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Intrahepatic cholangiocarcinoma (iCCA) has emerged as a promising candidate for precision medicine, especially in the case of activating FGFR2 gene fusions. In addition to fusions, a considerable fraction of iCCA patients reveals FGFR2 mutations, which might lead to uncontrolled activation of the FGFR2 pathway but are mostly of unknown functional significance. A current challenge for molecular tumor boards (MTB) is to predict the functional consequences of such FGFR2 alterations to guide potential treatment decisions. We report two iCCA patients with extracellular and juxtamembrane FGFR2 mutations. After in silico investigation of the alterations and identification of activated FGFR2 downstream targets in tumor specimens by immunohistochemistry and transcriptome analysis, the MTB recommended treatment with an FGFR-inhibiting tyrosine kinase inhibitor. Both patients developed a rapidly detectable and prolonged partial response to treatment. These two cases suggest an approach to characterize further detected FGFR2 mutations in iCCA to enable patients´ selection for a successful application of the FGFR -inhibiting drugs., (© 2021. The Author(s).)

Hepatocellular carcinoma (HCC) can have viral or non-viral causes 1-5 . Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need 6,7 . Here we report the progressive accumulation of exhausted, unconventionally activated CD8 + PD1 + T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8 + PD1 + T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8 + PD1 + CXCR6 + , TOX + , and TNF + T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8 + T cells or TNF neutralization, suggesting that CD8 + T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8 + PD1 + T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Immune checkpoint inhibitors (ICIs) belong to the therapeutic armamentarium in advanced hepatocellular carcinoma (HCC). However, only a minority of patients benefit from immunotherapy. Therefore, we aimed to identify indicators of therapy response. This multicenter analysis included 99 HCC patients. Progression-free (PFS) and overall survival (OS) were studied by Kaplan-Meier analyses for clinical parameters using weighted log-rank testing. Next-generation sequencing (NGS) was performed in a subset of 15 patients. The objective response (OR) rate was 19% median OS (mOS)16.7 months. Forty-one percent reached a PFS > 6 months; these patients had a significantly longer mOS (32.0 vs. 8.5 months). Child-Pugh (CP) A and B patients showed a mOS of 22.1 and 12.1 months, respectively. Ten of thirty CP-B patients reached PFS > 6 months, including 3 patients with an OR. Tumor mutational burden (TMB) could not predict responders. Of note, antibiotic treatment within 30 days around ICI initiation was associated with significantly shorter mOS (8.5 vs. 17.4 months). Taken together, this study shows favorable outcomes for OS with low AFP, OR, and PFS > 6 months. No specific genetic pattern, including TMB, could identify responders. Antibiotics around treatment initiation were associated with worse outcome, suggesting an influence of the host microbiome on therapy success.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually gated channels that are operated by voltage and by neurotransmitters via the cAMP system. cAMP-dependent HCN regulation has been proposed to play a key role in regulating circuit behavior in the thalamus. By analyzing a knockin mouse model (HCN2EA), in which binding of cAMP to HCN2 was abolished by 2 amino acid exchanges (R591E, T592A), we found that cAMP gating of HCN2 is essential for regulating the transition between the burst and tonic modes of firing in thalamic dorsal-lateral geniculate (dLGN) and ventrobasal (VB) nuclei. HCN2EA mice display impaired visual learning, generalized seizures of thalamic origin, and altered NREM sleep properties. VB-specific deletion of HCN2, but not of HCN4, also induced these generalized seizures of the absence type, corroborating a key role of HCN2 in this particular nucleus for controlling consciousness. Together, our data define distinct pathological phenotypes resulting from the loss of cAMP-mediated gating of a neuronal HCN channel.

Background: Extrahepatic manifestations of Echinococcus multilocularis are very rare, especially in the adrenal glands. To the best of our knowledge, only seven cases of adrenal alveolar echinococcosis have been reported, all from the Far East. All of these occurred exclusively in the right adrenal gland., Case Presentation: We report a rare case of an extrahepatic alveolar echinococcosis in an asymptomatic 78-year-old white man with an incidentaloma of his right adrenal gland. After surgical resection and medical treatment with albendazole no recurrence of the disease appeared at 1-year follow-up., Conclusions: As the occurrence of Echinococcus multilocularis in Europe increases, alveolar echinococcosis should be included in the differential diagnosis of cystic adrenal incidentalomas.

