The ABCG8 polymorphism increases the risk of gallbladder cancer in the general population and gallstones in obese patients from Poland.

BACKGROUND: Gallstone disease (GD) is common but remains asymptomatic in most cases. However, gallstones can lead to complications like choledocholithiasis or gallbladder cancer. In this study, we analyse the common genetic risk factor for GD, the p.D19H variant in the sterol transporter ABCG8, in Polish patients with gallstones and gallbladder cancer. METHODS: Three adult cohorts were prospectively recruited: 65 patients with gallbladder cancer, 170 obese individuals scheduled for bariatric surgery and 72 patients who underwent endoscopic retrograde cholangiopancreatography due to recurrent choledocholithiasis. The control cohort consisted of 172 gallstone-free adults. The ABCG8 p.D19H (rs11887534) polymorphism was genotyped using TaqMan assays. RESULTS: The minor allele frequency (MAF) of the ABCG8 p.D19H polymorphism was significantly (p = .02) higher among cases with either gallstones or gallbladder cancer (MAF = 8.4%) as compared to controls (MAF = 4.0%). The highest frequency of the risk allele was detected in patients with gallbladder cancer (18.5%) and obese patients with GD (17.5%), followed by individuals with choledocholithiasis (13.9%). Notably, the p.19H variant was associated with an increased risk of developing gallbladder cancer (OR 2.76, 95% CI 1.16-6.54, p = .01) and an increased risk of GD in obese individuals scheduled for bariatric surgery (OR = 2.70, 95% CI 1.05-6.49, p = .03), but did not significantly affect the risk of choledocholithiasis. CONCLUSIONS: The ABCG8 p.D19H common risk variant increases the risk of developing gallbladder cancer in Central Europeans and enhances the risk of gallstones in the obese. Carriers of the p.D19H variant might benefit from personalized preventive strategies, particularly regarding gallbladder cancer.

Stacked probability plots of the extended illness-death model using constant transition hazards - an easy to use shiny app.

BACKGROUND: Extended illness-death models (a specific class of multistate models) are a useful tool to analyse situations like hospital-acquired infections, ventilation-associated pneumonia, and transfers between hospitals. The main components of these models are hazard rates and transition probabilities. Calculation of different measures and their interpretation can be challenging due to their complexity. METHODS: By assuming time-constant hazards, the complexity of these models becomes manageable and closed mathematical forms for transition probabilities can be derived. Using these forms, we created a tool in R to visualize transition probabilities via stacked probability plots. RESULTS: In this article, we present this tool and give some insights into its theoretical background. Using published examples, we give guidelines on how this tool can be used. Our goal is to provide an instrument that helps obtain a deeper understanding of a complex multistate setting. CONCLUSION: While multistate models (in particular extended illness-death models), can be highly complex, this tool can be used in studies to both understand assumptions, which have been made during planning and as a first step in analysing complex data structures. An online version of this tool can be found at https://eidm.imbi.uni-freiburg.de/ .

Skin advanced glycation end-products as indicators of the metabolic profile in diabetes mellitus: correlations with glycemic control, liver phenotypes and metabolic biomarkers.

INTRODUCTION: The production of advanced glycation end-products (AGEs) is a key pathomechanism related to the complications of diabetes mellitus. The measurement of HbA1c as one of the AGEs is widely used in the clinic, but also other proteins undergo glycation in the course of diabetes. Here, we measure skin AGEs (SAGEs) in patients with diabetes type 1 (DM1) and type 2 (DM2) and correlate them with metabolic markers as well as non-invasively measured liver fibrosis and steatosis. PATIENTS AND METHODS: In this cross-sectional study, a total of 64 patients with either DM1 or DM2 and 28 healthy controls were recruited. SAGEs were measured using autofluorescence (AGE Reader). Liver fibrosis and steatosis were quantified using transient elastography, which determines liver stiffness measurement (LSM) and controlled attenuation parameter (CAP). FGF19, FGF21 and GDF-15 were measured in blood samples using ELISA. RESULTS: SAGEs were elevated in both groups of patients with diabetes as compared to healthy controls (both p < 0.001) and were higher in patients with DM2 in comparison to DM1 (p = 0.006). SAGEs correlated positively with HbA1c (r = 0.404, p < 0.001), CAP (r = 0.260, p = 0.016) and LSM (r = 0.356, p < 0.001), and negatively with insulin growth factor binding protein 3 (p < 0.001). We also detected a positive correlation between GDF15 and SAGEs (r = 0.469, p < 0.001). CONCLUSIONS: SAGEs are significantly elevated in patients with both DM types 1 and 2 and correlate with metabolic markers, including HbA1c and GDF15. They might also help to detect patients with advanced liver injury in the setting of diabetes.

