Primary sarcopenia is associated with elevated spontaneous NET formation.

Introduction: Sarcopenia is a frequent complication of liver cirrhosis, but it can also occur independently as a result of any underlying cause. The immune system plays an important role in the pathogenesis of both liver cirrhosis and sarcopenia. Neutrophil function, including neutrophil extracellular trap (NET) formation, is linked to chronic inflammation; however, it has not been extensively studied in patients with sarcopenia. Here, we aim to study if main neutrophil functions, such as phagocytosis, reactive oxygen species (ROS) production, and NET formation, are altered in patients with sarcopenia with or without liver cirrhosis. Methods: Neutrophils from 92 patients (52 patients with liver cirrhosis and sarcopenia, 25 patients with liver cirrhosis without sarcopenia, and 15 patients with sarcopenia without liver cirrhosis) and 10 healthy controls were isolated and stimulated with heat-inactivated E. coli (250 bacteria/cell), phorbol 12-myristate 13-acetate (PMA) (100 nM), or incubation medium in duplicates for 2 h at 37°C. Cells were fixed with paraformaldehyde and stained with 4',6-diamidino-2-phenylindole (DAPI). Pictures of 10 random fields of vision per slide were taken with an Olympus BX51 fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) at 600x total magnification. The DNA Area and NETosis Analysis (DANA) algorithm was used to quantify the percentage of NET formation per patient. Phagocytosis and ROS production were assessed with the PhagotestTM kit and PhagoburstTM kit (Glycotope, Heidelberg, Germany) in 92 patients and 21 healthy controls, respectively. Results: Spontaneous NET formation was significantly elevated in patients with only sarcopenia compared to patients with cirrhosis and sarcopenia (p = 0.008) and healthy controls (p = 0.039). NET formation in response to PMA was significantly decreased in patients with cirrhosis (p = 0.007), cirrhosis and sarcopenia (p < 0.001), and sarcopenia (p = 0.002) compared to healthy controls. There was no significant difference in NET formation in response to E. coli between the groups. The DANA algorithm was successfully optimized and validated for assessment of clinical samples. There were no significant changes in neutrophil phagocytosis between patients' groups compared to healthy controls. A significantly lower percentage of neutrophils produced ROS in response to N-formylmethionine-leucyl-phenylalanine (fMLF) in patients compared to healthy controls. Discussion: Spontaneous NET formation might contribute to chronic inflammation and sarcopenia pathogenesis. This, however, does not result in the impairment of the NET formation function of neutrophils in response to a bacterial stimulus and, therefore, cannot be not linked with the increased risk of bacterial infections neither in sarcopenia nor in cirrhosis. The semi-automated NET formation analysis can be successfully implemented to analyze the vast amount of data generated within clinical studies. This approach opens up the possibilities to develop an NET formation-based biomarker in different diseases including sarcopenia and implement NET formation analysis into clinical settings. Phagocytosis and ROS production were not affected in patients with sarcopenia. Further research is needed to explore the mechanism of NET formation in patients with sarcopenia and its potential as a biomarker in sarcopenia.

Effect of albumin infusion on oxidative albumin modification and albumin binding capacity in chronic liver failure.

Oxidative albumin modification and impaired albumin binding function have been described both in chronic liver failure and for therapeutic albumin solutions. The aim of the present study was to evaluate the effect of albumin infusion on redox state and binding function of circulating albumin. We studied 20 patients with cirrhosis who routinely received albumin infusions for prevention of post-paracentesis circulatory dysfunction or treatment of hepatorenal syndrome. We measured albumin fractions by redox state of cysteine-34 and albumin binding properties using dansylsarcosine as site II ligand. Therapeutic albumin solutions showed high contents of human nonmercaptalbumin-1 and human nonmercaptalbumin-2, exceeding the respective values in our patients with decompensated cirrhosis. An initial protocol for the first nine patients sampled at baseline, 24 h and 48 h after albumin infusion revealed no significant changes of oxidized albumin species or albumin binding properties. However, a modified protocol for the remaining 11 patients sampled at baseline, <1 h after and 24 h after albumin infusion revealed short-lived changes of oxidized albumin species while no changes in albumin binding properties were observed. In conclusion, therapeutic albumin infusion transiently changed albumin redox state but did not improve binding function of circulating albumin in chronic liver failure.

