayoría de los casos beneficiaron del tratamiento después de la confeción de un ostomía de protección para evitar más complicaciones sépticas.OBJETIVO:Demostrar la efectividad de la terapia endoscópica por aspiración al vacío en casos de fuga anastomótica recto-cólica izquierda sin ostomía de protección comparada con los casos que tuvieron la creación de una ostomía secundaria.DISEÑO:Análisis de cohortes de tipo retrospectivo.AJUSTE:Hospital universitario, unicéntrico.PACIENTES:Aquellos pacientes sometidos a una resección sigmoidea o rectal sin ostomía de protección durante una cirugía primaria, y que fueron tratados con terapia endoscópica por aspiración al vacío en caso de fuga anastomótica clínicamente relevante.PRINCIPALES MEDIDAS DE RESULTADO:Tratamiento exitoso (control de la sepsis, granulación y cierre de la cavidad de la fuga, sin requerir procedimiento quirúrgico o intervención ulteterior); duración del tratamiento; complicaciones asociadas con la terapia endoscópica por aspiración al vacío; tratamiento ambulatorio; restablecimiento de la continuidad intestinal en los pacientes portadores de ostomía.RESULTADOS:Se incluyeron 57 pacientes. En 20 pacientes (35%), se inició la terapia endoscópica por aspiración al vacío sin derivación secundaria, ya que la fuga era extraperitoneal y la esponja podía colocarse en la cavidad de la fuga con un sellado adecuado hacia el lumen. En 18 pacientes (90%), este enfoque fue exitoso. Ninguno de estos pacientes requirió una derivación posterior durante la evolución de la enfermedad. En dos pacientes, fue necesaria una derivación secundaria debido al fracaso del tratamiento. Se requirió dilatación con balón por estenosis luminal en dos pacientes. Al comparar las características de los pacientes y del tratamiento con y sin ostomía, incluido el éxito y la duración del tratamiento, no se encontraron diferencias significativas. El restablecimiento de la continuidad intestinal se logró en el 69% de los pacientes derivados.LIMITACIONES:Diseño de estudio retrospectivo no aleatorio; factores de confusión en la asignación del tratamiento; escaso número de pacientes y seguimiento a corto plazo de los pacientes ostomizados; bajo poder estadístico.CONCLUSIÓN:En este estudio de una sola institución, la terapia al vacío por vía endoscópica en casos de fuga anastomótica rectal fue exitosa en el 90% de los pacientes sin derivación con respecto al control de la sepsis, granulación de la cavidad de la fuga, como se evitó la cirugía y la sobrevida sin ostomía a largo plazo. Consulte Video Resumen en http://links.lww.com/DCR/B737. (Traducción-Dr. Xavier Delgadillo).

It is not NOD2 - genetic and clinical risk factors for postoperative complications following ileocolic resection in Crohn's disease.

PURPOSE: To evaluate the role of the nucleotide oligomerization domain 2 (NOD2) mutation status and other risk factors for the incidence of postoperative complications after ileocolic resection for Crohn's disease (CD). METHODS: Data of 138 patients consecutively undergoing ileocolic resection for CD at a tertiary academic referral center were retrospectively analyzed including single nucleotide polymorphism (SNP) data of the NOD2 gene. Uni- and multivariate regression analysis was performed to identify factors associated with increased risk of severe postoperative complications. RESULTS: From 114 patients (83%), the NOD2 mutation status was available. Of these, 60 (53%) had a NOD2 wildtype, whereas eleven (10%) were homozygous for the high risk p.Leu1007fsX1008 (rs2066847) variant. Major postoperative complications occurred in 28 patients (20%). Twenty-seven of these (96%) were intraabdominal septic complications such as anastomotic leakage or abscess. Male gender (P = 0.029; OR 3.052, the duration of CD (time [months] from initial diagnosis of CD to surgery; P = 0.001; OR 1.009), previous abdominal surgery for CD (P = 0.017; OR 3.49), and the presence of enteric fistulas (P = 0.023; OR 3.21) were identified as independent risk factors for major postoperative complications. Homozygosity for the NOD2 high-risk variant p.Leu1007fsX1008 did not show increased postoperative morbidity in the short and long-term outcome. CONCLUSIONS: We could detect independent risk factors for major postoperative complications after ileocolic resection for Crohn's disease. However, patients with the high-risk variant p.Leu1007fsX1008 of the NOD2 gene did not show increased postoperative morbidity.