Recent studies identified the SLC26A9 Cl(-) channel as a modifier and potential therapeutic target in cystic fibrosis (CF). However, understanding of the regulation of SLC26A9 in epithelia remains limited and cellular models with stable expression for biochemical and functional studies are missing. We, therefore, generated Fisher rat thyroid (FRT) epithelial cells with stable expression of HA-tagged SLC26A9 via retroviral transfection and characterized SLC26A9 expression and function using Western blotting, immunolocalization, whole cell patch-clamp, and transepithelial bioelectric studies in Ussing chambers. We demonstrate stable expression of SLC26A9 in transfected FRT (SLC26A9-FRT) cells on the mRNA and protein level. Immunolocalization and Western blotting detected SLC26A9 in different intracellular compartments and to a lesser extent at the cell surface. Whole cell patch-clamp recordings demonstrated significantly increased constitutive Cl(-) currents in SLC26A9-FRT compared with control-transduced FRT (Control-FRT) cells (P < 0.01). Similar, transepithelial measurements showed that the basal short circuit current was significantly increased in SLC26A9-FRT vs. Control-FRT cell monolayers (P < 0.01). SLC26A9-mediated Cl(-) currents were increased by cAMP-dependent stimulation (IBMX and forskolin) and inhibited by GlyH-101, niflumic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), as well as RNAi knockdown of WNK1 implicated in epithelial osmoregulation. Our results support that these novel epithelial cells with stable expression of SLC26A9 will be a useful model for studies of pharmacological regulation including the identification of activators of SLC26A9 Cl(-) channels that may compensate deficient cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion and serve as an alternative therapeutic target in patients with CF and potentially other muco-obstructive lung diseases., (Copyright © 2016 the American Physiological Society.)

The anaerobic, acetogenic bacterium Acetobacterium woodii grows on hydrogen and carbon dioxide and uses the Wood-Ljungdahl pathway to fix carbon but also to synthesize ATP. The free energy change of acetogenesis from H2 + CO2 allows for synthesis of only a fraction of an ATP under environmental conditions, and A. woodii is clearly a paradigm for microbial life under extreme energy limitation. However, it was unknown how much energy is required to make ATP under these conditions. In the present study, we determined the phosphorylation potential in cells metabolizing three different acetogenic substrates. It accounts to 37.9 ± 1.3 kJ/mol ATP during acetogenesis from fructose, 32.1 ± 0.3 kJ/mol ATP during acetogenesis from H2 + CO2 and 30.2 ± 0.9 kJ/mol ATP during acetogenesis from CO, the lowest phosphorylation potential ever described. The physiological consequences in terms of energy conservation under extreme energy limitation are discussed.

Background: We hypothesized that patients undergoing nonoperative treatment for asymptomatic or minimally symptomatic osteochondral lesions of the talus (OLTs) would not deteriorate clinically or radiologically over time., Methods: Forty-eight patients (mean age = 48 years; range, 13-78 years) with an OLT confirmed by magnetic resonance imaging (MRI) who had not undergone ankle joint surgery were retrospectively reviewed. All patients were evaluated after a minimum follow-up of 2 years (mean = 52 months; range, 27-124 months). All patients filled out an individual questionnaire and underwent a physical and radiographic assessment (radiograph and hindfoot MRI)., Results: At final follow-up, 43 ankles (86%) in 41 patients were pain-free (visual analogue scale [VAS] 0, n = 12) or less painful (VAS 1-3, n = 31). Radiographically, osteoarthritis was absent in 47%, and grade 1 and 2 osteoarthritis each were found in 27% (van Dijk classification). Magnetic resonance imaging revealed no substantial progression in staging or lesion size. Pain at time of follow-up correlated with the depth of the lesion at initial MRI (P < .05) and with subchondral cyst formation and presence or change of bone marrow edema at follow-up MRI (P < .05)., Conclusion: Minimally symptomatic OLTs did not appear to progress or worsen over time when treated nonoperatively., Level of Evidence: Level IV, case series., (© The Author(s) 2014.)

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites.