Altered gut microbiome, bile acid composition and metabolome in sarcopenia in liver cirrhosis.

BACKGROUND: Sarcopenia in liver cirrhosis is associated with low quality of life and high mortality risk. The pathogenesis has yet to be fully understood. We hypothesized that gut microbiome, bile acid (BA) composition and metabolites differ between cirrhotic patients with and without sarcopenia and contribute to pathogenesis. METHODS: Cirrhotic patients with (n = 78) and without (n = 38) sarcopenia and non-cirrhotic controls with (n = 39) and without (n = 20) sarcopenia were included in this study. Faecal microbiome composition was studied by 16S rDNA sequencing, serum and faecal BA composition by ultra-high-performance liquid chromatography-tandem mass spectrometry, and metabolite composition in serum, faeces and urine by nuclear magnetic resonance. RESULTS: Bacteroides fragilis, Blautia marseille, Sutterella spp. and Veillonella parvula were associated with cirrhotic patients with sarcopenia, whereas Bacteroides ovatus was more abundant in cirrhotic patients without sarcopenia. We observed significantly elevated secondary BAs, deoxycholic acid (DCA; P = 0.01) and lithocholic acid (LCA; P = 0.02), and the ratios of deoxycholic acid to cholic acid (DCA:CA; P = 0.04), lithocholic acid to chenodeoxycholic acid (LCA:CDCA; P = 0.03) and 12 alpha-hydroxylated to non-12 alpha-hydroxylated BAs (12-α-OH:non-12-α-OH BAs; P = 0.04) in serum of cirrhotic patients with sarcopenia compared with cirrhotic patients without sarcopenia, indicating an enhanced transformation of primary to secondary BAs by the gut microbiome. CA (P = 0.02) and the ratios of CA:CDCA (P = 0.03) and total ursodeoxycholic acid to total secondary BAs (T-UDCA:total-sec-BAs, P = 0.03) were significantly reduced in the stool of cirrhotic patients with sarcopenia compared with cirrhotic patients without sarcopenia. Also, valine and acetate were significantly reduced in the serum of cirrhotic patients with sarcopenia compared with cirrhotic patients without sarcopenia (P = 0.01 and P = 0.03, respectively). Multivariate logistic regression further confirmed the association of B. ovatus (P = 0.01, odds ratio [OR]: 12.8, 95% confidence interval [CI]: 168.1; 2.2), the ratios of 12-α-OH:non-12-α-OH BAs (P = 0.03, OR: 2.54, 95% CI: 0.99; 6.55) and T-UDCA:total-sec-BAs (P = 0.04, OR: 0.25, 95% CI: 0.06; 0.98) in serum and stool CA:CDCA (P = 0.04, OR: 0.79, 95% CI: 0.62; 0.99), and serum valine (P = 0.04, OR: 1.00, 95% CI: 1.02; 1.00) with sarcopenia in cirrhosis after correcting for the severity of liver disease and sex. CONCLUSIONS: Our study suggests a potential functional gut microbiome-host interaction linking sarcopenia with the altered gut microbiomes, BA profiles and amino acids pointing towards a potential mechanistic interplay in understanding sarcopenia pathogenesis.

Custodiol® Supplemented with Synthetic Human Relaxin Decreases Ischemia-Reperfusion Injury after Porcine Kidney Transplantation.