Cross-reactivity of a pathogenic autoantibody to a tumor antigen in GABAA receptor encephalitis.

Encephalitis associated with antibodies against the neuronal gamma-aminobutyric acid A receptor (GABAA-R) is a rare form of autoimmune encephalitis. The pathogenesis is still unknown but autoimmune mechanisms were surmised. Here we identified a strongly expanded B cell clone in the cerebrospinal fluid of a patient with GABAA-R encephalitis. We expressed the antibody produced by it and showed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry that it recognizes the GABAA-R. Patch-clamp recordings revealed that it tones down inhibitory synaptic transmission and causes increased excitability of hippocampal CA1 pyramidal neurons. Thus, the antibody likely contributed to clinical disease symptoms. Hybridization to a protein array revealed the cross-reactive protein LIM-domain-only protein 5 (LMO5), which is related to cell-cycle regulation and tumor growth. We confirmed LMO5 recognition by immunoprecipitation and ELISA and showed that cerebrospinal fluid samples from two other patients with GABAA-R encephalitis also recognized LMO5. This suggests that cross-reactivity between GABAA-R and LMO5 is frequent in GABAA-R encephalitis and supports the hypothesis of a paraneoplastic etiology.

Effects of MBOAT7 polymorphism and steatosis on liver function assessed by methacetin breath test.

BACKGROUND: MBOAT7 rs641738 variant is a risk factor for nonalcoholic fatty liver disease (NAFLD) and liver fibrosis, but the relationship between this polymorphism and early liver dysfunction remains uncertain. METHODS: Eighty outpatients underwent blood analyses, liver imaging by ultrasound and acoustic radiation force impulse shear wave elastography and were genotyped for MBOAT7 (wild-type [WT], rs641738 heterozygous or homozygous) polymorphism using TaqMan assays. RESULTS: NAFLD was confirmed in 53 patients. Portal uptake and hepatocyte microsomal metabolization of (13 C)-methacetin were explored by measuring 13 CO2 appearance in exhaled air. The distribution of the MBOAT7 genotypes was comparable in subjects with or without NAFLD. The majority of subjects with or without NAFLD had fibrosis ≤ F1 but decreased portal extraction of (13 C)-methacetin, i.e. 78.6% in homozygous, 45.0% in heterozygous and 46.2% in WT for the MBOAT7 variant. Both substrate extraction and microsomal metabolization were mostly defective in the homozygous carriers. The extraction efficiency from portal blood flow was minimal in subjects with both homozygous rs641738 polymorphism and NAFLD, as compared to those with WT/heterozygous polymorphism, with or without NAFLD. CONCLUSIONS: The homozygous MBOAT7 rs641738 polymorphism per se is associated with a reduced extraction efficiency of (13 C)-methacetin from the portal flow pointing to subclinical liver dysfunction independently from liver fibrosis. Liver steatosis worsens (13 C)-methacetin extraction efficiency. We urge to better explore the mechanisms of interaction between external factors and multiple gene polymorphisms (including MBOAT7), paving the road to primary prevention and novel therapeutic strategies.

Influence of NOD2 risk variants on hepatic encephalopathy and association with inflammation, bacterial translocation and immune activation.