Objective: Multicystic dysplastic kidney (MCDK) is the most common antenatally diagnosed cystic renal pathology. It refers to the presence of multiple kidney cysts surrounded by dysplastic parenchymal tissue. The typical MCDK ultrasound finding shows multiple nonconnecting thin-walled cysts within an irregularly outlined kidney. We want to present our own cases as an addition to the relatively scarce literature about MCDK., Method: We retrospectively review 54 cases (21 bilateral and 33 unilateral) with antenatally diagnosed MCDK between the years 1998 and 2012. We compare our data with the published literature and discuss different parameters., Results: Our findings show a male predominance of 1.8 : 1 and a left side/right side ratio of 0.73 : 1. Most of the cases were isolated findings, but 20.3% were associated with anomalies. One case was a Goldenhar syndrome. Of the bilateral MCDK cases, 14.3% had chromosomal defects. The mean gestational age at time of diagnosis was 20-21 weeks. Renal length was above the 90th normal percentile in 96.2% of the cases., Conclusion: Isolated unilateral MCDK has good prognosis. Bad prognosis is linked to association with other anomalies as well as to bilateral MCDK., (© 2013 John Wiley & Sons, Ltd.)

Background: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet., Methods: Gene expression profiling of two substrains of the Han:SPRD rat, namely PKD/Mhm and PKD/US, both harboring the same mutation, was conducted in 36-day-old animals. Catechol-O-methyltransferase (Comt) was identified as a potential modifier gene. A 3-month treatment with tolcapone, a selective inhibitor of Comt, was carried out in PKD/Mhm and PKD/US (cy/+) animals., Results: Comt is localized within a known modifier locus of PKD (MOP2). The enzyme encoding gene was found upregulated in the more severely affected PKD/Mhm substrain and was hence presumed to be a putative modifier gene of PKD. The treatment with tolcapone markedly attenuated the loss of renal function, inhibited renal enlargement, shifted the size distribution of renal cysts and retarded cell proliferation, apoptosis, inflammation and fibrosis development in affected (cy/+) male and female PKD/Mhm and PKD/US rats., Conclusions: Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD.

Objective: To determine if changes in natriuretic peptide levels [propeptide of atrial natriuretic peptide (proANP) and N-terminal propeptide of brain natriuretic peptide (NT-proBNP)] can be demonstrated in fetuses with serious growth restriction and Doppler ultrasonographic findings of hemodynamic compromise., Method: Natriuretic peptides of the umbilical vein (proANP and NT-proBNP), arterial and venous blood gases, as well as lactate levels of the umbilical vein were determined in 38 appropriate for gestational age (AGA) fetuses, and 24 intrauterine growth-restricted (IUGR) fetuses with absent (zero-flow) and 17 IUGR fetuses with reversed end-diastolic flow (reverse-flow) of the umbilical artery., Results: A continuous increase in natriuretic peptides in the presence of progressive deterioration was shown. ANP concentrations were significantly lower in the AGA group (mean 818 fmol/mL; range 508-991 fmol/mL) than the zero-flow group (mean 19,680 fmol/mL; range 13,680-25,080 fmol/mL) and the reverse-flow group (mean 33,880 fmol/mL; range 24,365-41,786 fmol/mL). Significant differences were further demonstrated in BNP concentrations of the AGA group (mean 32 fmol/L; range 25-38 fmol/L), zero-flow group (mean 1191 fmol/L; range 908-1655 fmol/L), and reverse-flow group (mean 3063 fmol/L; range 1281-4968 fmol/L). Additionally, significant differences in lactate concentrations were observed for the three groups: AGA, 2.6 mmol/L (range 2-3 mmol/L); zero-flow group, 4.1 mmol/L (range 3.3-5 mmol/L); and reverse-flow group, 7.7 mmol/L (range 5.9-13.7 mmol/L). Increased ANP levels correlated with the arterial base excess and lactate concentrations, as well as with the flow velocities in the ductus venosus during atrial contraction (a-wave), and both ductus venosus indices (S-a)/D and (S-a)/V(mean). No correlations were established for BNP., Conclusions: As components of a compensatory mechanism, natriuretic peptides exert an influence on the cardiovascular function of the fetus.

Effect of chronic thyrotropin-releasing hormone (TRH) treatment on the properties of spinal cord TRH-receptor was evaluated by Scatchard analysis of data on the saturation of specific binding of [methyl-His3-3H]TRH. Continuous subcutaneous administration of TRH (0.51 +/- 0.02 mg/kg/day) via an Alzetmini osmotic pump for ten days led to a substantial increase in both Bmax (control: 20.0 +/- 2.9, treated: 36.1 +/- 2.8 fmol/mg protein, P = 0.0018) and Kd (control: 7.6 +/- 0.8 treated: 11.7 +/- 1.8 nM, P = 0.0349) of the binding sites. These data suggest that chronic TRH treatment may not induce desensitization of physiologic/pharmacologic responses of TRH in the spinal cord.