Objective. Ischemia-reperfusion injury (IRI) is inevitable after kidney transplantation (KT), impairing outcomes. Relaxin-2 (RLX) is a promising insulin-related peptide hormone that protects against renal IRI in rodents, although large animal models are needed before RLX can be tested in a human setting. Methods. In this blinded, randomized, and placebo-controlled experimental study kidneys from 19 donor pigs were retrieved after perfusion with Custodiol® ± RLX (5 or 20 nmol/L) and underwent static cold storage (SCS) for 24 and 48 h, respectively. Subsequently, KT was performed after unilateral right nephrectomy. Study outcomes included markers for kidney function, oxidative stress, lipid peroxidation, and endothelial cell damage. PCR analysis for oxidative stress and apoptosis-related gene panels as well as immunohistochemistry were performed. Results. RLX upregulated SOD2 and NFKB expression to 135% (p = 0.042) and 125% (p = 0.019), respectively, while RIPK1 expression was downregulated to 82% (p = 0.016) of corresponding controls. Further RLX significantly downregulated RIPK1 and MLKL expression and decreased the number of Caspase 3- and MPO-positive cells in grafts after SCS. Conclusions. RLX supplemented Custodiol® significantly decreased IRI via both antioxidant and anti-apoptotic mechanisms. Clinical trials are warranted to implement synthetic human RLX as a novel additive to preservation solutions against IRI.

Fibroscan® probe selection for lean adults.

BACKGROUND AND AIM: Fibroscan® is used to assess fibrosis and steatosis of the liver noninvasively. The company suggests to use the S+-probe in people <18 years with a thoracic circumference (TC) between 45 and 75 cm and the M+-probe in children with a TC >75 cm and adults with a skin-liver capsule distance <2.5 cm. For lean adults with a TC ≤75 cm, no comparative studies have been performed. Furthermore, it is unclear whether lean adults need to be fasted before assessment. METHODS: We compared liver stiffness (LS) using Fibroscan® S+- and M+-probes and controlled attenuation parameter (CAP; only available for M+-probe) in healthy volunteers with a TC ≤75 cm compared with those with a TC >75 cm in fasting state and after intake of a standardized light meal (300 kcal). RESULTS: We examined 50 volunteers (26 female, 24 ± 3 years). Twenty-two participants were in the TC ≤75 cm group and 28 in TC >75 cm group. LS values with the S+-probe were 15% higher than with the M+-probe in both groups (median difference 0.6 kPa, P < 0.001). Both probes showed good agreement with minimal bias (Spearman correlation r = 0.754, P < 0.001; Interclass Correlation Coefficient 0.843, P < 0.001; Bland-Altman bias 0.6 ± 0.9 kPa, linear regression r 2 = 0.557, P < 0.001). Intake of a light meal had no relevant influence on LS (S+- and M+-probes) or CAP measurements (M+-probe) in both groups. CONCLUSION: Lean adults with a TC below 75 cm can be assessed with either the S+-probe or the M+-probe and may take a light meal before assessment.

Direct acting antiviral therapy rescues neutrophil dysfunction and reduces hemolysis in hepatitis C infection.

Chronic hepatitis C virus infection is characterized by multiple extra-hepatic manifestations. Innate immune dysfunction and hemolysis are symptoms which might be associated with each other. We investigated the impact of direct acting antivirals on neutrophil function and its connection to hemolysis. In this prospective study, 85 patients with or without cirrhosis and 21 healthy controls were included. Patients' blood samples were taken at baseline, at the end of therapy and at follow-up 12 weeks after end of therapy. Neutrophil phagocytosis, oxidative burst, and hemolysis parameters were studied. Multivariate analysis was performed to decipher the relationship between hemolysis and neutrophil function. Ex vivo cross-incubation experiments with neutrophils and serum fractions were done. Impaired neutrophil phagocytosis and mild hemolysis were observed in patients with and without cirrhosis. A proteome approach revealed different expression of hemolysis-related serum proteins in patients and controls. Direct acting antiviral therapy restored neutrophil function irrespective of severity of liver disease, achievement of sustained virologic response or type of drug and reduced hemolysis. Treatment with ribavirin delayed the improvement of neutrophil function. Statistical analysis revealed associations of haptoglobin with neutrophil phagocytic capacity. Neutrophil dysfunction could be transferred to healthy cells by incubation with patients' serum fractions (>30 kDa) ex vivo. Neutrophil dysfunction and hemolysis represent extrahepatic manifestations of chronic hepatitis C virus infection and simultaneously improve during direct acting antiviral therapy independently of therapy-related liver function recovery. Therefore, large-scale treatment would not only drive viral eradication but also improve patients' immune system and may reduce susceptibility to infections.