BACKGROUND: Nucleotide-binding oligomerization domain containing 2 (NOD2) risk variants lead to impaired mucosal barrier function, increased bacterial translocation (BT), and systemic inflammation. AIM: To evaluate the association between the presence of NOD2 risk variants, BT, inflammation, and hepatic encephalopathy (HE). PATIENTS AND METHODS: This prospective multicenter study included patients with cirrhosis and testing for NOD2 risk variants (p.R702W, p.G908R, c.3020insC, N289S, and c.-958T>C). Patients were evaluated for covert (C) and overt (O) HE. Markers of systemic inflammation (leukocytes, CRP, IL-6, LBP) and immune activation (soluble CD14) as well as bacterial endotoxin (hTRL4 activation) were determined in serum. RESULTS: Overall, 172 patients (70% men; median age 60 [IQR 54-66] years; MELD 12 [IQR 9-16]; 72% ascites) were included, of whom 53 (31%) carried a NOD2 risk variant. In this cohort, 11% presented with OHE and 27% and CHE. Presence and severity of HE and surrogates of inflammation, BT, and immune activation did not differ between patients with and without a NOD2 risk variant, also not after adjustment for MELD. HE was associated with increased ammonia and systemic inflammation, as indicated by elevated CRP (w/o HE: 7.2 [2.7-16.7]; with HE 12.6 [4.5-29.7] mg/dL; p < 0.001) and elevated soluble CD14 (w/o HE 2592 [2275-3033]; with HE 2755 [2410-3456] ng/mL; p = 0.025). CONCLUSIONS: The presence of NOD2 risk variants in patients with cirrhosis is not associated with HE and has no marked impact on inflammation, BT, or immune activation. In contrast, the presence of HE was linked to ammonia, the acute phase response, and myeloid cell activation.

Liver phenotypes in PCOS: Analysis of exogenous and inherited risk factors for liver injury in two European cohorts.

BACKGROUND & AIMS: Fatty liver disease (FLD) is common in women with polycystic ovary syndrome (PCOS). Here, we use non-invasive tests to quantify liver injury in women with PCOS and analyse whether FLD-associated genetic variants contribute to liver phenotypes in PCOS. METHODS: Prospectively, we recruited women with PCOS and controls at two university centres in Germany and Poland. Alcohol abuse was regarded as an exclusion criterion. Genotyping of variants associated with FLD was performed using TaqMan assays. Liver stiffness measurements (LSM), controlled attenuation parameters (CAP) and non-invasive HSI, FLI, FIB-4 scores were determined to assess hepatic steatosis and fibrosis. RESULTS: A total of 42 German (age range 18-53 years) and 143 Polish (age range 18-40 years) women with PCOS, as well as 245 German and 289 Polish controls were recruited. In contrast to Polish patients, Germans were older, presented with more severe metabolic profiles and had significantly higher LSM (median 5.9 kPa vs. 3.8 kPa). In the German cohort, carriers of the PNPLA3 p.I148M risk variant had an increased LSM (p = .01). In the Polish cohort, the minor MTARC1 allele was linked with significantly lower serum aminotransferases activities, whereas the HSD17B13 polymorphism was associated with lower concentrations of 17-OH progesterone, total testosterone, and androstenedione (all p < .05). CONCLUSIONS: FLD is common in women with PCOS. Its extent is modulated by both genetic and metabolic risk factors. Genotyping of variants associated with FLD might help to stratify the risk of liver disease progression in women suffering from PCOS.

Surface plasmon resonance assays for the therapeutic drug monitoring of infliximab indicate clinical relevance of anti-infliximab antibody binding properties.

OBJECTIVES: The therapeutic antibody infliximab (IFX) has improved the life quality of numerous autoinflammatory disease patients. However, IFX can trigger the generation of anti-drug antibodies (ADA), whose optimal evaluation and management are currently subject of controversial discussions. We present two novel surface plasmon resonance (SPR) biosensor assays for therapeutic drug monitoring of IFX and characterization of ADA and investigated the diagnostic value of ADA binding properties. METHODS: IFX and ADA were quantified via developed SPR biosensor assays (IFXmon and ADAmon, respectively) and diagnostics-approved ELISA in sera from inflammatory bowel disease patients. Pre-analytic ADA enrichment with magnetic beads enabled analytical drug tolerance of the ADAmon assay. The dissociation ratio (DissR) as an index for ADA:IFX binding stability was calculated from the SPR sensorgrams of ADA quantification runs. RESULTS: IFX levels determined by IFXmon assay and ELISA showed high agreement, whereas ADA quantification concordance between ADAmon assay and ELISA was poor. In patients, DissR was predominantly constant over time and differed significantly between therapy outcomes. A DissR cut-off of 1.524 indicated undetectable IFX levels with 71.4% sensitivity and 88.9% specificity. Additionally, the SPR reference surface was exploited as serum-individual negative control to check result plausibility within multi-sample run sequences. CONCLUSIONS: Overall, both SPR biosensor assays exhibited reliable quantitative performance with accuracies superior to their ELISA counterparts and precision inferior to ELISA only for ADAmon. DissR presented itself as promising ADA binding parameter and could contribute to both earlier and more tailored therapeutic decisions.