An accelerated loss of dopaminergic neurons with advancing age leads to Parkinson's disease. The incidence of this disease is suggested to be lower in nicotine users. As the body ages, the number of dopamine receptors in the striatum decrease, an effect that has been shown to be slowed by reduction in body weight via dietary manipulations. To investigate the potential role of nicotine in aging of dopaminergic neurons, therefore, using a dose of nicotine that has no effect on body weight becomes essential. The results presented here show that administration at up to 1.0 mg% nicotinewater to rats for one year does not alter their body weight.

Many biologic effects of TRH seem to be mediated via a dopaminergic mechanism. The present study examined the effects of chronic TRH administration on the properties of nigrostriatal dopaminergic neurons. Ten days, continuous subcutaneous TRH administration via an osmotic minipump led to a significant rise in striatal level of 3,4-dihydroxyphenylacetic acid, but not of homovanillic acid or dopamine. These treatments also did not elicit any significant changes in the maximal binding capacity (Bmax) or affinity (KD) of either D1- or D2-dopamine receptor. By contrast, TRH administration led to a significant increase in both Bmax and KD of striatal mazindol binding. This effect of TRH, however, was not observed in in vitro studies. In conclusion, these data suggest that in vivo administration of TRH may modulate dopaminergic activities by altering, directly or indirectly, dopamine release and reuptake.

The influence of partial replacement of starch by sucrose on dietary cholesterol-induced serum lipoprotein responses was examined in 10 male cynomolgus monkeys (Macaca fascicularis). In a crossover design two semipurified diets provided either starch or starch and sucrose (1:1) as carbohydrate (49% by calories) with 0.4 mg cholesterol/kcal. Six weeks of starch + sucrose diet resulted in significantly reduced levels (mean +/- SE, mg/dl) of serum total cholesterol (264 +/- 9 vs 244 +/- 8) and apo B (110 +/- 6 vs 96 +/- 6) when compared with starch diet, whereas serum triglyceride levels remained similar between diets. With respect to changes in lipids and apolipoproteins (A-I or B) of very low (VLDL), low (LDL), intermediate (IDL), and high (HDL) density lipoproteins, starch + sucrose diet significantly increased VLDL-apo B (+34%), and decreased LDL-cholesterol (-18%) and LDL-apo B (-15%) as compared with starch alone; no differences were found in IDL and HDL between diets. The relative proportion of starch to sucrose in a diet appears to influence the magnitude of response of lipoproteins to dietary cholesterol.

Epidemiologic studies have suggested a positive association in man between nicotine use and the incidence of tardive dyskinesia, a disease characterized by dopaminergic supersensitivity after chronic neuroleptic therapy. In rats, repeated administration of neuroleptics results into dopaminergic supersensitivity and increased density of striatal D2-dopamine receptors. We investigated the effects of 6-week continuous nicotine intake on the neuroleptic (haloperidol)-induced increase in murine striatal D2-dopamine receptor density. Contrary to expectations, our data show that nicotine blocked the increase in D2-dopamine receptor density after neuroleptic administration.

The relationship of carbohydrate type to cholesterol-induced hypercholesterolemia and the potential role of intestinal flora in the above process were examined in 12 male cynomolgus monkeys (M. fascicularis). Semipurified diets provided two types of carbohydrates (starch or sucrose, 49% by calorie) with 0.4 mg cholesterol/kcal. Six weeks of the starch diet resulted in significantly enhanced hypercholesterolemia when compared to sucrose diet. Starch in relation to sucrose produced cholesterol enrichment of intermediate density lipoproteins and increase in low density lipoprotein particles, whereas sucrose increased high density lipoprotein constituents (phospholipids, cholesterol, and apoA-I) and triglyceride content of very low density lipoproteins. Fecal Escherichia counts were high during the starch diet as contrasted with sucrose diet, but the Escherichia, Streptococcus, and Bacteroides groups did not show differences by diet following each consecutive 4-week period of oral neomycin (107 mg/kg body wt) treatment and withdrawal. The magnitude of hypercholesterolemia during these periods also remained similar between starch and sucrose, suggesting formation of germfree metabolic characteristics. Thus, the magnitude of cholesterol-induced hypercholesterolemia can be affected by the type of carbohydrate, which may be in part determined by intestinal flora metabolism.