Secondary Sclerosing Cholangitis in Critically Ill Patients Alters the Gut-Liver Axis: A Case Control Study.

Secondary sclerosing cholangitis in critically ill patients (SC-CIP) occurs after long-term intensive care treatment. This study aimed to assess the gut-liver axis in SC-CIP. Stool microbiome composition, gut permeability, bacterial translocation and serum bile acid profiles of 18 SC-CIP patients compared to 11 patients after critical illness without liver disease (CIP controls), 21 patients with cirrhosis and 21 healthy controls were studied. 16S rDNA was isolated from stool and sequenced using the Illumina technique. Diamine oxidase, zonulin, soluble CD14 (sCD14) and lipopolysaccharide binding protein were measured in serum and calprotectin in stool. Serum bile acids were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Reduced microbiome alpha diversity and altered beta diversity were seen in SC-CIP, CIP controls and cirrhosis compared to healthy controls. SC-CIP patients showed a shift towards pathogenic taxa and an oralization. SC-CIP, CIP controls and cirrhotic patients presented with impaired gut permeability, and biomarkers of bacterial translocation were increased in SC-CIP and cirrhosis. Total serum bile acids were elevated in SC-CIP and cirrhosis and the bile acid profile was altered in SC-CIP, CIP controls and cirrhosis. In conclusions, observed alterations of the gut-liver axis in SC-CIP cannot solely be attributed to liver disease, but may also be secondary to long-term intensive care treatment.

Dietary Glycine Prevents FOLFOX Chemotherapy-Induced Heart Injury: A Colorectal Cancer Liver Metastasis Treatment Model in Rats.

INTRODUCTION: FOLFOX chemotherapy (CTx) is used for the treatment of colorectal liver metastasis (CRLM). Side effects include rare cardiotoxicity, which may limit the application of FOLFOX. Currently, there is no effective strategy to prevent FOLFOX-induced cardiotoxicity. Glycine has been shown to protect livers from CTx-induced injury and oxidative stress, and it reduces platelet aggregation and improves microperfusion. This study tested the hypothesis of glycine being cardioprotective in a rat model of FOLFOX in combination with CRLM. MATERIALS AND METHODS: The effect of glycine was tested in vitro on human cardiac myocytes (HCMs). To test glycine in vivo Wag/Rij rats with induced CRLM were treated with FOLFOX ±5% dietary glycine. Left ventricle ejection fraction (LVEF), myocardial fibrosis, and apoptosis, also heart fatty acid binding protein (h-FABP) and brain natriuretic peptide levels were monitored. PCR analysis for Collagen type I, II, and brain natriuretic peptide (BNP) in the heart muscle was performed. RESULTS: In vitro glycine had no effect on HCM cell viability. Treatment with FOLFOX resulted in a significant increase of h-FABP levels, increased myocardial fibrosis, and apoptosis as well as increased expression of type I Collagen. Furthermore, FOLFOX caused a decrease of LVEF by 10% (p = 0.028). Dietary glycine prevented FOLFOX-induced myocardial injury by preserving the LVEF and reducing the levels of fibrosis (p = 0.012) and apoptosis (p = 0.015) in vivo. CONCLUSIONS: Data presented here demonstrate for the first time that dietary glycine protects the heart against FOLFOX-induced injury during treatment for CRLM.

Dysbiosis, gut barrier dysfunction and inflammation in dementia: a pilot study.

