Auto/Paracrine C-Type Natriuretic Peptide/Cyclic GMP Signaling Prevents Endothelial Dysfunction.

Endothelial dysfunction is cause and consequence of cardiovascular diseases. The endothelial hormone C-type natriuretic peptide (CNP) regulates vascular tone and the vascular barrier. Its cGMP-synthesizing guanylyl cyclase-B (GC-B) receptor is expressed in endothelial cells themselves. To characterize the role of endothelial CNP/cGMP signaling, we studied mice with endothelial-selective GC-B deletion. Endothelial EC GC-B KO mice had thicker, stiffer aortae and isolated systolic hypertension. This was associated with increased proinflammatory E-selectin and VCAM-1 expression and impaired nitric oxide bioavailability. Atherosclerosis susceptibility was evaluated in such KO and control littermates on Ldlr (low-density lipoprotein receptor)-deficient background fed a Western diet for 10 weeks. Notably, the plaque areas and heights within the aortic roots were markedly increased in the double EC GC-B/Ldlr KO mice. This was accompanied by enhanced macrophage infiltration and greater necrotic cores, indicating unstable plaques. Finally, we found that EC GC-B KO mice had diminished vascular regeneration after critical hind-limb ischemia. Remarkably, all these genotype-dependent changes were only observed in female and not in male mice. Auto/paracrine endothelial CNP/GC-B/cGMP signaling protects from arterial stiffness, systolic hypertension, and atherosclerosis and improves reparative angiogenesis. Interestingly, our data indicate a sex disparity in the connection of diminished CNP/GC-B activity to endothelial dysfunction.

Prognostic value of clinical and radiomic parameters in patients with liver metastases from uveal melanoma.

Approximately every second patient with uveal melanoma develops distant metastases, with the liver as the predominant target organ. While the median survival after diagnosis of distant metastases is limited to a year, yet-to-be-defined subgroups of patients experience a more favorable outcome. Therefore, prognostic biomarkers could help identify distinct risk groups to guide patient counseling, therapeutic decision-making, and stratification of study populations. To this end, we retrospectively analyzed a cohort of 101 patients with newly diagnosed hepatic metastases from uveal melanoma by using Cox-Lasso regression machine learning, adapted to a high-dimensional input parameter space. We show that substantial binary risk stratification can be performed, based on (i) clinical and laboratory parameters, (ii) measures of quantitative overall hepatic tumor burden, and (iii) radiomic parameters. Yet, combining two or all three domains failed to improve prognostic separation of patients. Additionally, we identified highly relevant clinical parameters (including lactate dehydrogenase, thrombocyte counts, aspartate transaminase, and the metastasis-free interval) at first diagnosis of metastatic disease as predictors for time-to-treatment failure and overall survival. Taken together, the risk stratification models, built by our machine-learning algorithm, identified a comparable and independent prognostic value of clinical, radiological, and radiomic parameters in uveal melanoma patients with hepatic metastases.

Hepatitis B surface antigen expression impairs endoplasmic reticulum stress-related autophagic flux by decreasing LAMP2.