Reprograming of sRNA target specificity by the leader peptide peTrpL in response to antibiotic exposure.

Trans-acting regulatory RNAs have the capacity to base pair with more mRNAs than generally detected under defined conditions, raising the possibility that sRNA target specificities vary depending on the specific metabolic or environmental conditions. In Sinorhizobium meliloti, the sRNA rnTrpL is derived from a tryptophan (Trp) transcription attenuator located upstream of the Trp biosynthesis gene trpE(G). The sRNA rnTrpL contains a small ORF, trpL, encoding the 14-aa leader peptide peTrpL. If Trp is available, efficient trpL translation causes transcription termination and liberation of rnTrpL, which subsequently acts to downregulate the trpDC operon, while peTrpL is known to have a Trp-independent role in posttranscriptional regulation of antibiotic resistance mechanisms. Here, we show that tetracycline (Tc) causes rnTrpL accumulation independently of Trp availability. In the presence of Tc, rnTrpL and peTrpL act collectively to destabilize rplUrpmA mRNA encoding ribosomal proteins L21 and L27. The three molecules, rnTrpL, peTrpL, and rplUrpmA mRNA, form an antibiotic-dependent ribonucleoprotein complex (ARNP). In vitro reconstitution of this ARNP in the presence of competing trpD and rplU transcripts revealed that peTrpL and Tc cause a shift of rnTrpL specificity towards rplU, suggesting that sRNA target prioritization may be readjusted in response to changing environmental conditions.

Common variant p.D19H of the hepatobiliary sterol transporter ABCG8 increases the risk of gallstones in children.

INTRODUCTION: Gallstones are increasingly common in children. Genetic analyses of adult cohorts demonstrated that the sterol transporter ABCG8 p.D19H and Gilbert UGT1A1*28 variants enhance the odds of developing gallstones. The genetic background of common lithiasis in children remains unknown. METHODS: Overall, 214 children with gallstone disease (1 month-17 years, 107 boys) were inclueded. The control cohorts comprised 214 children (age 6-17 years, 115 boys) and 172 adults (age 40-92 years, 70 men) without gallstones. The ABCG8 p.D19H and UGT1A1*28 polymorphisms as well as ABCB4 (c.504C>T rs1202283, c.711A>T rs2109505) and NPC1L1 variants (p.V1296V rs217434, c.-18C>A rs41279633) were genotyped using TaqMan assays. Serum concentrations of plant sterols and cholesterol precursors were measured by gas chromatography/mass spectrometry. RESULTS: The ABCG8 risk allele was associated with an increased risk of stones (OR = 1.82, p = .03). Children carrying the p.19H allele presented with lower serum concentrations of surrogate markers of intestinal cholesterol absorption and decreased ratios of phytosterols to the cholesterol precursor desmosterol. Carriers of the common NPC1L1 rs217434 allele had an increased gallstone risk compared with stone-free adults (OR 1.90, p < .01). This variant also affected the ratio of phytosterols to cholesterol precursors (p = .03). Other tested variants were not associated with gallstone risk. CONCLUSIONS: The p.D19H ABCG8 and, to a lesser extent, NPC1L1 rs217434 variants increase the risk of early-onset gallstone formation. These results point to the presence of a common lithogenic pathway in children and adults.

An integrated device for fast and sensitive immunosuppressant detection.

The present paper describes a compact point of care (POC) optical device for therapeutic drug monitoring (TDM). The core of the device is a disposable plastic chip where an immunoassay for the determination of immunosuppressants takes place. The chip is designed in order to have ten parallel microchannels allowing the simultaneous detection of more than one analyte with replicate measurements. The device is equipped with a microfluidic system, which provides sample mixing with the necessary chemicals and pumping samples, reagents and buffers into the measurement chip, and with integrated thin film amorphous silicon photodiodes for the fluorescence detection. Submicrometric fluorescent magnetic particles are used as support in the immunoassay in order to improve the efficiency of the assay. In particular, the magnetic feature is used to concentrate the antibody onto the sensing layer leading to a much faster implementation of the assay, while the fluorescent feature is used to increase the optical signal leading to a larger optical dynamic change and consequently a better sensitivity and a lower limit of detection. The design and development of the whole integrated optical device are here illustrated. In addition, detection of mycophenolic acid and cyclosporine A in spiked solutions and in microdialysate samples from patient blood with the implemented device are reported.