BACKGROUND: Dementia is an increasing public health threat worldwide. The pathogenesis of dementia has not been fully elucidated yet. Inflammatory processes are hypothesized to play an important role as a driver for cognitive decline but the origin of inflammation is not clear. We hypothesize that disturbances in gut microbiome composition, gut barrier dysfunction, bacterial translocation and resulting inflammation are associated with cognitive dysfunction in dementia. METHODS: To test this hypothesis, a cohort of 23 patients with dementia and 18 age and sex matched controls without cognitive impairments were studied. Gut microbiome composition, gut barrier dysfunction, bacterial translocation and inflammation were assessed from stool and serum samples. Malnutrition was assessed by Mini Nutritional Assessment Short Form (MNA-SF), detailed information on drug use was collected. Microbiome composition was assessed by 16S rRNA sequencing, QIIME 2 and Calypso 7.14 tools. RESULTS: Dementia was associated with dysbiosis characterized by differences in beta diversity and changes in taxonomic composition. Gut permeability was increased as evidenced by increased serum diamine oxidase (DAO) levels and systemic inflammation was confirmed by increased soluble cluster of differentiation 14 levels (sCD14). BMI and statin use had the strongest impact on microbiome composition. CONCLUSION: Dementia is associated with changes in gut microbiome composition and increased biomarkers of gut permeability and inflammation. Lachnospiraceae NK4A136 group as potential butyrate producer was reduced in dementia. Malnutrition and drug intake were factors, that impact on microbiome composition. Increasing butyrate producing bacteria and targeting malnutrition may be promising therapeutic targets in dementia. TRIAL REGISTRATION: NCT03167983 .

The effects of a multispecies synbiotic on microbiome-related side effects of long-term proton pump inhibitor use: A pilot study.

Side effects of proton pump inhibitors (PPI) can be linked to the changes in the intestinal microbiome that occur during therapy, especially in long-term users. Therefore, the microbiome might also be a key player in the reduction of PPI side effects. We tested the effects of a three-month intervention with a multispecies synbiotic on intestinal inflammation, gut barrier function, microbiome composition, routine laboratory parameters and quality of life in patients with long-term PPI therapy. Thirty-six patients received a daily dose of a multispecies synbiotic for three months and were clinically observed without intervention for another three months. After intervention 17% of patients reached normal calprotectin levels; the overall reduction did not reach statistical significance (-18.8 ng/mg; 95%CI: -50.5; 12.9, p = 0.2). Elevated zonulin levels could be significantly reduced (-46.3 ng/mg; 95%CI: -71.4; -21.2; p < 0.001). The abundance of Stomatobaculum in the microbiome was reduced and Bacillus increased during the intervention. Furthermore, albumin, alkaline phosphatase and thrombocyte count were significantly increased and aspartate transaminase was significantly decreased during intervention. Gastrointestinal quality of life showed significant improvements. In conclusion, microbiome-related side effects of long-term PPI use can be substantially reduced by synbiotic intervention. Further studies are warranted to optimize dosage and duration of the intervention.

Effects of a multispecies synbiotic on glucose metabolism, lipid marker, gut microbiome composition, gut permeability, and quality of life in diabesity: a randomized, double-blind, placebo-controlled pilot study.