Background & Aims: Hepatitis B surface antigen (HBsAg) drives hepatocarcinogenesis. Factors and mechanisms involved in this progression remain poorly defined, hindering the development of effective therapeutic strategies. Therefore, the mechanisms involved in the HBsAg-induced transformation of normal liver into hepatocellular carcinoma (HCC) were investigated. Methods: Hemizygous Tg(Alb1HBV)44Bri/J mice were examined for HBsAg-induced carcinogenic events. Gene set-enrichment analysis identified significant signatures in HBsAg-transgenic mice that correlated with endoplasmic reticulum (ER) stress, unfolded protein response, autophagy and proliferation. These events were investigated by western blotting, immunohistochemical and immunocytochemical staining in 2-, 8- and 12-month-old HBsAg-transgenic mice. The results were verified in HBsAg-overexpressing Hepa1-6 cells and validated in human HBV-related HCC samples. Results: Increased BiP expression in HBsAg-transgenic mice indicated induction of the unfolded protein response. In addition, early-phase autophagy was enhanced (increased BECN1 and LC3B) and late-phase autophagy blocked (increased p62) in HBsAg-transgenic mice. Finally, HBsAg altered lysosomal acidification via ATF4- and ATF6-mediated downregulation of lysosome-associated membrane protein 2 (LAMP2) expression. In patients, HBV-related HCC and adjacent tissues showed increased BiP, p62 and downregulated LAMP2 compared to uninfected controls. In vitro, the use of ER stress inhibitors reversed the HBsAg-related suppression of LAMP2. Furthermore, HBsAg promoted hepatocellular proliferation as indicated by Ki67, cleaved caspase-3 and AFP staining in paraffin-embedded liver sections from HBsAg-transgenic mice. These results were further verified by colony formation assays in HBsAg-expressing Hepa1-6 cells. Interestingly, inhibition of ER stress in HBsAg-overexpressing Hepa1-6 cells suppressed HBsAg-mediated cell proliferation. Conclusions: These data showed that HBsAg directly induces ER stress, impairs autophagy and promotes proliferation, thereby driving hepatocarcinogenesis. In addition, this study expanded the understanding of HBsAg-mediated intracellular events in carcinogenesis. Impact and implications: Factors and mechanisms involved in hepatocarcinogenesis driven by hepatitis B surface antigen (HBsAg) are poorly defined, hindering the development of effective therapeutic strategies. This study showed that HBsAg-induced endoplasmic reticulum stress suppressed LAMP2, thereby mediating autophagic injury. The present data suggest that restoring LAMP2 function in chronic HBV infection may have both antiviral and anti-cancer effects. This study has provided insights into the role of HBsAg-mediated intracellular events in carcinogenesis and thereby has relevance for future drug development.

Sporadic noninvasive medullary thyroid neoplasm: A desmoplasia-negative unifocal nonmetastatic tumor cured by hemithyroidectomy.

BACKGROUND: The absence of primary tumor desmoplasia, a marker of node metastases, on frozen section may help reduce the extent of surgery without compromising the biochemical cure. We aimed to clarify whether hemithyroidectomy with diagnostic ipsilateral central neck dissection can replace total thyroidectomy with routine central neck dissection in patients with sporadic medullary thyroid cancer. METHODS: We retrospectively evaluated data collected from patients who had undergone primary neck surgery for hypercalcitoninemic sporadic medullary thyroid cancer between January 2017 and December 2022 at one institution. RESULTS: Of the 25 patients we examined, 19 had desmoplasia-negative and 6 desmoplasia-positive primary thyroid tumors on frozen section. The desmoplasia-negative patients had undergone less surgery with fewer nodes removed than the desmoplasia-positive patients (medians of 6 vs 31 nodes, P < .001). The desmoplasia-negative patients had predominantly undergone hemithyroidectomy with ipsilateral central neck dissection. None of the desmoplasia-negative tumors was multifocal (0 of 19 desmoplasia-negative vs 2 of 6 desmoplasia-positive or 0% vs 33%, P = .050) or node-positive (0 of 19 vs 6 of 6 patients or 0% vs 100%; medians of 0 vs 3.5 node metastases; both P < .001). Despite limited surgery, all desmoplasia-negative patients attained and maintained biochemical cure. CONCLUSION: Hemithyroidectomy combined with diagnostic ipsilateral central neck dissection is a viable risk-reducing and curative strategy for desmoplasia-negative and node-negative, nonmetastatic unifocal tumors, for which we propose the term sporadic noninvasive medullary thyroid neoplasm (SNMTP).

Characteristics and Long-Term Outcome of 535 Patients with Autoimmune Hepatitis-The 20-Year Experience of a High-Volume Tertiary Center.

