Publications by authors named "Srinivasan Dasarathy"

136 Publications

Ex Vivo Normothermic Preservation of Amputated Limbs with a Hemoglobin-Based Oxygen Carrier (HBOC-201) Perfusate.

J Trauma Acute Care Surg 2021 Sep 10. Epub 2021 Sep 10.

Department of Plastic Surgery, Cleveland Clinic, Cleveland, OH, USA Department of Nutrition, School of Medicine, Case Western Reserve University, Cleveland, OH Department of Gastroenterology Cleveland Clinic, Cleveland, OH, USA Hemoglobin Oxygen Therapeutics, LLC, Souderton, PA, USA.

Background: Ex vivo normothermic limb perfusion (EVNLP) preserves amputated limbs under near-physiologic conditions. Perfusates containing red blood cells (RBCs) have shown to improve outcomes during ex vivo normothermic organ perfusion, when compared to acellular perfusates. To avoid limitations associated with use of blood-based products, we evaluated the feasibility of EVNLP utilizing a polymerized Hemoglobin-Based Oxygen Carrier-201 (HBOC-201).

Methods: Twenty-four porcine forelimbs were procured from Yorkshire pigs. Six forelimbs underwent EVNLP with an HBOC-201-based perfusate, six with an RBC-based perfusate, and twelve served as static cold storage controls (SCS). EVNLP was terminated in presence of systolic arterial pressure ≥ 115 mmHg, fullness of compartments, or drop of tissue oxygen saturation by 20%. Limb contractility, weight change, compartment pressure, tissue oxygen saturation, oxygen uptake rates (OUR) were assessed. Perfusate fluid-dynamics, gases, electrolytes, metabolites, methemoglobin (MetHb), creatine kinase (CK) and myoglobin concentration were measured. Uniformity of skin perfusion was assessed with indocyanine green (ICG) angiography and infrared thermography (IRT).

Results: Warm ischemia time before EVNLP was 35.50 ± 8.62 min (HBOC-201), 30.17 ± 8.03 min (RBC) and 37.82 ± 10.45 (SCS) (p = 0.09). EVNLP duration was 22.5 ± 1.7 (HBOC-201) and 28.2 ± 7.3 (RBC) hours (p = 0.04). Vascular flow (325 ± 25 vs. 444.7 ± 50.6 ml/min; p = 0.39), OUR (2.0 ± 1.45 vs. 1.3 ± 0.92 mlO2/min*g of tissue; p = 0.80), lactate (14.66 ± 4.26 vs. 13.11 ± 6.68 mmol/L; p = 0.32), perfusate pH (7.53 ± 0.25 HBOC-201; 7.50 ± 0.23 RBC; p = 0.82), flexor (28.3 ± 22.0 vs. 27.5 ± 10.6; p = 0.99) and extensor (31.5 ± 22.9 vs. 28.8 ± 14.5; p = 0.82) compartment pressures, and weight changes (23.1 ± 3.0% vs. 13.2 ± 22.7; p = 0.07) were not significantly different between HBOC-201 and RBC groups, respectively. In HBOC-201 perfused limbs, MetHb levels increased, reaching 47.8 ± 12.1% at endpoint. Methemoglobin saturation did not affect OUR (ρ = -0.15, r2 = 0.022; p = 0.45). A significantly greater number of necrotic myocytes was found in the SCS group at endpoint (SCS: 127 ± 17 cells; HBOC-201: 72 ± 30 cells; RBC-based: 56 ± 40 cells; vs. p = 0.003).

Conclusion: HBOC-201- and RBC-based perfusates similarly support isolated limb physiology, metabolism, and function.

Level Of Evidence: N/A.
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http://dx.doi.org/10.1097/TA.0000000000003395DOI Listing
September 2021

Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes.

J Biol Chem 2021 Sep 31;297(3):101023. Epub 2021 Jul 31.

Department of Inflammation & Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA; Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, Ohio, USA. Electronic address:

Ammonia is a cytotoxic molecule generated during normal cellular functions. Dysregulated ammonia metabolism, which is evident in many chronic diseases such as liver cirrhosis, heart failure, and chronic obstructive pulmonary disease, initiates a hyperammonemic stress response in tissues including skeletal muscle and in myotubes. Perturbations in levels of specific regulatory molecules have been reported, but the global responses to hyperammonemia are unclear. In this study, we used a multiomics approach to vertically integrate unbiased data generated using an assay for transposase-accessible chromatin with high-throughput sequencing, RNA-Seq, and proteomics. We then horizontally integrated these data across different models of hyperammonemia, including myotubes and mouse and human muscle tissues. Changes in chromatin accessibility and/or expression of genes resulted in distinct clusters of temporal molecular changes including transient, persistent, and delayed responses during hyperammonemia in myotubes. Known responses to hyperammonemia, including mitochondrial and oxidative dysfunction, protein homeostasis disruption, and oxidative stress pathway activation, were enriched in our datasets. During hyperammonemia, pathways that impact skeletal muscle structure and function that were consistently enriched were those that contribute to mitochondrial dysfunction, oxidative stress, and senescence. We made several novel observations, including an enrichment in antiapoptotic B-cell leukemia/lymphoma 2 family protein expression, increased calcium flux, and increased protein glycosylation in myotubes and muscle tissue upon hyperammonemia. Critical molecules in these pathways were validated experimentally. Human skeletal muscle from patients with cirrhosis displayed similar responses, establishing translational relevance. These data demonstrate complex molecular interactions during adaptive and maladaptive responses during the cellular stress response to hyperammonemia.
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http://dx.doi.org/10.1016/j.jbc.2021.101023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8424232PMC
September 2021

Alcohol Consumption Is Associated with Poor Prognosis in Obese Patients with COVID-19: A Mendelian Randomization Study Using UK Biobank.

Nutrients 2021 May 10;13(5). Epub 2021 May 10.

Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH 44195, USA.

Background: Acute and chronic alcohol abuse has adverse impacts on both the innate and adaptive immune response, which may result in reduced resistance to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and promote the progression of coronavirus disease 2019 (COVID-19). However, there are no large population-based data evaluating potential causal associations between alcohol consumption and COVID-19.

