Although hepatic encephalopathy (HE) is linked to the gut microbiota, stool microbiome analysis h... more Although hepatic encephalopathy (HE) is linked to the gut microbiota, stool microbiome analysis has not found differences between HE and no-HE patients. This study aimed to compare sigmoid mucosal microbiome of cirrhotic patients to controls, between HE vs. no-HE patients, and to study their linkage with cognition and inflammation. Sixty cirrhotic patients (36 HE and 24 no-HE) underwent cognitive testing, stool collection, cytokine (Th1, Th2, Th17, and innate immunity), and endotoxin analysis. Thirty-six patients (19 HE and 17 no-HE) and 17 age-matched controls underwent sigmoid biopsies. Multitag pyrosequencing (including autochthonous genera, i.e., Blautia, Roseburia, Fecalibacterium, Dorea) was performed on stool and mucosa. Stool and mucosal microbiome differences within/between groups and correlation network analyses were performed. Controls had significantly higher autochthonous and lower pathogenic genera compared with cirrhotic patients, especially HE patients. HE patients had worse MELD (model for end-stage liver disease) score and cognition and higher IL-6 and endotoxin than no-HE. Mucosal microbiota was different from stool within both HE/no-HE groups. Between HE/no-HE patients, there was no difference in stool microbiota but mucosal microbiome was different with lower Roseburia and higher Enterococcus, Veillonella, Megasphaera, and Burkholderia abundance in HE. On network analysis, autochthonous genera (Blautia, Fecalibacterium, Roseburia, and Dorea) were associated with good cognition and decreased inflammation in both HE/no-HE, whereas genera overrepresented in HE (Enterococcus, Megasphaera, and Burkholderia) were linked to poor cognition and inflammation. Sigmoid mucosal microbiome differs significantly from stool microbiome in cirrhosis. Cirrhotic, especially HE, patients' mucosal microbiota is significantly different from controls with a lack of potentially beneficial autochthonous and overgrowth of potentially pathogenic genera, which are associated with poor cognition and inflammation.
Hepatic encephalopathy (HE) has been related to gut bacteria and inflammation in the setting of i... more Hepatic encephalopathy (HE) has been related to gut bacteria and inflammation in the setting of intestinal barrier dysfunction. We aimed to link the gut microbiome with cognition and inflammation in HE using a systems biology approach. Multitag pyrosequencing (MTPS) was performed on stool of cirrhotics and age-matched controls. Cirrhotics with/without HE underwent cognitive testing, inflammatory cytokines, and endotoxin analysis. Patients with HE were compared with those without HE using a correlation-network analysis. A select group of patients with HE (n = 7) on lactulose underwent stool MTPS before and after lactulose withdrawal over 14 days. Twenty-five patients [17 HE (all on lactulose, 6 also on rifaximin) and 8 without HE, age 56 ± 6 yr, model for end-stage liver disease score 16 ± 6] and ten controls were included. Fecal microbiota in cirrhotics were significantly different (higher Enterobacteriaceae, Alcaligeneceae, and Fusobacteriaceae and lower Ruminococcaceae and Lachnospiraceae) compared with controls. We found altered flora (higher Veillonellaceae), poor cognition, endotoxemia, and inflammation (IL-6, TNF-α, IL-2, and IL-13) in HE compared with cirrhotics without HE. In the cirrhosis group, Alcaligeneceae and Porphyromonadaceae were positively correlated with cognitive impairment. Fusobacteriaceae, Veillonellaceae, and Enterobacteriaceae were positively and Ruminococcaceae negatively related to inflammation. Network-analysis comparison showed robust correlations (all P < 1E-5) only in the HE group between the microbiome, cognition, and IL-23, IL-2, and IL-13. Lactulose withdrawal did not change the microbiome significantly beyond Fecalibacterium reduction. We concluded that cirrhosis, especially when complicated with HE, is associated with significant alterations in the stool microbiome compared with healthy individuals. Specific bacterial families (Alcaligeneceae, Porphyromonadaceae, Enterobacteriaceae) are strongly associated with cognition and inflammation in HE.