Background and aims: Autoimmune hepatitis (AIH) is a complex and progressive inflammatory liver disease characterized by immune-mediated destruction of the liver parenchyma, hypergammaglobulinemia, the presence of circulating autoantibodies, and good response to immunosuppressive therapy. Since the prevalence of AIH is relatively rare, data on the clinical course and the long-term outcome are scarce. Patients and methods: We retrospectively analyzed the data of 535 well-documented AIH patients treated at the University Hospital Essen between 2000 and 2020. Results: The majority of patients were middle-aged females (75% women, mean age 45 years) with AIH type 1 (97%). Approximately 32% of patients were diagnosed with cirrhosis due to AIH, 29% had concomitant autoimmune (predominantly autoimmune thyroiditis), and 10% had psychiatric diseases, respectively. Skin tumors were the most common malignant diseases (47% of all tumors), while hepatocellular carcinoma rarely occurred (only six cases). Overall long-term mortality and liver-associated mortality were 9.16% and 4.67%, respectively. However, long-term survival was strongly associated with disease remission. Conclusions: Although AIH is a silent disease and cirrhosis is present in many cases, a favorable long-term prognosis can be achieved by consequent immunosuppressive therapy. The incidence of (liver-associated) complications seems to be lower in comparison to other etiologies, such as viral hepatitis or NASH, and mainly depends on the long-term side effects of immunosuppressive therapy.

Nuclear translocation of YAP drives BMI-associated hepatocarcinogenesis in hepatitis B virus infection.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) development and progression. The aim of this study was to mechanistically investigate the involvement of Hippo signalling in HBV surface antigen (HBsAg)-dependent neoplastic transformation. METHODS: Liver tissue and hepatocytes from HBsAg-transgenic mice were examined for the Hippo cascade and proliferative events. Functional experiments in mouse hepatoma cells included knockdown, overexpression, luciferase reporter assays and chromatin immunoprecipitation. Results were validated in HBV-related HCC biopsies. RESULTS: Hepatic expression signatures in HBsAg-transgenic mice correlated with YAP responses, cell cycle control, DNA damage and spindle events. Polyploidy and aneuploidy occurred in HBsAg-transgenic hepatocytes. Suppression and inactivation of MST1/2 led to the loss of YAP phosphorylation and the induction of BMI1 expression in vivo and in vitro. Increased BMI1 directly mediated cell proliferation associated with decreased level of p16INK4a , p19ARF , p53 and Caspase 3 as well as increased Cyclin D1 and γ-H2AX expression. Chromatin immunoprecipitation and the analysis of mutated binding sites in dual-luciferase reporter assays confirmed that the YAP/TEAD4 transcription factor complex bound and activated the Bmi1 promoter. In chronic hepatitis B patients, paired liver biopsies of non-tumour and tumour tissue indicated a correlation between YAP expression and the abundance of BMI1. In a proof-of-concept, treatment of HBsAg-transgenic mice with YAP inhibitor verteporfin directly suppressed the BMI1-related cell cycle. CONCLUSION: HBV-associated proliferative HCC might be related to the HBsAg-YAP-BMI1 axis and offer a potential target for the development of new therapeutic approaches.

Liquid biopsy can cure early colorectal cancer recurrence - Case Report.

In the context of colorectal cancer (CRC), circulating tumor DNA (ctDNA) is frequently used to monitor the minimal residual disease (MRD). ctDNA has become an excellent biomarker to predict which patients with CRC are likely to relapse due to the persistence of micrometastases. MRD diagnosis via analysis of ctDNA may allow much earlier detection of relapse compared with conventional diagnosis during follow-up. It should lead to an increased rate of curative-intended complete resection of an asymptomatic relapse. Besides, ctDNA can provide crucial information on whether and how intensively adjuvant or additive therapy should be administered. In the present case, analysis of ctDNA gave us a crucial hint to the use of more intensive diagnostics (MRI and Positron emission tomography-computed tomography PET-CT) which led to earlier detection of CRC relapse. Metastasis detected early are more likely to be completely resectable with curative intent.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

Correlation between Imaging Markers Derived from PET/MRI and Invasive Acquired Biomarkers in Newly Diagnosed Breast Cancer.