Methods: We conducted a Mendelian randomization study using data from UK Biobank to explore the association between alcohol consumption and risk of SARS-CoV-2 infection and serious clinical outcomes in patients with COVID-19. A total of 12,937 participants aged 50-83 who tested for SARS-CoV-2 between 16 March to 27 July 2020 (12.1% tested positive) were included in the analysis. The exposure factor was alcohol consumption. Main outcomes were SARS-CoV-2 positivity and death in COVID-19 patients. We generated allele scores using three genetic variants (rs1229984 (Alcohol Dehydrogenase 1B, ), rs1260326 (Glucokinase Regulator, ), and rs13107325 (Solute Carrier Family 39 Member 8, )) and applied the allele scores as the instrumental variables to assess the effect of alcohol consumption on outcomes. Analyses were conducted separately for white participants with and without obesity.

Results: Of the 12,937 participants, 4496 were never or infrequent drinkers and 8441 were frequent drinkers. Both logistic regression and Mendelian randomization analyses found no evidence that alcohol consumption was associated with risk of SARS-CoV-2 infection in participants either with or without obesity (All > 0.10). However, frequent drinking, especially heavy drinking (HR = 2.07, 95%CI 1.24-3.47; = 0.054), was associated with higher risk of death in patients with obesity and COVID-19, but not in patients without obesity. Notably, the risk of death in frequent drinkers with obesity increased slightly with the average amount of alcohol consumed weekly (All < 0.10).

Conclusions: Our findings suggest that alcohol consumption has adverse effects on the progression of COVID-19 in white participants with obesity, but was not associated with susceptibility to SARS-CoV-2 infection.
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http://dx.doi.org/10.3390/nu13051592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8152000PMC
May 2021

Sarcopenia and frailty in decompensated cirrhosis.

J Hepatol 2021 07;75 Suppl 1:S147-S162

Department of Clinical Medicine, Gastroenterology, Sapienza University of Rome, Italy. Electronic address:

In patients with decompensated cirrhosis, sarcopenia and frailty are prevalent. Although several definitions exist for these terms, in the field of hepatology, sarcopenia has commonly been defined as loss of muscle mass, and frailty has been broadly defined as the phenotypic manifestation of the loss of muscle function. Prompt recognition and accurate assessment of these conditions are critical as they are both strongly associated with morbidity, mortality, poor quality of life and worse post-liver transplant outcomes in patients with cirrhosis. In this review, we describe the complex pathophysiology that underlies the clinical phenotypes of sarcopenia and frailty, their association with decompensation, and provide an overview of tools to assess these conditions in patients with cirrhosis. When available, we highlight data focusing on patients with acutely decompensated cirrhosis, such as inpatients, as this is an area of unmet clinical need. Finally, we discuss management strategies to reverse and/or prevent the development of sarcopenia and frailty, which include adequate nutritional intake of calories and protein, as well as regular exercise of at least moderate intensity, with a mix of aerobic and resistance training. Key knowledge gaps in our understanding of sarcopenia and frailty in decompensated cirrhosis remain, including best methods to measure muscle mass and function in the inpatient setting, racial/ethnic variation in the development and presentation of sarcopenia and frailty, and optimal clinical metrics to assess response to therapeutic interventions that translate into a reduction in adverse outcomes associated with these conditions.
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http://dx.doi.org/10.1016/j.jhep.2021.01.025DOI Listing
July 2021

Hepatocellular Carcinoma in Patients Without Cirrhosis: The Fibrosis Stage Distribution, Characteristics and Survival.

Dig Dis Sci 2021 May 23. Epub 2021 May 23.

Department of Gastroenterology, Hepatology and Nutrition, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195, USA.

Background: The data on hepatocellular carcinoma (HCC) patients without liver cirrhosis is scarce.

Aims: To study the epidemiology, underlying etiology and fibrosis distribution in noncirrhotic HCC and compare the survival outcomes to cirrhotic HCC.

Methods: We conducted a retrospective study including all adult patients diagnosed with HCC at two US tertiary academic centers from 2000 to 2015. Univariable and multivariable Cox regression analyses were performed to evaluate the variables associated with patient survival.

Results: Two thousand two hundred and thirty-seven HCC patients were included in the final analysis, of which, 13% had no liver cirrhosis. The most common underlying liver disease in non-cirrhotic patients was cryptogenic cause (40%), followed by nonalcoholic fatty liver disease (NAFLD) (25.2%) and hepatitis C (19%). The percentage of F0-F1, F2, and F3 was 72%, 17%, and 11% (cryptogenic cause); 69%, 12%, and 19% (NAFLD); 50%, 17%, and 33% (alcohol); 33%, 39%, and 28% (hepatitis B); 20%, 40%, and 40% (hemochromatosis); and 12%, 40%, and 48% (hepatitis C), respectively. In non-cirrhotic compared to cirrhotic patients, the tumor was more likely to be larger and fell outside Milan criteria (all p < 0.001). Cirrhotic patients had significant shorter survival than non-cirrhotic patients (p < 0.001). On the multivariable analysis, having liver cirrhosis (HR 1.48; 1.21-1.82, p < 0.001), combined viral hepatitis and alcohol use (HR 1.51; 1.23-1.88, p < 0.001), morbid obesity (HR 1.31; 1.01-1.69, p = 0.040) and underweight (HR 2.06; 1.27-3.34, p = 0.004) were associated with worse patient survival.

Conclusions: The fibrosis distribution in non-cirrhotic HCC differed among each etiology of liver diseases. Despite more advanced HCC, patients without cirrhosis had significantly longer survival than those with cirrhosis.
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http://dx.doi.org/10.1007/s10620-021-07048-5DOI Listing
May 2021

Acute Responses to Oxygen Delivery via High Flow Nasal Cannula in Patients with Severe Chronic Obstructive Pulmonary Disease-HFNC and Severe COPD.

J Clin Med 2021 Apr 21;10(9). Epub 2021 Apr 21.

Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, NE4 208, Cleveland, OH 44195, USA.