The rs12532734 Polymorphism Near the Solute Carrier 26A3 Gene Locus Is Associated With Gallstone Disease in Children.

Gallstones are increasingly frequent in children. In this candidate gene study, we genotyped 5 gene variants ( ANO1 , SPTLC3 , TMEM147 , TNRC6B , rs12532734) from a recent gallstone genome-wide association study (GWAS) in a cohort of 214 children with gallstones and 172 gallstone-free adult controls. In total, 138 genotyped children presented with symptomatic gallstone disease, 47 underwent cholecystectomy, and 126 received ursodeoxycholic acid (UDCA) as therapy for stones. Among 5 tested variants, the rs12532734 polymorphism modulated the gallstone risk in the studied cohort. Its genotype distribution significantly ( P = 0.025) departed from the Hardy-Weinberg equilibrium among cases, and the common allele was associated with increased odds of developing gallstones at young age (OR = 1.69, P = 0.014). SLC26A3 is the nearest gene to rs12532734 and is involved in the transepithelial bicarbonate and chloride transport. The association of rs12532734 with pediatric gallstones is a novel finding warranting further investigations also with regard to biliary bicarbonate flux and bile composition.

Genetically determined NLRP3 inflammasome activation associates with systemic inflammation and cardiovascular mortality.

AIMS: Inflammation plays an important role in cardiovascular disease (CVD) development. The NOD-like receptor protein-3 (NLRP3) inflammasome contributes to the development of atherosclerosis in animal models. Components of the NLRP3 inflammasome pathway such as interleukin-1ß can therapeutically be targeted. Associations of genetically determined inflammasome-mediated systemic inflammation with CVD and mortality in humans are unknown. METHODS AND RESULTS: We explored the association of genetic NLRP3 variants with prevalent CVD and cardiovascular mortality in 538 167 subjects on the individual participant level in an explorative gene-centric approach without performing multiple testing. Functional relevance of single-nucleotide polymorphisms on NLRP3 inflammasome activation has been evaluated in monocyte-enriched peripheral blood mononuclear cells (PBMCs). Genetic analyses identified the highly prevalent (minor allele frequency 39.9%) intronic NLRP3 variant rs10754555 to affect NLRP3 gene expression. rs10754555 carriers showed significantly higher C-reactive protein and serum amyloid A plasma levels. Carriers of the G allele showed higher NLRP3 inflammasome activation in isolated human PBMCs. In carriers of the rs10754555 variant, the prevalence of coronary artery disease was significantly higher as compared to non-carriers with a significant interaction between rs10754555 and age. Importantly, rs10754555 carriers had significantly higher risk for cardiovascular mortality during follow-up. Inflammasome inducers (e.g. urate, triglycerides, apolipoprotein C3) modulated the association between rs10754555 and mortality. CONCLUSION: The NLRP3 intronic variant rs10754555 is associated with increased systemic inflammation, inflammasome activation, prevalent coronary artery disease, and mortality. This study provides evidence for a substantial role of genetically driven systemic inflammation in CVD and highlights the NLRP3 inflammasome as a therapeutic target.

MTARC1 and HSD17B13 Variants Have Protective Effects on Non-Alcoholic Fatty Liver Disease in Patients Undergoing Bariatric Surgery.