PURPOSE: Evaluate the diagnostic potential of [18F]FDG-PET/MRI data compared with invasive acquired biomarkers in newly diagnosed early breast cancer (BC). METHODS: Altogether 169 women with newly diagnosed BC were included. All underwent a breast- and whole-body [18F]FDG-PET/MRI for initial staging. A tumor-adapted volume of interest was placed in the primaries and defined bone regions on each standard uptake value (SUV)/apparent diffusion coefficient (ADC) dataset. Immunohistochemical markers, molecular subtype, tumor grading, and disseminated tumor cells (DTCs) of each patient were assessed after ultrasound-guided biopsy of the primaries and bone marrow (BM) aspiration. Correlation analysis and group comparisons were assessed. RESULTS: A significant inverse correlation of estrogen-receptor (ER) expression and progesterone-receptor (PR) expression towards SUVmax was found (ER: r = 0.27, p < 0.01; PR: r = 0.19, p < 0.05). HER2-receptor expression showed no significant correlation towards SUV and ADC values. A significant positive correlation between Ki67 and SUVmax and SUVmean (r = 0.42 p < 0.01; r = 0.19 p < 0.05) was shown. Tumor grading significantly correlated with SUVmax and SUVmean (ρ = 0.36 and ρ = 0.39, both p's < 0.01). There were no group differences between SUV/ADC values of DTC-positive/-negative patients. CONCLUSIONS: [18F]FDG-PET/MRI may give a first impression of BC-receptor status and BC-tumor biology during initial staging by measuring glucose metabolism but cannot distinguish between DTC-positive/-negative patients and replace biopsy.

CARS Imaging Advances Early Diagnosis of Cardiac Manifestation of Fabry Disease.

Vibrational spectroscopy can detect characteristic biomolecular signatures and thus has the potential to support diagnostics. Fabry disease (FD) is a lipid disorder disease that leads to accumulations of globotriaosylceramide in different organs, including the heart, which is particularly critical for the patient's prognosis. Effective treatment options are available if initiated at early disease stages, but many patients are late- or under-diagnosed. Since Coherent anti-Stokes Raman (CARS) imaging has a high sensitivity for lipid/protein shifts, we applied CARS as a diagnostic tool to assess cardiac FD manifestation in an FD mouse model. CARS measurements combined with multivariate data analysis, including image preprocessing followed by image clustering and data-driven modeling, allowed for differentiation between FD and control groups. Indeed, CARS identified shifts of lipid/protein content between the two groups in cardiac tissue visually and by subsequent automated bioinformatic discrimination with a mean sensitivity of 90-96%. Of note, this genotype differentiation was successful at a very early time point during disease development when only kidneys are visibly affected by globotriaosylceramide depositions. Altogether, the sensitivity of CARS combined with multivariate analysis allows reliable diagnostic support of early FD organ manifestation and may thus improve diagnosis, prognosis, and possibly therapeutic monitoring of FD.

Antiviral Toll-like Receptor Signaling in Non-Parenchymal Liver Cells Is Restricted to TLR3.

The role of non-parenchymal liver cells as part of the hepatic, innate immune system in the defense against hepatotropic viruses is not well understood. Here, primary human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells were isolated from liver tissue obtained after tumor resections or liver transplantations. Cells were stimulated with Toll-like receptor 1-9 ligands for 6-24 h. Non-parenchymal liver cells expressed and secreted inflammatory cytokines (IL6, TNF and IL10). Toll-like receptor- and cell type-specific downstream signals included the phosphorylation of NF-κB, AKT, JNK, p38 and ERK1/2. However, only supernatants of TLR3-activated Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells contained type I and type III interferons and mediated an antiviral activity in the interferon-sensitive subgenomic hepatitis C virus replicon system. The antiviral effect could not be neutralized by antibodies against IFNA, IFNB nor IFNL, but could be abrogated using an interferon alpha receptor 2-specific neutralization. Interestingly, TLR3 responsiveness was enhanced in liver sinusoidal endothelial cells isolated from hepatitis C virus-positive donors, compared to uninfected controls. In conclusion, non-parenchymal liver cells are potent activators of the hepatic immune system by mediating inflammatory responses. Furthermore, liver sinusoidal endothelial cells were identified to be hyperresponsive to viral stimuli in chronic hepatitis C virus infection.