Differences in oxygen delivery methods to treat hypoxemia have the potential to worsen CO retention in chronic obstructive lung disease (COPD). Oxygen administration using high flow nasal cannula (HFNC) has multiple physiological benefits in treating respiratory failure including reductions in PaCO in a flow-dependent manner. We hypothesized that patients with COPD would develop worsening hypercapnia if oxygen fraction was increased without increasing flow rate. We evaluated the acute response to HFNC in subjects with severe COPD when flow remained constant and inspired oxygen was increased. In total, 11 subjects with severe COPD (FEV1 < 50%) on supplemental oxygen with baseline normocapnia (PaCO < 45 mm Hg; = 5) and hypercapnia (PaCO ≥ 45 mm Hg; = 6) were studied. Arterial blood gas responses were studied at three timepoints: Baseline, HFNC at a flow rate of 30 L/min at resting oxygen supplementation for 1 h, and FiO 30% above baseline with the same flow rate for the next hour. The primary endpoint was the change in PaCO from baseline. No significant changes in PaCO were noted in response to HFNC applied at baseline FiO in the normocapnic and hypercapnic group. At HFNC with FiO 30% above baseline, the normocapnic group did not show a change in PaCO (baseline: 38.9 ± 1.8 mm Hg; HFNC at higher FiO: 38.8 ± 3.1 mm Hg; = 0.93), but the hypercapnic group demonstrated significant increase in PaCO (baseline: 58.2 ± 9.3 mm Hg; HFNC at higher FiO: 63.3 ± 10.9 mm Hg; = 0.025). We observed worsening hypercapnia in severe COPD patients and baseline hypercapnia who received increased oxygen fraction when flow remained constant. These data show the need for careful titration of oxygen therapy in COPD patients, particularly those with baseline hypercapnia when flow rate is unchanged.
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http://dx.doi.org/10.3390/jcm10091814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122595PMC
April 2021

Intestinal function is impaired in patients with Chronic Obstructive Pulmonary Disease.

Clin Nutr 2021 04 10;40(4):2270-2277. Epub 2020 Oct 10.

Center for Translational Research in Aging & Longevity, Department of Health & Kinesiology, Texas A&M University, College Station, TX, USA. Electronic address:

Background & Aims: Gastrointestinal symptoms are prevalent extrapulmonary systemic manifestations of Chronic Obstructive Pulmonary Disease (COPD), but have been rarely studied. We dissected the perturbations in intestinal function in human patients with COPD using comprehensive metabolic and physiological approaches.

Methods: In this observational study, small intestinal membrane integrity and active carrier-mediated glucose transport were quantified by sugar permeability test in 21 clinically stable patients with moderate to severe COPD (mean FEV, 41.2 (3.2) % of predicted) and 16 healthy control subjects. Protein digestion and absorption was analyzed using stable tracer kinetic methods. Plasma acetate, propionate, and butyrate concentrations were measured as markers of intestinal microbial metabolism.

Results: Compared with healthy controls, non carrier-mediated permeability was higher (0.062 (95% CI [0.046, 0.078]) vs. 0.037 (95% CI [0.029, 0.045]), P = 0.009) and active glucose transport lower in COPD (31.4 (95% CI [23.4, 39.4])% vs. 48.0 (95% CI [37.8, 58.3])%, P = 0.010). Protein digestion and absorption was lower in COPD (0.647 (95% CI [0.588, 0.705]) vs. 0.823 (95% CI [0.737, 0.909]), P = 0007), and impairment greater in patients with dyspnea (P = 0.038), exacerbations in preceding year (P = 0.052), and reduced transcutaneous oxygen saturation (P = 0.051), and was associated with reduced physical activity score (P = 0.016) and lower quality of life (P = 0.0007). Plasma acetate concentration was reduced in COPD (41.54 (95% CI [35.17, 47.91]) vs. 80.44 (95% CI [54.59, 106.30]) μmol/L, P = 0.001) suggesting perturbed intestinal microbial metabolism.

Conclusions: We conclude that intestinal dysfunction is present in COPD, worsens with increasing disease severity, and is associated with reduced quality of life.
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http://dx.doi.org/10.1016/j.clnu.2020.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058450PMC
April 2021

Quantitative Computed Tomography Assessment of Pectoralis and Erector Spinae Muscle Area and Disease Severity in Chronic Obstructive Pulmonary Disease Referred for Lung Volume Reduction.

COPD 2021 04 19;18(2):191-200. Epub 2021 Mar 19.

Departments of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, USA.

Patients with advanced chronic obstructive pulmonary disease (COPD) develop skeletal muscle loss (sarcopenia) that is associated with adverse clinical outcomes including mortality. We evaluated if thoracic muscle area is associated with clinical outcomes in patients with severe COPD. We analyzed consecutive patients with severe COPD undergoing evaluation for lung volume reduction from 2015 to 2019 ( = 117) compared to current and former smoking controls undergoing lung cancer screening with normal lung function ( = 41). Quantitative assessments of pectoralis muscle (PM) and erector spinae muscle (ESM) cross sectional area (CSA) were related to clinical outcomes including composite endpoints. Our results showed a reduction in PM CSA but not ESM CSA was associated with the severity of GOLD stage of COPD. Current smokers demonstrated reduced PM CSA which was similar to that in COPD patients who were GOLD stages 3 and 4. PM CSA was associated positively with FEV1, FEV% predicted, FVC, DLCO, and FEV/FVC ratio, and was associated negatively with the degree of radiologic emphysema. ESM correlated positively with DLCO, RV/TLC (a marker of hyperinflation), and correlated negatively with radiologic severity of emphysema. Kaplan-Meier analysis showed that reductions in PM but not ESM CSA was associated with the composite end point of mortality, need for lung volume reduction, or lung transplant. In conclusion, in well-characterized patients with severe COPD referred for lung volume reduction, PM CSA correlated with severity of lung disease, mortality, and need for advanced therapies. In addition to predicting clinical outcomes, targeting sarcopenia is a potential therapeutic approach in patients with severe COPD.
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http://dx.doi.org/10.1080/15412555.2021.1897560DOI Listing
April 2021

Clinical impact of compound sarcopenia in hospitalized older adult patients with heart failure.

J Am Geriatr Soc 2021 07 18;69(7):1815-1825. Epub 2021 Mar 18.

Inflammation and Immunity, Cleveland Clinic, Cleveland, Ohio, USA.

Objectives: Skeletal muscle loss or sarcopenia is a frequent complication in heart failure (HF) and contributes to adverse clinical outcomes. We evaluated if age (primary) and chronic disease (secondary) related sarcopenia, that we refer to as compound sarcopenia, impacts clinical outcomes in hospitalized patients with HF.

Design: Cross-sectional study using hospitalized patient data.

Setting: Data from the Agency for Healthcare Research and Quality through the Healthcare Cost and Utilization Project (HCUP).

Participants: Hospitalized adult patients with a primary or secondary diagnosis of HF (n = 64,476) and a concurrent random 2% sample of general medical population (GMP; n = 322,217) stratified by age (<50 years of age [y], 51-65y, >65y) from the Nationwide Inpatient Sample (NIS) database (years 2010-2014).

Measurements: In-hospital mortality, length of stay (LoS), cost of hospitalization per admission (CoH), comorbidities and discharge disposition, with and without muscle loss phenotype, were analyzed. Muscle loss phenotype was defined using a comprehensive code set from international classification of diseases-9 (ICD-9).