Endoscopic vacuum therapy for the treatment of colorectal leaks - a systematic review and meta-analysis.

BACKGROUND: During the last two decades, vacuum-assisted wound therapy has been successfully transferred to an endoscopic treatment approach of various upper and lower gastrointestinal leaks called endoscopic vacuum therapy (EVT). As mostly small case series are published in this field, the aim of our systematic review and meta-analysis was to evaluate the efficacy and safety of EVT in the treatment of colorectal leaks. METHODS: A systematic search of MEDLINE/PubMed and Cochrane databases was performed using search terms related to EVT and colorectal defects (anastomotic leakage, rectal stump insufficiency) according to the PRISMA guidelines. Randomized controlled trials (RCTs), observational studies, and case series published by December 2020 were eligible for inclusion. A meta-analysis was conducted on the success of EVT, stoma reversal rate after EVT as well as procedure-related complications. Statistical interferences were based on pooled estimates from random effects models using DerSimonian-Laird estimator. RESULTS: Only data from observational studies and case series were available. Twenty-four studies reporting on 690 patients with colorectal defects undergoing EVT were included. The mean rate of success was 81.4% (95% CI: 74.0%-87.1%). The proportion of diverted patients was 76.4% (95% CI: 64.9%-85.0%). The mean rate of ostomy reversal across the studies was 66.7% (95% CI: 58.0%-74.4%). Sixty-four patients were reported with EVT-associated complications, the weighted mean complication rate across the studies was 12.1% (95% CI: 9.7%-15.2%). CONCLUSIONS: Current medical evidence on EVT in patients with colorectal leaks lacks high quality data from RCTs. Based on the data available, EVT can be seen as a feasible treatment option with manageable risks for selected patients with colorectal leaks.

Nelfinavir and Its Active Metabolite M8 Are Partial Agonists and Competitive Antagonists of the Human Pregnane X Receptor.

The HIV protease inhibitor nelfinavir is currently being analyzed for repurposing as an anticancer drug for many different cancers because it exerts manifold off-target protein interactions, finally resulting in cancer cell death. Xenosensing pregnane X receptor (PXR), which also participates in the control of cancer cell proliferation and apoptosis, was previously shown to be activated by nelfinavir; however, the exact molecular mechanism is still unknown. The present study addresses the effects of nelfinavir and its major and pharmacologically active metabolite nelfinavir hydroxy-tert-butylamide (M8) on PXR to elucidate the underlying molecular mechanism. Molecular docking suggested direct binding to the PXR ligand-binding domain, which was confirmed experimentally by limited proteolytic digestion and competitive ligand-binding assays. Concentration-response analyses using cellular transactivation assays identified nelfinavir and M8 as partial agonists with EC50 values of 0.9 and 7.3 µM and competitive antagonists of rifampin-dependent induction with IC50 values of 7.5 and 25.3 µM, respectively. Antagonism exclusively resulted from binding into the PXR ligand-binding pocket. Impaired coactivator recruitment by nelfinavir as compared with the full agonist rifampin proved to be the underlying mechanism of both effects on PXR. Physiologic relevance of nelfinavir-dependent modulation of PXR activity was investigated in respectively treated primary human hepatocytes, which showed differential induction of PXR target genes and antagonism of rifampin-induced ABCB1 and CYP3A4 gene expression. In conclusion, we elucidate here the molecular mechanism of nelfinavir interaction with PXR. It is hypothesized that modulation of PXR activity may impact the anticancer effects of nelfinavir. SIGNIFICANCE STATEMENT: Nelfinavir, which is being investigated for repurposing as an anticancer medication, is shown here to directly bind to human pregnane X receptor (PXR) and thereby act as a partial agonist and competitive antagonist. Its major metabolite nelfinavir hydroxy-tert-butylamide exerts the same effects, which are based on impaired coactivator recruitment. Nelfinavir anticancer activity may involve modulation of PXR, which itself is discussed as a therapeutic target in cancer therapy and for the reversal of chemoresistance.

Kupffer cell activation by different microbial lysates: Toll-like receptor-2 plays pivotal role on thromboxane A2 production in mice and humans.