Association of cell death mechanisms and fibrosis in visceral white adipose tissue with pathological alterations in the liver of morbidly obese patients with NAFLD.

The role of visceral white adipose tissue (vWAT) in the progression of non-alcoholic liver disease (NAFLD) with its sub entities non-alcoholic fatty liver and steatohepatitis (NAFL; NASH) is underinvestigated. We thus explored mechanisms of fibrosis and regulated cell death in vWAT and liver tissue. In NAFLD, women displayed significantly more fibrosis in vWAT than men, and collagen 1α mRNA expression was significantly upregulated. The degrees of fibrosis in vWAT and liver tissue correlated significantly. The size of vWAT-resident adipocytes in NAFLD correlated negatively with the local degree of fibrosis. The extent of apoptosis, as measured by circulating M30, positively correlated with the degree of fibrosis in vWAT; necrosis-associated HMGB1 mRNA expression was significantly downregulated in vWAT and liver tissue; (iii) necroptosis-related RIPK-3 mRNA expression was significantly upregulated in vWAT; and autophagy-related LC3 mRNA expression was significantly downregulated in vWAT, while upregulated in the liver. Thus, the different cell death mechanisms in the vWAT in NAFLD are regulated independently while not ruling out their interaction. Fibrosis in vWAT may be associated with reduced adipocyte size and thus partially protective against NAFLD progression.Abbreviations: ATG5: autophagy related 5; BAS: bariatric surgery; BMI: body mass index; ELISA: enzyme-linked immunosorbent assay; EtOH: ethanol; FFAs: free fatty acids; HCC: hepatocellular carcinoma; HMGB1: high-mobility group box 1 protein; IHC: immunohistochemistry; IL: interleukin; LC3: microtubule-associated proteins 1A/1B light chain 3B; M30: neoepitope K18Asp396-NE displayed on the caspase-cleaved keratin 18 fragment; M65: epitope present on both caspase-cleaved and intact keratin 18; NAFL: non-alcoholic fatty liver; NAFLD: non-alcoholic fatty liver disease; NAS: NAFLD activity score; NASH: non-alcoholic steatohepatitis; NLRP3: nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3; qRT-PCR: quantitative real-time polymerase-chain reaction; r: Pearson's correlation coefficient (r); rs: Spearman's rank correlation coefficient; RIPK3: receptor-interacting serine/threonine-protein kinase 3; T2DM: type 2 diabetes mellitus (T2DM); TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; vWAT: visceral WAT; WAT: white adipose tissue.

Pexidartinib Long-Term Hepatic Safety Profile in Patients with Tenosynovial Giant Cell Tumors.

BACKGROUND: Pexidartinib is approved in the U.S. for tenosynovial giant cell tumors (TGCTs). Herein, we assessed the hepatic safety profile of pexidartinib across patients with TGCTs receiving pexidartinib. MATERIALS, AND METHODS: Hepatic adverse reactions (ARs) were assessed by type and magnitude of liver test abnormalities, classified as (a) isolated aminotransferase elevations (alanine [ALT] or aspartate [AST], without significant alkaline phosphatase [ALP] or bilirubin elevations), or (b) mixed or cholestatic hepatotoxicity (increase in ALP with or without ALT/AST and bilirubin elevations, based on adjudication). Median follow-up from initial pexidartinib treatment was 39 months (range, 32-82) in 140 patients with TGCTs across clinical studies NCT01004861, NCT02371369, NCT02734433, and NCT03291288. RESULTS: In total, 95% of patients with TGCTs (133/140) treated with pexidartinib (median duration of exposure, 19 months [range, 1-76]), experienced a hepatic AR. A total of 128 patients (91%) had reversible, low-grade dose-dependent isolated AST/ALT elevations without significant ALP elevations. Five patients (4%) experienced serious mixed or cholestatic injury. No case met Hy's law criteria. Onset of hepatic ARs was predominantly in the first 2 months. All five serious hepatic AR cases recovered 1-7 months following pexidartinib discontinuation. Five patients from the non-TGCT population (N = 658) experienced serious hepatic ARs, two irreversible cases. CONCLUSION: This pooled analysis provides information to help form the basis for the treating physician's risk assessment for patients with TCGTs, a locally aggressive but typically nonmetastatic tumor. In particular, long-term treatment with pexidartinib has a predictable effect on hepatic aminotransferases and unpredictable risk of serious cholestatic or mixed liver injury. IMPLICATIONS FOR PRACTICE: This is the first long-term pooled analysis to report on the long-term hepatic safety of pexidartinib in patients with tenosynovial giant cell tumors associated with severe morbidity or functional limitations and not amenable to improvement with surgery. These findings extend beyond what has been previously published, describing the observed instances of hepatic toxicity following pexidartinib treatment across the clinical development program. This information is highly relevant for medical oncologists and orthopedic oncologists and provides guidance for its proper use for appropriate patients within the Pexidartinib Risk Evaluation and Mitigation Safety program.