Results: Muscle loss phenotype was observed in 8673 (13.5%) patients with HF compared to 5213 (1.6%) GMP across all age strata. In patients with HF, muscle loss phenotype was associated with higher mortality, LoS, and CoH. Patients with HF (>65y) and muscle loss phenotype had higher mortality (adjusted OR: 1.81; 95% CI 1.56-2.10), CoH (adjusted OR 1.48; 95% CI 1.44-1.1.52), and LoS (adjusted OR 1.40; 95% CI 1.37-1.43) compared to >65y GMP with muscle loss phenotype.

Conclusion: Muscle loss phenotype is more commonly associated with increasing age in hospitalized patients with HF. Clinical outcomes were significantly worse in patients with HF aged >65y compared to younger patients with HF and all age strata in GMP with and without a muscle loss phenotype.
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http://dx.doi.org/10.1111/jgs.17108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8273144PMC
July 2021

Compound Sarcopenia in Hospitalized Patients with Cirrhosis Worsens Outcomes with Increasing Age.

Nutrients 2021 Feb 18;13(2). Epub 2021 Feb 18.

Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH 44195, USA.

Background: There are limited data on outcomes of older patients with chronic diseases. Skeletal muscle loss of aging (primary sarcopenia) has been extensively studied but the impact of secondary sarcopenia of chronic disease is not as well evaluated. Older patients with chronic diseases have both primary and secondary sarcopenia that we term compound sarcopenia. We evaluated the clinical impact of compound sarcopenia in hospitalized patients with cirrhosis given the increasing number of patients and high prevalence of sarcopenia in these patients.

Design: The Nationwide Inpatients Sample (NIS) database (years 2010-2014) was analyzed to study older patients with cirrhosis. Since there is no universal hospital diagnosis code for "muscle loss", we used a comprehensive array of codes for "muscle loss phenotype" in the international classification of diseases-9 (ICD-9). A randomly selected 2% sample of hospitalized general medical population (GMP) and inpatients with cirrhosis were stratified into 3 age groups based on age-related changes in muscle mass. In-hospital mortality, length of stay (LoS), cost of hospitalization (CoH), comorbidities and discharge disposition were analyzed.

Results: Of 517,605 hospitalizations for GMP and 106,835 hospitalizations for treatment of cirrhosis or a cirrhosis-related complication, 207,266 (40.4%) GMP and 29,018 (27.7%) patients with cirrhosis were >65 years old, respectively. Muscle loss phenotype in both GMP and inpatients with cirrhosis 51-65 years old and >65 years old was significantly ( < 0.001 for all) associated with higher mortality, LoS, and CoH compared to those ≤50 years old. Patients >65 years old with cirrhosis and muscle loss phenotype had higher mortality (adjusted OR: 1.06, 95% CI [1.04, 1.08] and CoH (adjusted odds ratio (OR): 1.10, 95% confidence interval (CI) [1.04, 1.08])) when compared to >65 years old GMP with muscle loss phenotype. Muscle loss in younger patients with cirrhosis (≤50 years old) was associated with worse outcomes compared to GMP >65 years old. Non-home discharges (nursing, skilled, long-term care) were more frequent with increasing age to a greater extent in patients with cirrhosis with muscle loss phenotype for each age stratum.

Conclusion: Muscle loss is more frequent in older patients with cirrhosis than younger patients with cirrhosis and older GMP. Younger patients with cirrhosis had clinical outcomes similar to those of older GMP, suggesting an accelerated senescence in cirrhosis. Compound sarcopenia in older patients with cirrhosis is associated with higher inpatient mortality, increased LoS, and CoH compared to GMP with sarcopenia.
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http://dx.doi.org/10.3390/nu13020659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923160PMC
February 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Differential role of MLKL in alcohol-associated and non-alcohol-associated fatty liver diseases in mice and humans.

JCI Insight 2021 02 22;6(4). Epub 2021 Feb 22.

Northern Ohio Alcohol Center, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, Ohio, USA.

Hepatocellular death contributes to progression of alcohol-associated (ALD-associated) and non-alcohol-associated (NAFL/NASH) liver diseases. However, receptor-interaction protein kinase 3 (RIP3), an intermediate in necroptotic cell death, contributes to injury in murine models of ALD but not NAFL/NASH. We show here that a differential role for mixed-lineage kinase domain-like protein (MLKL), the downstream effector of RIP3, in murine models of ALD versus NAFL/NASH and that RIP1-RIP3-MLKL can be used as biomarkers to distinguish alcohol-associated hepatitis (AH) from NASH. Phospho-MLKL was higher in livers of patients with NASH compared with AH or healthy controls (HCs). MLKL expression, phosphorylation, oligomerization, and translocation to plasma membrane were induced in WT mice fed diets high in fat, fructose, and cholesterol but not in response to Gao-binge (acute on chronic) ethanol exposure. Mlkl-/- mice were not protected from ethanol-induced hepatocellular injury, which was associated with increased expression of chemokines and neutrophil recruitment. Circulating concentrations of RIP1 and RIP3, but not MLKL, distinguished patients with AH from HCs or patients with NASH. Taken together, these data indicate that MLKL is differentially activated in ALD/AH compared with NAFL/NASH in both murine models and patients. Furthermore, plasma RIP1 and RIP3 may be promising biomarkers for distinguishing AH and NASH.
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http://dx.doi.org/10.1172/jci.insight.140180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934930PMC
February 2021

Multiomics-Identified Intervention to Restore Ethanol-Induced Dysregulated Proteostasis and Secondary Sarcopenia in Alcoholic Liver Disease.

Cell Physiol Biochem 2021 Feb;55(1):91-116

Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, USA,

Background/aims: Signaling and metabolic perturbations contribute to dysregulated skeletal muscle protein homeostasis and secondary sarcopenia in response to a number of cellular stressors including ethanol exposure. Using an innovative multiomics-based curating of unbiased data, we identified molecular and metabolic therapeutic targets and experimentally validated restoration of protein homeostasis in an ethanol-fed mouse model of liver disease.

Methods: Studies were performed in ethanol-treated differentiated C2C12 myotubes and physiological relevance established in an ethanol-fed mouse model of alcohol-related liver disease (mALD) or pair-fed control C57BL/6 mice. Transcriptome and proteome from ethanol treated-myotubes and gastrocnemius muscle from mALD and pair-fed mice were analyzed to identify target pathways and molecules. Readouts including signaling responses and autophagy markers by immunoblots, mitochondrial oxidative function and free radical generation, and metabolic studies by gas chromatography-mass spectrometry and sarcopenic phenotype by imaging.