Thromboxane (TX) A2 has been identified as an important intrahepatic vasoconstrictor upon Kupffer cell (KC) activation during infections such as spontaneous bacterial peritonitis (SBP). The study aimed to investigate the role of TLRs in the TXA2 increase in liver nonparenchymal cells and their related mechanisms. Here, we identified TLR-2 as a common pathway for different microbials: microbial lysates including Gram-positive bacteria, Gram-negative bacteria, and fungi all increased TXA2 secretion via activation of TLR-2 in human KCs, accompanied by increased expression and phosphorylation of Myd88-related pathway. Of all TLR agonists, only TLR-1, -2, and -4 agonists increased TXA2 in human KCs. These results were further confirmed by mouse liver nonparenchymal cells. Comparing the effects of TLR-1, -2, and -4 antagonists, only TLR-2 antagonist showed inhibitory effects with all tested microbial lysates. Pretreatment with TLR-2 antagonist in human KCs blocked the secretion of IL-10, CXCL-10, TNF-α, and IL-6 induced by Gram-positive and Gram-negative bacterial stimulation. IL-23 and IL-1ß were only induced by Gram-negative bacteria. Thus, TLR-2 might be a potential marker and an attractive target for future treatment of patients with SBP. In addition, IL-23 and IL-1ß might distinguish early between Gram-positive and Gram-negative SBP.

Chances, challenges and outcomes of patients with complex bilobar colorectal liver metastases approached by a combined multimodal therapy with liver resection and local ablative therapy.

BACKGROUND AND OBJECTIVES: In treatment of colorectal liver metastases (CRC-LM), liver surgery combined with systemic therapies and local ablation (LAT) allows improved survival. This study aims at the outcomes of patients with complex bilobar CRC-LM who were intended to undergo multimodal therapy with liver resection and LAT. METHODS: Forty-three CRC-LM patients with recommendation for multimodal treament were extracted from 5878 tumor board decisions between 2014 and 2017. Outcome variables included patient survival, as well as completion of hepatic clearance. Prognostic factors were identified by correlation and a Cox proportional hazards model. RESULTS: Out of 43 patients only 23 achieved complete clearance of CRC-LM. One- and 3-year overall survival of patients with cleared liver disease was 100% and 91.7%, respectively, as compared to 83.8% and 12.1%. Incomplete hepatic clearance was the strongest independent risk factor for overall survival (hazards ratio [HR], 5.86; p = .009). Risk factors for incomplete clearance were higher age (r = .34; p = .026), comorbidities (r = .40; p = .008), major complications (r = .34; p = .024), and prolonged intensive care unit stay (r = .41; p = .017). CONCLUSION: Completion of hepatic clearance is crucial to achieve long-term survival in patients with complex bilobar CRC-LM. Careful patient selection and treatment planning should avoid treatment failure before completing the intended therapy plan when multimodal treatments are planned.

Endoscopic vacuum therapy for in- and outpatient treatment of colorectal defects.

BACKGROUND: Evidence for endoscopic vacuum therapy (EVT) for colorectal defects is still based on small patient series from various institutions, employing different treatment algorithms and methods. As EVT was invented at our institution 20 years ago, the aim was to report the efficacy and safety of EVT for colorectal defects as well as to analyze factors associated with efficacy, therapy duration, and outpatient treatment. METHODS: Cohort study with analysis of prospectively collected data of patients receiving EVT for colorectal defects at a tertiary referral center in Germany (n = 281). RESULTS: The majority of patients had malignant disease (83%) and an American Society of Anesthesiologists classification of III/IV (81%). Most frequent indications for EVT were anastomotic leakage after sigmoid or rectal resection (67%) followed by rectal stump leakage (20%). EVT was successful in 256 out of 281 patients (91%). EVT following multi-visceral resection (P = 0.037) and recent surgical revision after primary surgery (P = 0.009) were risk factors for EVT failure. EVT-associated adverse events occurred in 27 patients (10%). Median treatment duration was 25 days. Previous chemo-radiation (P = 0.006) was associated with a significant longer duration of EVT. Outpatient treatment was conducted in 49% of patients with a median hospital stay reduction of 15 days and 98% treatment success. Younger patient age (P = 0.044) was associated with the possibility of outpatient treatment. Restoration of intestinal continuity was achieved in 60% of patients where technically possible with a 12-month rate of 52%. CONCLUSIONS: In patients with colorectal defects, EVT appears to be a safe and effective, minimally invasive option for in- and outpatient treatment.

Endoscopic vacuum therapy for treatment of rectal stump leakage.