[Histopathological diagnosis and differential diagnosis of nonalcoholic fatty liver disease]. / Histopathologische Diagnostik und Differenzialdiagnostik der nichtalkoholischen Fettlebererkrankung.

Fatty liver disease is a rising problem worldwide, particularly due to metabolic syndrome. The current prevalence is 20-30%, but a further increase is expected whereby children will also be increasingly affected. The presence of fat in hepatocytes is known as steatosis or, in the case of nonalcoholic origin, nonalcoholic fatty liver (NAFL). It is basically reversible, but can progress to steatohepatitis (NASH) as an active and progressive form of fatty liver disease due to continuous cell damage. This leads to progressive liver fibrosis up to end-stage liver cirrhosis. The gold standard of diagnosis is liver biopsy, in which obesity, inflammation, and hepatocellular damage (hepatocellular ballooning) are assessed for the distinction between NAFL and NASH. The extent of fibrosis indicates the progress of the disease. Childhood and adult fatty liver diseases differ morphologically, particularly in the location and amount of fat, inflammation, and fibrosis. Alcoholic and nonalcoholic fatty liver disease/steatohepatitis cannot be reliably differentiated by histology. Clinical parameters must also be taken into consideration for the differential diagnosis of other diseases associated with fatty liver. The main therapeutic goal is to reduce insulin resistance, which can be achieved through weight loss and lifestyle changes. Recently, however, drug therapies have also become available as a promising therapeutic option.

Hepatocyte KLF6 expression affects FXR signalling and the clinical course of primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is characterized by chronic cholestasis and inflammation, which promotes cirrhosis and an increased risk of cholangiocellular carcinoma (CCA). The transcription factor Krueppel-like-factor-6 (KLF6) is a mediator of liver regeneration, steatosis, and hepatocellular carcinoma (HCC), but no data are yet available on its potential role in cholestasis. Here, we aimed to identify the impact of hepatic KLF6 expression on cholestatic liver injury and PSC and identify potential effects on farnesoid-X-receptor (FXR) signalling. METHODS: Hepatocellular KLF6 expression was quantified by immunohistochemistry (IHC) in liver biopsies of PSC patients and correlated with serum parameters and clinical outcome. Liver injury was analysed in hepatocyte-specific Klf6-knockout mice following bile duct ligation (BDL). Chromatin-immunoprecipitation-assays (ChIP) and KLF6-overexpressing HepG2 cells were used to analyse the interaction of KLF6 and FXR target genes such as NR0B2. RESULTS: Based on IHC, PSC patients could be subdivided into two groups showing either low (<80%) or high (>80%) hepatocellular KLF6 expression. In patients with high KLF6 expression, we observed a superior survival in Kaplan-Meier analysis. Klf6-knockout mice showed reduced hepatic necrosis following BDL when compared to controls. KLF6 suppressed NR0B2 expression in HepG2 cells mediated through binding of KLF6 to the NR0B2 promoter region. CONCLUSION: Here, we show an association between KLF6 expression and the clinical course and overall survival in PSC patients. Mechanistically, we identified a direct interaction of KLF6 with the FXR target gene NR0B2.