Results: Multiomics analyses showed that ethanol impaired skeletal muscle mTORC1 signaling, mitochondrial oxidative pathways, including intermediary metabolite regulatory genes, interleukin-6, and amino acid degradation pathways are β-hydroxymethyl-butyrate targets. Ethanol decreased mTORC1 signaling, increased autophagy flux, impaired mitochondrial oxidative function with decreased tricarboxylic acid cycle intermediary metabolites, ATP synthesis, protein synthesis and myotube diameter that were reversed by HMB. Consistently, skeletal muscle from mALD had decreased mTORC1 signaling, reduced fractional and total muscle protein synthesis rates, increased autophagy markers, lower intermediary metabolite concentrations, and lower muscle mass and fiber diameter that were reversed by β-hydroxymethyl-butyrate treatment.

Conclusion: An innovative multiomics approach followed by experimental validation showed that β-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.
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http://dx.doi.org/10.33594/000000327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195260PMC
February 2021

Erratum to: "MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcoholic-associated fatty liver and steatohepatitis (J Hepatol 2020; 73: 616-627)".

J Hepatol 2021 Apr 30;74(4):1002. Epub 2021 Jan 30.

Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States; Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH, United States; Department of Molecular Medicine, Case Western Reserve University, Cleveland, OH, United States. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2021.01.006DOI Listing
April 2021

Metabolic Profiling of Skeletal Muscle During Ex-Vivo Normothermic Limb Perfusion.

Mil Med 2021 01;186(Suppl 1):358-363

Cleveland Clinic Department of Plastic Surgery, Cleveland, OH 44195, USA.

Introduction: Ex vivo normothermic limb perfusion (EVNLP) provides several advantages for the preservation of limbs following amputation: the ability to maintain oxygenation and temperature of the limb close to physiological values, a perfusion solution providing all necessary nutrients at optimal concentrations, and the ability to maintain physiological pH and electrolytes. However, EVNLP cannot preserve the organ viability infinitely. We identified evidence of mitochondrial injury (swelling, elongation, and membrane disruption) after 24 hours of EVNLP of human upper extremities. The goal of this study was to identify metabolic derangements in the skeletal muscle during EVNLP.

Materials And Methods: Fourteen human upper extremities were procured from organ donors after family consent. Seven limbs underwent EVNLP for an average of 41.6 ± 9.4 hours, and seven contralateral limbs were preserved at 4°C for the same amount of time. Muscle biopsies were performed at 24 hours of perfusion, both from the EVNLP and control limbs. Perturbations in the metabolic profiles of the muscle during EVNLP were determined via untargeted liquid chromatography-mass spectrometry (MS) operated in positive and negative electrospray ionization modes, over a mass range of 50 to 750 Da. The data were deconvoluted using the XCMS software and further statistically analyzed using the in-house statistical package, MetaboLyzer. Putative identification of metabolites using exact mass within ±7 ppm mass error and MS/MS spectral matching to the mzCloud spectral library were performed via Compound Discoverer v.2.1 (Thermo Scientific, Fremont, CA, USA). We further validated the identity of candidate metabolites by matching the fragmentation pattern of these metabolites to those of their reference pure chemicals. A nonparametric Mann-Whitney U-test was used to compare EVNLP and control group spectral features. Differences were considered significantly different when P-value < 0.05.

Results: We detected over 13,000 spectral features of which 58 met the significance criteria with biologically relevant putative identifications. Furthermore we were able to confirm the identities of the ions taurine (P-value: 0.002) and tryptophan (P-value: 0.002), which were among the most significantly perturbed ions at 24 hours between the experimental and control groups. Metabolites belonging to the following pathways were the most perturbed at 24 hours: neuroactive ligand-receptor interaction (P-values: 0.031 and 0.036) and amino acid metabolism, including tyrosine and tryptophan metabolism (P-values: 0.015, 0.002, and 0.017). Taurine abundance decreased and tryptophan abundance increased at 24 hours. Other metabolites also identified at 24 hours included phenylalanine, xanthosine, and citric acid (P-values: 0.002, 0.002, and 0.0152).

Discussion: This study showed presence of active metabolism during EVNLP and metabolic derangement toward the end of perfusion, which correlated with detection of altered mitochondrial structure, swelling, and elongation.
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http://dx.doi.org/10.1093/milmed/usaa268DOI Listing
January 2021

Bile acids profile, histopathological indices and genetic variants for non-alcoholic fatty liver disease progression.

Metabolism 2021 03 1;116:154457. Epub 2020 Dec 1.

Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, USA; Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland Clinic, Cleveland, OH 44195, USA; Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA. Electronic address:

Objective: Metabolomic studies suggest plasma levels of bile acids (BAs) are elevated amongst subjects with non-alcoholic fatty liver disease (NAFLD) compared to healthy controls. However, it remains unclear whether or not specific BAs are associated with the clinically relevant transition from nonalcoholic fatty liver (i.e. simple steatosis) to non-alcoholic steatohepatitis (NASH), or enhanced progression of hepatic fibrosis, or genetic determinants of NAFLD/NASH.

Methods: Among sequential subjects (n=102) undergoing diagnostic liver biopsy, we examined the associations of a broad panel of BAs with distinct histopathological features of NAFLD, the presence of NASH, and their associations with genetic variants linked to NAFLD and NASH.

Results: Plasma BA alterations were observed through the entire spectrum of NAFLD, with several glycine conjugated forms of the BAs demonstrating significant associations with higher grades of inflammation and fibrosis. Plasma 7-Keto-DCA levels showed the strongest associations with advanced stages of hepatic fibrosis [odds ratio(95% confidence interval)], 4.2(1.2-16.4), NASH 24.5(4.1-473), and ballooning 18.7(4.8-91.9). Plasma 7-Keto-LCA levels were associated with NASH 9.4(1.5-185) and ballooning 5.9(1.4-28.8). Genetic variants at several NAFLD/NASH loci were nominally associated with increased levels of 7-Keto- and glycine-conjugated forms of BAs, and the NAFLD risk allele at the TRIB1 locus showed strong tendency toward increased plasma levels of GCA (p=0.02) and GUDCA (p=0.009).