The severity of hepatic steatosis is modulated by genetic variants, such as patatin-like phospholipase domain containing 3 (PNPLA3) rs738409, transmembrane 6 superfamily member 2 (TM6SF2) rs58542926, and membrane-bound O-acyltransferase domain containing 7 (MBOAT7) rs641738. Recently, mitochondrial amidoxime reducing component 1 (MTARC1) rs2642438 and hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) rs72613567 polymorphisms were shown to have protective effects on liver diseases. Here, we evaluate these variants in patients undergoing bariatric surgery. A total of 165 patients who underwent laparoscopic sleeve gastrectomy and intraoperative liver biopsies and 314 controls were prospectively recruited. Genotyping was performed using TaqMan assays. Overall, 70.3% of operated patients presented with hepatic steatosis. NASH (non-alcoholic steatohepatitis) was detected in 28.5% of patients; none had cirrhosis. The increment of liver fibrosis stage was associated with decreasing frequency of the MTARC1 minor allele (p = 0.03). In multivariate analysis MTARC1 was an independent protective factor against fibrosis ≥ 1b (OR = 0.52, p = 0.03) and ≥ 1c (OR = 0.51, p = 0.04). The PNPLA3 risk allele was associated with increased hepatic steatosis, fibrosis, and NASH (OR = 2.22, p = 0.04). The HSD17B13 polymorphism was protective against liver injury as reflected by lower AST (p = 0.04) and ALT (p = 0.03) activities. The TM6SF2 polymorphism was associated with increased ALT (p = 0.04). In conclusion, hepatic steatosis is common among patients scheduled for bariatric surgery, but the MTARC1 and HSD17B13 polymorphisms lower liver injury in these individuals.

Immunosuppressant quantification in intravenous microdialysate - towards novel quasi-continuous therapeutic drug monitoring in transplanted patients.

OBJECTIVES: Therapeutic drug monitoring (TDM) plays a crucial role in personalized medicine. It helps clinicians to tailor drug dosage for optimized therapy through understanding the underlying complex pharmacokinetics and pharmacodynamics. Conventional, non-continuous TDM fails to provide real-time information, which is particularly important for the initial phase of immunosuppressant therapy, e.g., with cyclosporine (CsA) and mycophenolic acid (MPA). METHODS: We analyzed the time course over 8 h of total and free of immunosuppressive drug (CsA and MPA) concentrations measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 16 kidney transplant patients. Besides repeated blood sampling, intravenous microdialysis was used for continuous sampling. Free drug concentrations were determined from ultracentrifuged EDTA-plasma (UC) and compared with the drug concentrations in the respective microdialysate (µD). µDs were additionally analyzed for free CsA using a novel immunosensor chip integrated into a fluorescence detection platform. The potential of microdialysis coupled with an optical immunosensor for the TDM of immunosuppressants was assessed. RESULTS: Using LC-MS/MS, the free concentrations of CsA (fCsA) and MPA (fMPA) were detectable and the time courses of total and free CsA comparable. fCsA and fMPA and area-under-the-curves (AUCs) in µDs correlated well with those determined in UCs (r≥0.79 and r≥0.88, respectively). Moreover, fCsA in µDs measured with the immunosensor correlated clearly with those determined by LC-MS/MS (r=0.82). CONCLUSIONS: The new microdialysis-supported immunosensor allows real-time analysis of immunosuppressants and tailor-made dosing according to the AUC concept. It readily lends itself to future applications as minimally invasive and continuous near-patient TDM.

Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans.

Ribosome-mediated transcription attenuation is a basic posttranscriptional regulation mechanism in bacteria. Liberated attenuator RNAs arising in this process are generally considered nonfunctional. In Sinorhizobium meliloti, the tryptophan (Trp) biosynthesis genes are organized into three operons, trpE(G), ppiD-trpDC-moaC-moeA, and trpFBA-accD-folC, of which only the first one, trpE(G), contains a short ORF (trpL) in the 5'-UTR and is regulated by transcription attenuation. Under conditions of Trp sufficiency, transcription is terminated between trpL and trpE(G), and a small attenuator RNA, rnTrpL, is produced. Here, we show that rnTrpL base-pairs with trpD and destabilizes the polycistronic trpDC mRNA, indicating rnTrpL-mediated downregulation of the trpDC operon in trans. Although all three trp operons are regulated in response to Trp availability, only in the two operons trpE(G) and trpDC the Trp-mediated regulation is controlled by rnTrpL. Together, our data show that the trp attenuator coordinates trpE(G) and trpDC expression posttranscriptionally by two fundamentally different mechanisms: ribosome-mediated transcription attenuation in cis and base-pairing in trans. Also, we present evidence that rnTrpL-mediated regulation of trpDC genes expression in trans is conserved in Agrobacterium and Bradyrhizobium, suggesting that the small attenuator RNAs may have additional conserved functions in the control of bacterial gene expression.