Stabilization of Perivascular Mast Cells by Endothelial CNP (C-Type Natriuretic Peptide).

OBJECTIVE: Activated perivascular mast cells (MCs) participate in different cardiovascular diseases. Many factors provoking MC degranulation have been described, while physiological counterregulators are barely known. Endothelial CNP (C-type natriuretic peptide) participates in the maintenance of vascular barrier integrity, but the target cells and mechanisms are unclear. Here, we studied whether MCs are regulated by CNP. Approach and Results: In cultured human and murine MCs, CNP activated its specific GC (guanylyl cyclase)-B receptor and cyclic GMP signaling. This enhanced cyclic GMP-dependent phosphorylation of the cytoskeleton-associated VASP (vasodilator-stimulated phosphoprotein) and inhibited ATP-evoked degranulation. To elucidate the relevance in vivo, mice with a floxed GC-B (Npr2) gene were interbred with a Mcpt5-CreTG line to generate mice lacking GC-B in connective tissue MCs (MC GC-B knockout). In anesthetized mice, acute ischemia-reperfusion of the cremaster muscle microcirculation provoked extensive MC degranulation and macromolecule extravasation. Superfusion of CNP markedly prevented MC activation and endothelial barrier disruption in control but not in MC GC-B knockout mice. Notably, already under resting conditions, such knockout mice had increased numbers of degranulated MCs in different tissues, together with elevated plasma chymase levels. After transient coronary occlusion, their myocardial areas at risk and with infarction were enlarged. Moreover, MC GC-B knockout mice showed augmented perivascular neutrophil infiltration and deep vein thrombosis in a model of inferior vena cava ligation. CONCLUSIONS: CNP, via GC-B/cyclic GMP signaling, stabilizes resident perivascular MCs at baseline and prevents their excessive activation under pathological conditions. Thereby CNP contributes to the maintenance of vascular integrity in physiology and disease.

Hepatitis B Virus Particles Activate Toll-Like Receptor 2 Signaling Initially Upon Infection of Primary Human Hepatocytes.

BACKGROUND AND AIMS: To date, conflicting data exist as to whether hepatitis B virus (HBV) has the ability to induce innate immune responses. Here, we investigated cellular changes after the first contact between HBV and primary human hepatocytes (PHH) in vitro and in vivo. APPROACH AND RESULTS: The exposure of PHH to HBV particles resulted in nuclear translocation of NFκB, followed by the expression and secretion of inflammatory cytokines (IL [interleukin] 1B, IL6, and TNF [tumor necrosis factor]). Ultraviolet irradiation of viral particles suppressed HBV infectivity but not the induction of cytokines in PHH, suggesting that the inoculum contains the immune-inducing agent. Purified HBV particles on the whole, which were prepared from HBV DNA-positive and protein-rich fractions after heparin column separation, still had immune-inducing capacity in PHH. The HBV-induced gene expression profile was similar to that induced by toll-like receptor 2 (TLR2) ligand Pam3Cys, but different from those induced by the viral sensors TLR3 or TLR7-9. Treatment of PHH with both HBV particles and Pam3Cys led to phosphorylation of ERK (extracellular signal-regulated kinase), JNK, and p38 mitogen-activated protein kinases as well as NFκB (nuclear factor kappa B). Finally, HBV-induced gene expression could be neutralized by TLR2-specific antibodies. Of note, pretreatment with an HBV entry inhibitor attenuated the TLR2-mediated response to HBV, suggesting a receptor binding-related mechanism. In liver-humanized uPA/severe combined immunodeficient (SCID)/beige mice challenged with HBV in vivo, immune induction could only marginally be seen. CONCLUSIONS: PHHs are able to sense HBV particles through TLR2, leading to an activation of anti-HBV immune responses in vitro. These findings challenge the previously described stealth properties of HBV.

New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation.

BACKGROUND: Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function. METHODS: NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing. RESULTS: In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14-29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins. CONCLUSIONS: NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.