Conclusions: Circulating bile acid levels are associated with histopathological and genetic determinants of the transition from simple hepatic steatosis into NASH. Further studies exploring the potential involvement of bile acid metabolism in the development and/or progression of distinct histopathological features of NASH are warranted.
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http://dx.doi.org/10.1016/j.metabol.2020.154457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856026PMC
March 2021

Alcohol Consumption is Associated with Poor Prognosis in Obese Patients with COVID-19: a Mendelian Randomization Study using UK Biobank.

medRxiv 2020 Nov 30. Epub 2020 Nov 30.

Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH.

Background: Acute and chronic alcohol abuse have adverse impacts on both the innate and adaptive immune response, which may result in reduced resistance to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and promote the progression of coronavirus disease 2019 (COVID-19). However, there are no large population-based data evaluating potential causal associations between alcohol consumption and COVID-19.

Method: We conducted a Mendelian randomization study using data from UK Biobank to explore the association between alcohol consumption and risk of SARS-CoV-2 infection and serious clinical outcomes in patients with COVID-19. A total of 12,937 participants aged 50-83 who tested for SARS-CoV-2 between 16 March to 27 July 2020 (12.1% tested positive) were included in the analysis. The exposure factor was alcohol consumption. Main outcomes were SARS-CoV-2 positivity and death in COVID-19 patients. We generated weighted and unweighted allele scores using three genetic variants (rs1229984, rs1260326, and rs13107325) and applied the allele scores as the instrumental variables to assess the effect of alcohol consumption on outcomes. Analyses were conducted separately for white participates with and without obesity.

Results: Of the 12,937 participants, 4,496 were never or infrequent drinkers and 8,441 were frequent drinkers. (including 1,156 light drinkers, 3,795 moderate drinkers, and 3,490 heavy drinkers). Both logistic regression and Mendelian randomization analyses found no evidence that alcohol consumption was associated with risk of SARS-CoV-2 infection in participants either with (OR=0.963, 95%CI 0.800-1.159; =1.000) or without obesity (OR=0.891, 95%CI 0.755-1.053; =.319). However, frequent drinking (HR=1.565, 95%CI 1.012-2.419; =.079), especially heavy drinking (HR=2.071, 95%CI 1.235-3.472; =.054), was associated with higher risk of death in patients with obesity and COVID-19, but not in patients without obesity. Notably, the risk of death in frequent drinkers with obesity increased slightly with the average amount of alcohol consumed weekly (HR=1.480, 95%CI 1.059-2.069; =.099).

Conclusions: Our findings suggested alcohol consumption may had adverse effects on the progression of COVID-19 in white participants with obesity, but was not associate with susceptibility to SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2020.11.25.20238915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709191PMC
November 2020

ESPEN practical guideline: Clinical nutrition in liver disease.

Clin Nutr 2020 12 27;39(12):3533-3562. Epub 2020 Oct 27.

Department of Internal Medicine, Municipal Hospital of Dessau, Dessau, Germany.

Background: The Practical guideline is based on the current scientific ESPEN guideline on Clinical Nutrition in Liver Disease.

Methods: It has been shortened and transformed into flow charts for easier use in clinical practice. The guideline is dedicated to all professionals including physicians, dieticians, nutritionists and nurses working with patients with chronic liver disease.

Results: A total of 103 statements and recommendations are presented with short commentaries for the nutritional and metabolic management of patients with (i) acute liver failure, (ii) alcoholic steatohepatitis, (iii) non-alcoholic fatty liver disease, (iv) liver cirrhosis, and (v) liver surgery/transplantation. The disease-related recommendations are preceded by general recommendations on the diagnostics of nutritional status in liver patients and on liver complications associated with medical nutrition.

Conclusion: This practical guideline gives guidance to health care providers involved in the management of liver disease to offer optimal nutritional care.
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http://dx.doi.org/10.1016/j.clnu.2020.09.001DOI Listing
December 2020

Effect of Acid Suppressants on the Risk of COVID-19: A Propensity Score-Matched Study Using UK Biobank.

Gastroenterology 2021 01 24;160(1):455-458.e5. Epub 2020 Sep 24.

Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, Ohio; Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, Ohio; Department of Molecular Medicine, Case Western Reserve University, Cleveland, Ohio. Electronic address:

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http://dx.doi.org/10.1053/j.gastro.2020.09.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513758PMC
January 2021

Activated Protein Phosphatase 2A Disrupts Nutrient Sensing Balance Between Mechanistic Target of Rapamycin Complex 1 and Adenosine Monophosphate-Activated Protein Kinase, Causing Sarcopenia in Alcohol-Associated Liver Disease.

Hepatology 2021 May 20;73(5):1892-1908. Epub 2021 Apr 20.

Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH.

Background And Aims: Despite the high clinical significance of sarcopenia in alcohol-associated cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol-induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine monophosphate-activated protein kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown).

Approach And Results: Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from patients with alcohol-associated cirrhosis were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue adenosine triphosphate, indicating a "pseudofed" state. We identified protein phosphatase (PP) 2A as a key mediator of ethanol-induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A to PP2A, resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3-kinase-γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol-induced molecular and phenotypic perturbations in wild-type mice were observed in PI3Kγ mice even at baseline. Importantly, overexpressing kinase-active PI3Kγ but not the kinase-dead mutant reversed ethanol-induced molecular perturbations.

Conclusions: Our study describes the mechanistic underpinnings for ethanol-mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ-PP2A axis.
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http://dx.doi.org/10.1002/hep.31524DOI Listing
May 2021

Association of non-alcoholic fatty liver disease and polycystic ovarian syndrome.

BMJ Open Gastroenterol 2020 08;7(1)

Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, Ohio, USA.

Background: Polycystic ovarian syndrome (PCOS) is a common endocrine disorder in women. Women with PCOS have androgen excess as a defining feature. They also have increased insulin resistance and obesity, which are also risk factors for non-alcoholic fatty liver disease (NAFLD). However, published data regarding PCOS as independent risk factor for NAFLD remain controversial. Therefore, we conducted this study to evaluate the association between PCOS and NAFLD using a large national database.

Methods: We identified adult female patients (≥18 years) with PCOS using the National Inpatient Sample database between 2002 and 2014. The control group included patients who did not have a diagnosis of PCOS. Multivariate logistic regression analysis was performed to study the association of NAFLD with PCOS.

Results: Out of a total of 50 785 354 women, 77 415 (0.15%) had PCOS. These patients were younger (32.7 vs 54.8; p<0.001) and more likely to be obese (29.4% vs 8.6%; p<0.001) compared with non-PCOS patients. However, the PCOS group had less hypertension (23.2% vs 39.8%), dyslipidaemia (12% vs 17.8%) and diabetes mellitus (18.1% vs 18.3%) (p<0.001 for all). Using multivariate logistic regression, patients with PCOS had significantly higher rate of NAFLD (OR 4.30, 95% CI 4.11 to 4.50, p<0.001).