PURPOSE: Diabesity, the combination of obesity and type 2 diabetes, is an ever-growing global health burden. Diabesity-associated dysbiosis of the intestinal microbiome has gained attention as a potential driver of disease and, therefore, a possible therapeutic target by means of pro- or prebiotic supplementation. This study tested the effects of a multispecies synbiotic (i.e. a combination of probiotics and prebiotics) on glucose metabolism, gut microbiota, gut permeability, neutrophil function and quality of life in treatment-experienced diabesity patients. METHODS: A randomized, double-blind, placebo-controlled pilot study with 26 diabesity patients was conducted in which patients received a daily dose of a multispecies probiotic and a prebiotic (or a placebo) for 6 months. RESULTS: There were no changes in glucose metabolism or mixed meal tolerance test responses throughout the study. The analysis of secondary outcomes revealed beneficial effects on hip circumference [- 1 (95% CI - 4; 3) vs +3 (- 1; 8) cm, synbiotics vs. placebo, respectively, p = 0.04], serum zonulin [- 0.04 (- 0.2; 0.1) vs +0.3 (- 0.05; 0.6) ng/ml, p = 0.004)] and the physical role item of the SF36 quality of life assessment [+ 5.4 (- 1.7; 12.5) vs - 5.0 (- 10.1; 0.2) points, p = 0.02] after 3 months of intervention, and lipoprotein (a) [- 2.1 (- 5.7; 1.6) vs +3.4 (- 0.9; 7.9) mg/dl, p = 0.02] after 6 months. There were no significant differences in alpha or beta diversity of the microbiome between groups or time points. CONCLUSIONS: Glucose metabolism as the primary outcome was unchanged during the intervention with a multispecies synbiotic in patients with diabesity. Nevertheless, synbiotics improved some symptoms and biomarkers of type 2 diabetes and aspects of quality of life suggesting a potential role as adjuvant tool in the management of diabesity.

Dietary glycine decreases both tumor volume and vascularization in a combined colorectal liver metastasis and chemotherapy model.

Rationale: Chemotherapy (CTx) with FOLFOX is indicated prior to resection of liver metastases; however, its effect is limited due to chemoresistance and its toxicity prevents from aggressive surgery needed in some cases. Hepatoprotective glycine has been shown to have anti-tumorigenic properties in various cancers. Thus, this study was designed to evaluate the effects of glycine combined with FOLFOX on colorectal liver metastases (CRLM). Methods: The effect of glycine combined with 5-fluorouracil and oxaliplatin was investigated in vitro on colorectal cancer (CC531). Further, Wag/Rij rats with CRLM were treated with 5% dietary glycine ± FOLFOX. µCT liver scan, anti-Ki67, and anti-CD31 were compared. Results: Glycine alone and combined with CTx has no effect on both CC531 viability in vitro and tumor proliferation in vivo; however, glycine significantly decreased tumor volume to about 42-35% of controls in vivo (p<0.05) with a 60% decreased tumor microvascular density (MVD) (p=0.004). Further glycine doesn't counteract anti-tumor properties of CTx. Conclusions: This study nicely demonstrates that glycine inhibits the growth of CRLM and does not decrease CTx effectiveness. Underlying mechanisms most likely include a decreased tumor MVD. Clinical trials are warranted to implement non-toxic hepatoprotective glycine in novel anti-cancer strategies in humans.

A single alcohol binge impacts on neutrophil function without changes in gut barrier function and gut microbiome composition in healthy volunteers.

Alcohol binge drinking is a dangerous drinking habit, associated with neurological problems and inflammation. The impact of a single alcohol binge on innate immunity, gut barrier and gut microbiome was studied. In this cohort study 15 healthy volunteers received 2 ml vodka 40% v/v ethanol/kg body weight. Neutrophil function was studied by flow cytometry; markers of gut permeability and inflammation (lactulose/mannitol/sucrose test, zonulin, calprotectin, diamino-oxidase) were studied with NMR spectroscopy and enzyme-linked immunosorbent assay in urine, stool and serum respectively. Bacterial products in serum were quantified using different reporter cell lines. Gut microbiome composition was studied by 16S rDNA sequencing and bioinformatics analysis. After a single alcohol binge, neutrophils were transiently primed and the response to E.coli stimulation with reactive oxygen species (ROS) production was transiently increased, on the other hand the percentage of neutrophils that did not perform phagocytosis increased. No changes in gut permeability, inflammatory biomarker, bacterial translocation and microbiome composition could be detected up to 4 hours after a single alcohol binge or on the next day. A single alcohol binge in young, healthy volunteers transiently impacts on neutrophil function. Although the exact biological consequence of this finding is not clear yet, we believe that this strengthens the importance to avoid any alcohol binge drinking, even in young, otherwise healthy persons.