Validation of the German Version of Functional Oral Intake Scale (FOIS-G) for Flexible Endoscopic Evaluation of Swallowing (FEES).

The Functional Oral Intake Scale (FOIS) is the most frequently used scale for the evaluation of functional oral intake by dysphagia patients. FOIS was validated using data from Videofluoroscopic Swallowing Study (VFSS). Until now, a validated German version of FOIS for Flexible Endoscopic Evaluation of Swallowing (FEES) is lacking. The aim of this study was a cross-cultural validation of the German version of FOIS (FOIS-G) for FEES. The translation of the original FOIS was carried out according to the Translation, Review, Adjudication, Pretesting, Documentation (TRAPD) translation methodology. For the validation process, six experienced language therapists (SLT) retrospectively analyzed charts of 93 stroke patients. Inclusion criteria were comprised of stroke, clinical examination by an SLT within 24 h of admission, and FEES within 72 h of admission. The validity was calculated by comparison with Modified Rankin Scale (MRS), Barthel Index (BI), the Penetration-Aspiration-Scale (PAS), and a water swallow test. Spearman rank correlation of all paired raters ranged from rs = 0.96 to rs = 0.99, and percentage agreement ranged from 81 to 94%. The overall agreement between all raters was calculated by Fleiss kappa (0.83) (s.e. 0.02). There is a significant correlation between the BI and the MRS with the FOIS-G (rs = 0.301, p = 0.003 for BI; rs = - 0.366, p < 0.001 for MRS), between the PAS and the FOIS-G (rs = - 0.758, p < 0.001), as well as between the 70 ml-water-test and the FOIS-G (rs = 0.470, p < 0.001). FOIS-G is a valid instrument for the evaluation of the functional oral intake of food and liquids in dysphagia patients.

Fibroblast Growth Factor 21 Response in a Preclinical Alcohol Model of Acute-on-Chronic Liver Injury.

BACKGROUND AND AIMS: Fibroblast growth factor (FGF) 21 has recently been shown to play a potential role in bile acid metabolism. We aimed to investigate the FGF21 response in an ethanol-induced acute-on-chronic liver injury (ACLI) model in Abcb4-/- mice with deficiency of the hepatobiliary phospholipid transporter. METHODS: Total RNA was extracted from wild-type (WT, C57BL/6J) and Abcb4-/- (KO) mice, which were either fed a control diet (WT-Cont and KO-Cont groups; n = 28/group) or ethanol diet, followed by an acute ethanol binge (WT-EtOH and KO-EtOH groups; n = 28/group). A total of 58 human subjects were recruited into the study, including patients with alcohol-associated liver disease (AALD; n = 31) and healthy controls (n = 27). The hepatic and ileal expressions of genes involved in bile acid metabolism, plasma FGF levels, and bile acid and its precursors 7α- and 27-hydroxycholesterol (7α- and 27-OHC) concentrations were determined. Primary mouse hepatocytes were isolated for cell culture experiments. RESULTS: Alcohol feeding significantly induced plasma FGF21 and decreased hepatic Cyp7a1 levels. Hepatic expression levels of Fibroblast growth factor receptor 1 (Fgfr1), Fgfr4, Farnesoid X-activated receptor (Fxr), and Small heterodimer partner (Shp) and plasma FGF15/FGF19 levels did not differ with alcohol challenge. Exogenous FGF21 treatment suppressed Cyp7a1 in a dose-dependent manner in vitro. AALD patients showed markedly higher FGF21 and lower 7α-OHC plasma levels while FGF19 did not differ. CONCLUSIONS: The simultaneous upregulation of FGF21 and downregulation of Cyp7a1 expressions upon chronic plus binge alcohol feeding together with the invariant plasma FGF15 and hepatic Shp and Fxr levels suggest the presence of a direct regulatory mechanism of FGF21 on bile acid homeostasis through inhibition of CYP7A1 by an FGF15-independent pathway in this ACLI model. Lay Summary: Alcohol challenge results in the upregulation of FGF21 and repression of Cyp7a1 expressions while circulating FGF15 and hepatic Shp and Fxr levels remain constant both in healthy and pre-injured livers, suggesting the presence of an alternative FGF15-independent regulatory mechanism of FGF21 on bile acid homeostasis through the inhibition of Cyp7a1.