Conclusion: Our study showed that patients with PCOS have four times higher risk of developing NAFLD compared with women without PCOS. Further studies are needed to assess if specific PCOS treatments can affect NAFLD progression.
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http://dx.doi.org/10.1136/bmjgast-2019-000352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418668PMC
August 2020

Design and rationale of a multicenter defeat alcoholic steatohepatitis trial: (DASH) randomized clinical trial to treat alcohol-associated hepatitis.

Contemp Clin Trials 2020 09 31;96:106094. Epub 2020 Jul 31.

Cleveland Clinic, USA.

Background/aims: Despite high mortality of alcohol-associated hepatitis, there has been limited advancement in treatment strategies. Defeat Alcoholic Steatohepatitis (DASH) is a multicenter, randomized, double-blind controlled trial whose primary objective was to evaluate the safety and efficacy of a novel combination of 3 drugs targeting different perturbations in AH.

Methods: Severe AH was diagnosed by liver biopsy or clinical and biochemical criteria and model for end stage liver disease (MELD) score ≥ 20 stratified by MELD scores (20-25 and ≥ 26) and randomized to a combination of an interleukin receptor 1 antagonist, Anakinra(100 mg daily for 14 days) to suppress acute inflammation, pentoxifylline (400 mg three times a day for 28 days) to prevent hepatorenal syndrome, and zinc sulfate (220 mg orally once daily for 6 months) or the standard of care therapy including methylprednisolone 32 mg orally once daily for 28 days. The primary efficacy outcome was the unadjusted log-rank test of the Kaplan-Meier survival estimates for the two treatment groups at 180 days.

Results: Between July 2012 to March 2018, 500 subjects with severe AH were screened of which 104 subjects were enrolled with MELD score of 25.6 ± 3.2 (20.0-35.0) in the investigational arm and 25.8 ± 4.5 (20.0-40.0) in the standard of care arm. Causes of screen failures included not meeting eligibility criteria (n = 347), declining to participate (n = 39), and other reasons (n = 10).

Conclusions: Data from the DASH consortium studies will determine if a combination of drugs targeting multiple mechanisms of injury in the severe AH will improve clinical outcomes.
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http://dx.doi.org/10.1016/j.cct.2020.106094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494528PMC
September 2020

Myogenic Response to Increasing Concentrations of Ammonia Differs between Mammalian, Avian, and Fish Species: Cell Differentiation and Genetic Study.

Genes (Basel) 2020 07 24;11(8). Epub 2020 Jul 24.

Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC 27695, USA.

Ammonia is very toxic to the body and has detrimental effects on many different organ systems. Using cultured myoblast cells, we examined ammonia's effect on myostatin expression, a negative regulator of skeletal muscle growth, and myotube diameters. The objective of this study was to examine how murine, avian, and fish cells respond to increasing levels of ammonia up to 50 mM. The murine myoblast cell line (C2C12), primary chick, and primary tilapia myoblast cells were cultured and then exposed to 10, 25, and 50 mM ammonium acetate, sodium acetate, and an untreated control for 24 h. High levels of ammonia were detrimental to the C2C12 cells, causing increased Myostatin (MSTN) expression and decreased myotube diameters between 10 and 25 mM ( < 0.002). Ammonia at 10 mM continued the positive myogenic response in the chick, with lower MSTN expression than the C2C12 cells and larger myotube diameters, but the myotube diameter at 50 mM ammonium acetate was significantly smaller than those at 10 and 25 mM ( < 0.001). However, chick myotubes at 50 mM were still significantly larger than the sodium acetate-treated and untreated control ( < 0.001). The tilapia cells showed no significant difference in MSTN expression or myotube diameter in response to increasing the concentrations of ammonia. Overall, these results confirm that increasing concentrations of ammonia are detrimental to mammalian skeletal muscle, while chick cells responded positively at lower levels but began to exhibit a negative response at higher levels, as the tilapia experienced no detrimental effects. The differences in ammonia metabolism strategies between fish, avian, and mammalian species could potentially contribute to the differences between species in response to high levels of ammonia. Understanding how ammonia affects skeletal muscle is important for the treatment of muscle wasting observed in liver failure patients.
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http://dx.doi.org/10.3390/genes11080840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464490PMC
July 2020

Diagnostic and Prognostic Significance of Complement in Patients With Alcohol-Associated Hepatitis.

Hepatology 2021 03 22;73(3):983-997. Epub 2020 Nov 22.

Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, USA.

Background And Aims: Given the lack of effective therapies and high mortality in acute alcohol-associated hepatitis (AH), it is important to develop rationally designed biomarkers for effective disease management. Complement, a critical component of the innate immune system, contributes to uncontrolled inflammatory responses leading to liver injury, but is also involved in hepatic regeneration. Here, we investigated whether a panel of complement proteins and activation products would provide useful biomarkers for severity of AH and aid in predicting 90-day mortality.

Approach And Results: Plasma samples collected at time of diagnosis from 254 patients with moderate and severe AH recruited from four medical centers and 31 healthy persons were used to quantify complement proteins by enzyme-linked immunosorbent assay and Luminex arrays. Components of the classical and lectin pathways, including complement factors C2, C4b, and C4d, as well as complement factor I (CFI) and C5, were reduced in AH patients compared to healthy persons. In contrast, components of the alternative pathway, including complement factor Ba (CFBa) and factor D (CFD), were increased. Markers of complement activation were also differentially evident, with C5a increased and the soluble terminal complement complex (sC5b9) decreased in AH. Mannose-binding lectin, C4b, CFI, C5, and sC5b9 were negatively correlated with Model for End-Stage Liver Disease score, whereas CFBa and CFD were positively associated with disease severity. Lower CFI and sC5b9 were associated with increased 90-day mortality in AH.

Conclusions: Taken together, these data indicate that AH is associated with a profound disruption of complement. Inclusion of complement, especially CFI and sC5b9, along with other laboratory indicators, could improve diagnostic and prognostic indications of disease severity and risk of mortality for AH patients.
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http://dx.doi.org/10.1002/hep.31419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005264PMC
March 2021

Muscle loss contributes to higher morbidity and mortality in COPD: An analysis of national trends.

Respirology 2021 01 16;26(1):62-71. Epub 2020 Jun 16.

Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, USA.