BACKGROUND: Symptomatic rectal stump leakage (RSL) is a serious complication after discontinuity resection and requires immediate surgical, interventional, or endoscopic therapy. Re-operations are associated with high morbidity and mortality in these mostly very ill patients. Endoscopic vacuum therapy (EVT) has been established for management of anastomotic leakage; however, its effectiveness for RSL treatment has not been analyzed in detail yet. METHODS: A retrospective analysis of patients treated with EVT for RSL between 2001 and 2018 analyzing factors predicting therapy success and duration was carried out. RESULTS: Fifty-six patients with RSL at a median age of 66 years were included. Of these, 18 patients (32%) had been referred for EVT from external departments or institutions. RSL was associated with a relevant clinical deterioration in all patients, and 55 patients (98%) had been classified as ASA 3 and 4, preoperatively. In 9 patients (16%), additional surgical revision was necessary with initiation of EVT. In 47 patients (84%), EVT was successful and local control of the inflammatory focus was achieved. The median duration of therapy was 20 days. Two patients (4%) suffered from minor EVT-associated bleeding that was endoscopically controlled. Preoperative radiation of the pelvis was significantly associated with EVT failure (P = 0.035), whereas patient age represented a predictive factor for therapy length (P = 0.039). In 12 patients (21%), restoration of intestinal continuity was achieved in the further course. CONCLUSIONS: We present the first specific series on EVT for RSL. EVT for RSL was shown to be an effective and safe minimal-invasive treatment option, avoiding surgical revision in the majority of patients.

Experimental postoperative ileus: is Th2 immune response involved?

Rationale: Postoperative ileus (POI) is a frequent complication arising after gastrointestinal surgery but pathogenesis of POI is still not fully understood. While Th1 immune cells are implicated in POI, the involvement of Th2 cells has not yet been clarified. Given the impact of reactive oxygen species (ROS) in the regulation of Th1 and Th2 balance, we hypothesized that not only Th1 but also Th2 immune response can be involved in the development of experimental POI. Methods: The intestinal transit test was performed using carbon gum arabic. Electron microscopy was employed to assess tissue morphology and the presence of immune cells. Cytokines, IgE and ROS were measured. Immune cells from Peyer's patches were analyzed by Flow Cytometry and toluidine blue staining was used for detection of mast cells. Transcriptional factors were analyzed by Western blot. Results: POI is associated with an increase in both Th2 cytokines and Th2 cells. We have further demonstrated that POI induces a Th2-dependent activation of memory and non-memory B cells. This was accompanied by an increase in a number of mast cells in the colon of POI mice as well by an increased IgE and histamine plasma levels. We found that POI-induced accumulation of ROS was associated with an increased expression of the transcriptional factors HMBGI, NF-κB, and p38. This increased expression seemed to be associated with a Th2 response. Conclusion: Th2 immune response can be involved in the activation of mast cells in POI, which was associated with ROS mediated activation of NF-κB and p38 MAPK signaling pathway.

Infections after kidney transplantation: A comparison of mTOR-Is and CNIs as basic immunosuppressants. A systematic review and meta-analysis.

BACKGROUND: Side effects of the immunosuppressive therapy after solid organ transplantation are well known. Recently, significant benefits were shown for mTOR-Is with respect to certain viral infections in comparison with CNIs. However, reported total incidences of infections under mTOR-Is vs CNIs are usually not different. This raises the question to additional differences between these immunosuppressants regarding development and incidence of infections. METHODS: The current literature was searched for prospective randomized controlled trials in renal transplantation. There were 954 trials screened of which 19 could be included (9861 pts.). The 1-year incidence of infections, patient and graft survival were assessed in meta-analyses. RESULTS: Meta-analysis on 1-year incidence of infections showed a significant benefit of an mTOR-I based therapy when combined with a CNI vs CNI-based therapy alone (OR 0.76). There was no difference between mTOR-I w/o CNI and CNI therapy (OR 0.97). For pneumonia, a significant disadvantage was seen only for mTOR-I monotherapy compared to CNI's (OR 2.09). The incidence of CMV infections was significantly reduced under mTOR-I therapy (combination with CNI: OR 0.30; mTOR w/o CNI: OR: 0.46). There was no significant difference between mTOR-I and CNI therapy with respect to patient survival (mTOR-I w/o CNI vs CNI: OR 1.22; mTOR-I with CNI vs CNI: OR 0.86). Graft survival was negatively affected by mTOR-I monotherapy (OR 1.52) but not when combined with a CNI (OR 0.97). CONCLUSION: Following renal transplantation the incidence of infections is lower when mTOR-Is are combined with a CNI compared to a standard CNI therapy. Pneumonia occurs more often under mTOR-I w/o CNI.