Carotid geometry is an independent predictor of wall thickness - a 3D cardiovascular magnetic resonance study in patients with high cardiovascular risk.

BACKGROUND: The posterior wall of the proximal internal carotid artery (ICA) is the predilection site for the development of stenosis. To optimally prevent stroke, identification of new risk factors for plaque progression is of high interest. Therefore, we studied the impact of carotid geometry and wall shear stress on cardiovascular magnetic resonance (CMR)-depicted wall thickness in the ICA of patients with high cardiovascular disease risk. METHODS: One hundred twenty-one consecutive patients ≥50 years with hypertension, ≥1 additional cardiovascular risk factor and ICA plaque ≥1.5 mm thickness and < 50% stenosis were prospectively included. High-resolution 3D-multi-contrast (time of flight, T1, T2, proton density) and 4D flow CMR were performed for the assessment of morphological (bifurcation angle, ICA/common carotid artery (CCA) diameter ratio, tortuosity, and wall thickness) and hemodynamic parameters (absolute/systolic wall shear stress (WSS), oscillatory shear index (OSI)) in 242 carotid bifurcations. RESULTS: We found lower absolute/systolic WSS, higher OSI and increased wall thickness in the posterior compared to the anterior wall of the ICA bulb (p < 0.001), whereas this correlation disappeared in ≥10% stenosis. Higher carotid tortuosity (regression coefficient = 0.764; p < 0.001) and lower ICA/CCA diameter ratio (regression coefficient = - 0.302; p < 0.001) were independent predictors of increased wall thickness even after adjustment for cardiovascular risk factors. This association was not found for bifurcation angle, WSS or OSI in multivariate regression analysis. CONCLUSIONS: High carotid tortuosity and low ICA diameter were independent predictors for wall thickness of the ICA bulb in this cross-sectional study, whereas this association was not present for WSS or OSI. Thus, consideration of geometric parameters of the carotid bifurcation could be helpful to identify patients at increased risk of carotid plaque generation. However, this association and the potential benefit of WSS measurement need to be further explored in a longitudinal study.

Randomized, Double-Blind, Placebo-Controlled, Single-Ascending-Dose Study of the Penetration of a Monoclonal Antibody Combination (ASN100) Targeting Staphylococcus aureus Cytotoxins in the Lung Epithelial Lining Fluid of Healthy Volunteers.

ASN100 is a novel antibody combination of two fully human IgG1(κ) monoclonal antibodies (MAbs), ASN-1 and ASN-2, which neutralize six Staphylococcus aureus cytotoxins, alpha-hemolysin (Hla) and five bicomponent leukocidins. We assessed the safety, tolerability, and serum and lung pharmacokinetics of ASN100 in a randomized, double-blind, placebo-controlled single-dose-escalation first-in-human study. Fifty-two healthy volunteers were enrolled and randomized to receive either ASN-1, ASN-2, a combination of both MAbs (ASN100), or a corresponding placebo. Thirty-two subjects in the double-blind dose escalation portion of the study received ASN-1 or ASN-2 at a 200-, 600-, 1,800-, or 4,000-mg dose, or placebo. Eight subjects received both MAbs simultaneously in a 1:1 ratio (ASN100) at 3,600 or 8,000 mg, or they received placebos. Twelve additional subjects received open-label ASN100 at 3,600 or 8,000 mg to assess the pharmacokinetics of ASN-1 and ASN-2 in epithelial lining fluid (ELF) by bronchoalveolar lavage fluid sampling. Subjects were monitored for 98 days (double-blind cohorts) or 30 days (open-label cohorts) for safety assessment. No dose-limiting toxicities were observed, and all adverse events were mild and transient, with only two adverse events considered possibly related to the investigational product. ASN100 exhibited linear serum pharmacokinetics with a half-life of approximately 3 weeks and showed detectable penetration into the ELF. No treatment-emergent anti-drug antibody responses were detected. The toxin neutralizing potency of ASN100 in human serum was confirmed up to 58 days postdosing. The favorable safety profile, ELF penetration, and maintained functional activity in serum supported the further clinical development of ASN100.