Background And Objective: COPD is the third most common cause of death worldwide and fourth most common in the United States. In hospitalized patients with COPD, mortality, morbidity and healthcare resource utilization are high. Skeletal muscle loss is frequent in patients with COPD. However, the impact of muscle loss on adverse outcomes has not been systematically evaluated. We tested the hypothesis that patients hospitalized for COPD exacerbation with, compared to those without, a secondary diagnosis of muscle loss phenotype (all ICD-9 codes associated with muscle loss including cachexia) will have higher mortality and cost of care.

Methods: The NIS database of hospitalized patients in 2011 (1 January-31 December) in the United States was used. The impact of a muscle loss phenotype on in-hospital mortality, LOS and cost of care for each of the 174 808 hospitalizations for COPD exacerbations was analysed.

Results: Of the subjects admitted for a COPD exacerbation, 12 977 (7.4%) had a secondary diagnosis of muscle loss phenotype. A diagnosis of muscle loss phenotype was associated with significantly higher in-hospital mortality (14.6% vs 5.7%, P < 0.001), LOS (13.3 + 17.1 vs 5.7 + 7.6, P < 0.001) and median hospital charge per patient ($13 947 vs $6610, P < 0.001). Multivariate regression analysis showed that muscle loss phenotype increased mortality by 111% (95% CI: 2.0-2.2, P < 0.001), LOS by 68.4% (P < 0.001) and the direct cost of care by 83.7% (P < 0.001) compared to those without muscle loss.

Conclusion: In-hospital mortality, LOS and healthcare costs are higher in patients with COPD exacerbations and a muscle loss phenotype.
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http://dx.doi.org/10.1111/resp.13877DOI Listing
January 2021

Skeletal muscle loss phenotype in cirrhosis: A nationwide analysis of hospitalized patients.

Clin Nutr 2020 12 3;39(12):3711-3720. Epub 2020 Apr 3.

Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, USA; Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH, USA. Electronic address:

Background & Aims: There are very limited data on the healthcare burden of muscle loss, the most frequent complication in hospitalized cirrhotics. We determined the healthcare impact of a muscle loss phenotype in hospitalized cirrhotics.

Methods: The Nationwide Inpatient Sample (NIS) database (years 2010-2014) was analyzed. Search terms included cirrhosis and its complications, and an expanded definition of a muscle loss phenotype that included all conditions associated with muscle loss. In-hospital mortality, length of stay (LOS), post-discharge disposition, co-morbidities and cost during admission were analyzed. Univariate and multivariate analyses were performed to identify associations between a muscle loss phenotype and outcomes. Impact of muscle loss in cirrhotics was compared to that in a random sample (2%) of general medical inpatients.

Results: A total of 162,694 hospitalizations for cirrhosis were reported, of which 18,261 (11.2%) included secondary diagnosis codes for a muscle loss phenotype. A diagnosis of muscle loss was associated with a significantly (p < 0.001 for all) higher mortality (19.3% vs 8.2%), LOS (14.2 ± 15.8 vs. 4.6 ± 6.9 days), and median hospital charge per admission ($21,400 vs. $8573) and a lower likelihood of discharge to home (30.1% vs. 60.2%). All evaluated outcomes were more severe in cirrhotics than general medical patients (n = 534,687). Multivariate regression analysis showed that a diagnosis of muscle loss independently increased mortality by 130%, LOS by 80% and direct cost of care by 119% (p < 0.001 for all). Alcohol use, female gender, malignancies and other organ dysfunction were independently associated with muscle loss.

Conclusions: Muscle loss contributed to higher mortality, LOS, and direct healthcare costs in hospitalized cirrhotics.
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http://dx.doi.org/10.1016/j.clnu.2020.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541467PMC
December 2020

Exercise and physical activity in cirrhosis: opportunities or perils.

J Appl Physiol (1985) 2020 06 2;128(6):1547-1567. Epub 2020 Apr 2.

Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.

Reduced exercise capacity and impaired physical performance are observed in nearly all patients with liver cirrhosis. Physical activity and exercise are physiological anabolic stimuli that can reverse dysregulated protein homeostasis or proteostasis and potentially increase muscle mass and contractile function in healthy subjects. Cirrhosis is a state of anabolic resistance, and unlike the beneficial responses to exercise reported in physiological states, there are few systematic studies evaluating the response to exercise in cirrhosis. Hyperammonemia is a mediator of the liver-muscle axis with net skeletal muscle ammonia uptake in cirrhosis causing signaling perturbations, mitochondrial dysfunction with decreased ATP content, modifications of contractile proteins, and impaired ribosomal function, all of which contribute to anabolic resistance in cirrhosis and have the potential to impair the beneficial responses to exercise. English language-publications in peer-reviewed journals that specifically evaluated the impact of exercise in cirrhosis were reviewed. Most studies evaluated responses to endurance exercise, and readouts included peak or maximum oxygen utilization, grip strength, and functional capacity. Endurance exercise for up to 12 wk is clinically tolerated in well-compensated cirrhosis. Data on the safety of resistance exercise are conflicting. Nutritional supplements enhance the benefits of exercise in healthy subjects but have not been evaluated in cirrhosis. Whether the beneficial physiological responses with endurance exercise and increase in muscle mass with resistance exercise that occur in healthy subjects also occur in cirrhotics is not known. Specific organ-system responses, changes in body composition, or improved long-term clinical outcomes with exercise in cirrhosis need evaluation.
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http://dx.doi.org/10.1152/japplphysiol.00798.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311690PMC
June 2020

MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcohol-associated fatty liver and steatohepatitis.

J Hepatol 2020 09 24;73(3):616-627. Epub 2020 Mar 24.

Center for Liver Disease Research, Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH, United States; Department of Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH, United States; Department of Molecular Medicine, Case Western Reserve University, Cleveland, OH, United States. Electronic address:

Background & Aims: Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis. The pseudokinase mixed lineage kinase domain-like (MLKL) is a key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Herein, we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy.

Methods: Rip3, Rip3, Mlkl and Mlkl mice were fed a Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA).

Results: The FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency protected mice from FFC diet-induced liver injury. The FFC diet also induced accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress, in Mlkl but not Mlkl mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation, first to autophagosomes and then to the plasma membrane, independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to the plasma membrane in hepatocytes.

Conclusions: Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy.

Lay Summary: Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy contributes to cell injury and death, thus playing a critical role in the pathogenesis of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis. Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver disease.
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http://dx.doi.org/10.1016/j.jhep.2020.03.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438259PMC
September 2020
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