IL-18 but Not IL-1 Signaling Is Pivotal for the Initiation of Liver Injury in Murine Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is rising in prevalence, and a better pathophysiologic understanding of the transition to its inflammatory phenotype (NASH) is key to the development of effective therapies. To evaluate the contribution of the NLRP3 inflammasome and its downstream effectors IL-1 and IL-18 in this process, we applied the true-to-life "American lifestyle-induced obesity syndrome" (ALiOS) diet mouse model. Development of obesity, fatty liver and liver damage was investigated in mice fed for 24 weeks according to the ALiOS protocol. Lipidomic changes in mouse livers were compared to human NAFLD samples. Receptor knockout mice for IL-1 and IL-18 were used to dissect the impact of downstream signals of inflammasome activity on the development of NAFLD. The ALiOS diet induced obesity and liver steatosis. The lipidomic changes closely mimicked changes in human NAFLD. A pro-inflammatory gene expression pattern in liver tissue and increased serum liver transaminases indicated early liver damage in the absence of histological evidence of NASH. Mechanistically, Il-18r-/-- but not Il-1r-/- mice were protected from early liver damage, possibly due to silencing of the pro-inflammatory gene expression pattern. Our study identified NLRP3 activation and IL-18R-dependent signaling as potential modulators of early liver damage in NAFLD, preceding development of histologic NASH.

Improved survival after resection of colorectal liver metastases in patients with unresectable lung metastases.

BACKGROUND: Modern systemic therapies considerably improve tumour control and thus open the possibility of new surgical approaches in metastatic colorectal cancer. In this retrospective clinical cohort with a comparison group, we investigated whether liver resection in a combined liver-lung-metastasised stage is justified if pulmonary disease is not resected. METHODS: From 283 patients treated in our institution between 2000 and 2014 for combined colorectal liver- and lung metastases, 35 patients had their pulmonary metastases left in situ while they were eligible for both treatment options: resection versus non-resection of liver metastases. Effectively, 15 of these patients received whereas 20 did not receive a liver resection. In these patients, we compared overall survival and determined risk factors that are associated with poor survival, applying a Cox-Proportional Hazards model. RESULTS: Patients whose liver metastases were resected showed significantly longer median survival compared to patients who did not undergo hepatic surgery (median 2.6 vs 1.5 years, P = 0.0182). The Cox-Proportional Hazards model revealed hepatic metastasectomy to be the strongest determinant of patient survival (HR 5.27; CI: (1.89, 14.65)). CONCLUSION: Our results suggest that surgical removal of liver metastases may be beneficial in selected patients even if concomitant lung metastases cannot be resected.

A Novel Chimeric Oncolytic Virus Vector for Improved Safety and Efficacy as a Platform for the Treatment of Hepatocellular Carcinoma.

Oncolytic viruses represent an exciting new aspect of the evolving field of cancer immunotherapy. We have engineered a novel hybrid vector comprising vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV), named recombinant VSV-NDV (rVSV-NDV), wherein the VSV backbone is conserved but its glycoprotein has been replaced by the hemagglutinin-neuraminidase (HN) and the modified, hyperfusogenic fusion (F) envelope proteins of recombinant NDV. In comparison to wild-type VSV, which kills cells through a classical cytopathic effect, the recombinant virus is able to induce tumor-specific syncytium formation, allowing efficient cell-to-cell spread of the virus and a rapid onset of immunogenic cell death. Furthermore, the glycoprotein exchange substantially abrogates the off-target effects in brain and liver tissue associated with wild-type VSV, resulting in a markedly enhanced safety profile, even in immune-deficient NOD.CB17-prkdcscid/NCrCrl (NOD-SCID) mice, which are highly susceptible to wild-type VSV. Although NDV causes severe pathogenicity in its natural avian hosts, the incorporation of the envelope proteins in the chimeric rVSV-NDV vector is avirulent in embryonated chicken eggs. Finally, systemic administration of rVSV-NDV in orthotopic hepatocellular carcinoma (HCC)-bearing immune-competent mice resulted in significant survival prolongation. This strategy, therefore, combines the beneficial properties of the rapidly replicating VSV platform with the highly efficient spread and immunogenic cell death of a fusogenic virus without risking the safety and environmental threats associated with either parental vector. Taking the data together, rVSV-NDV represents an attractive vector platform for clinical translation as a safe and effective oncolytic virus.IMPORTANCE The therapeutic efficacy of oncolytic viral therapy often comes as a tradeoff with safety, such that potent vectors are often associated with toxicity, while safer viruses tend to have attenuated therapeutic effects. Despite promising preclinical data, the development of VSV as a clinical agent has been substantially hampered by the fact that severe neurotoxicity and hepatotoxicity have been observed in rodents and nonhuman primates in response to treatment with wild-type VSV. Although NDV has been shown to have an attractive safety profile in humans and to have promising oncolytic effects, its further development has been severely restricted due to the environmental risks that it poses. The hybrid rVSV-NDV vector, therefore, represents an extremely promising vector platform in that it has been rationally designed to be safe, with respect to both the recipient and the environment, while being simultaneously effective, both through its direct oncolytic actions and through induction of immunogenic cell death.

Identification of novel agonists by high-throughput screening and molecular modelling of human constitutive androstane receptor isoform 3.

Prediction of drug interactions, based on the induction of drug disposition, calls for the identification of chemicals, which activate xenosensing nuclear receptors. Constitutive androstane receptor (CAR) is one of the major human xenosensors; however, the constitutive activity of its reference variant CAR1 in immortalized cell lines complicates the identification of agonists. The exclusively ligand-dependent isoform CAR3 represents an obvious alternative for screening of CAR agonists. As CAR3 is even more abundant in human liver than CAR1, identification of its agonists is also of pharmacological value in its own right. We here established a cellular high-throughput screening assay for CAR3 to identify ligands of this isoform and to analyse its suitability for identifying CAR ligands in general. Proof-of-concept screening of 2054 drug-like compounds at 10 µM resulted in the identification of novel CAR3 agonists. The CAR3 assay proved to detect the previously described CAR1 ligands in the screened libraries. However, we failed to detect CAR3-selective compounds, as the four novel agonists, which were selected for further investigations, all proved to activate CAR1 in different cellular and in vitro assays. In primary human hepatocytes, the compounds preferentially induced the expression of the prototypical CAR target gene CYP2B6. Failure to identify CAR3-selective compounds was investigated by molecular modelling, which showed that the isoform-specific insertion of five amino acids did not impact on the ligand binding pocket but only on heterodimerization with retinoid X receptor. In conclusion, we demonstrate here the usability of CAR3 for screening compound libraries for the presence of CAR agonists.

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

Nonparametric change point estimation for survival distributions with a partially constant hazard rate.

We present a new method for estimating a change point in the hazard function of a survival distribution assuming a constant hazard rate after the change point and a decreasing hazard rate before the change point. Our method is based on fitting a stump regression to p values for testing hazard rates in small time intervals. We present three real data examples describing survival patterns of severely ill patients, whose excess mortality rates are known to persist far beyond hospital discharge. For designing survival studies in these patients and for the definition of hospital performance metrics (e.g. mortality), it is essential to define adequate and objective end points. The reliable estimation of a change point will help researchers to identify such end points. By precisely knowing this change point, clinicians can distinguish between the acute phase with high hazard (time elapsed after admission and before the change point was reached), and the chronic phase (time elapsed after the change point) in which hazard is fairly constant. We show in an extensive simulation study that maximum likelihood estimation is not robust in this setting, and we evaluate our new estimation strategy including bootstrap confidence intervals and finite sample bias correction.

Intraarterial indocyanine green (ICG) fluorescence augmentation by marking embryonal resection areas in colorectal surgery: a feasibility study in a porcine model.

Aim: In this pilot animal study we examined whether it is possible to visualize the embryonal resection layers by using intraarterial indocyanine green (ICG) staining when performing total mesorectal excision (TME) for carcinoma of the rectum. Material and methods: We injected ICG into the inferior mesenteric artery (AMI) of four swines to see whether the watershed area of the arterial supply zone can be sufficiently visualized by fluorescence imaging in order to mark the right dissection area along the fascia parietalis before and during resection. Results: We observed a fluorescence signal in all the supplied areas of AMI but not in other parts of the abdominal cavity or other organs. Additionally, the mesorectum also showed a sharp border between colored and non-colored tissue. Conclusion: In this study we present that resection borders may be determined before resection based on ICG-perfusion and we showed that intraoperative exclusive coloring of the rectum including the mesorectum is possible. Visualizing resection borders based on ICG-perfusion before settling the first cut may be a new approach in oncological surgery.