American journal of physiology. Gastrointestinal and liver physiology, Jan 15, 2014
Proton pump inhibitors (PPI) have been associated with infectious complications in cirrhosis, but... more Proton pump inhibitors (PPI) have been associated with infectious complications in cirrhosis, but their impact on distal gut microbiota composition and function is unclear. We aimed to evaluate changes in stool microbiota composition and function in patients with cirrhosis and healthy controls after omeprazole therapy. Both 15 compensated cirrhotic patients and 15 age-matched controls underwent serum gastrin measurement, stool microbiota profiling with multitagged pyrosequencing, and urinary metabolic profiling with NMR spectroscopy to assess microbial cometabolites before/after a 14-day course of 40 mg/day omeprazole under constant diet conditions. Results before (pre) and after PPI were compared in both groups, compared with baseline by systems biology techniques. Adherence was >95% without changes in diet or MELD (model for end-stage liver disease) score during the study. Serum gastrin concentrations significantly increased after PPI in cirrhosis (pre 38.3 ± 35.8 vs. 115.6 ± 7...
A picture is now starting to emerge regarding the liver-bile acid-microbiome axis. Increasing lev... more A picture is now starting to emerge regarding the liver-bile acid-microbiome axis. Increasing levels of the primary bile acid cholic acid (CA) causes a dramatic shift toward the Firmicutes, particularly Clostridium cluster XIVa and increasing production of the harmful secondary bile acid deoxycholic acid (DCA). During progression of cirrhosis, the microbiome, both through their metabolism, cell wall components (LPS) and translocation lead to inflammation. Inflammation suppresses synthesis of bile acids in the liver leading to a positive-feedback mechanism. Decrease in bile acids entering the intestines appears to favor overgrowth of pathogenic and pro-inflammatory members of the microbiome including Porphyromonadaceae and Enterobacteriaceae. Decreasing bile acid concentration in the colon in cirrhosis is also associated with decreases in Clostridium cluster XIVa, which includes bile acid 7α-dehydroxylating bacteria which produce DCA. Rifaximin treatment appears to act by suppressing DCA production, reducing endotoxemia and harmful metabolites without significantly altering microbiome structure. Taken together, the bile acid pool size and composition appear to be a major regulator of microbiome structure, which in turn appears to be an important regulator of bile acid pool size and composition. The balance between this equilibrium is critical for human health and disease.
The 7adehydroxylation of primary bile acids by Eubacterium sp- V.P.I. 12708 required a cell extra... more The 7adehydroxylation of primary bile acids by Eubacterium sp- V.P.I. 12708 required a cell extract prepared from a cholic acid-induced culture and NAD+. NADH (0.5 mM) inhibited bile acid 7-dehydroxylase activity more than 50% when added to reaction mixtures containing NAD+ (0.5 mM). Saturation kinetics and double reciprocal plots of NADH inhibition were consistent with negative cooperativity. 7-Dehydroxylase activity was
HIV protease inhibitor (PI), the core component of highly active antiretroviral treatment (HAART)... more HIV protease inhibitor (PI), the core component of highly active antiretroviral treatment (HAART) for HIV infection, has been implicated in HAART-associated cardiovascular complications. Our previous studies have demonstrated that activation of endoplasmic reticulum (ER) stress is linked to HIV PI-induced inflammation and foam cell formation in macrophages. Raltegravir is a first-in-its-class HIV integrase inhibitor, the newest class of anti-HIV agents. We have recently reported that raltegravir has less hepatic toxicity and could prevent HIV PI-induced dysregulation of hepatic lipid metabolism by inhibiting ER stress. However, little information is available as to whether raltegravir would also prevent HIV PI-induced inflammatory response and foam cell formation in macrophages. In this study, we examined the effect of raltegravir on ER stress activation and lipid accumulation in cultured mouse macrophages (J774A.1), primary mouse macrophages, and human THP-1-derived macrophages, an...
Retinoid X receptor (RXR) regulates key cellular responses such as cell growth and development, a... more Retinoid X receptor (RXR) regulates key cellular responses such as cell growth and development, and this regulation is frequently perturbed in various malignancies, including hepatocellular carcinoma (HCC). However, the molecule(s) that physically govern this deregulation are mostly unknown. Here, we identified RXR as an interacting partner of astrocyte-elevated gene-1 (AEG-1)/metadherin (MTDH), an oncogene upregulated in all cancers. Upon interaction, AEG-1 profoundly inhibited RXR/retinoic acid receptor (RAR)-mediated transcriptional activation. Consequently, AEG-1 markedly protected HCC and acute myelogenous leukemia (AML) cells from retinoid- and rexinoid-induced cell death. In nontumorigenic cells and primary hepatocytes, AEG-1/RXR colocalizes in the nucleus in which AEG-1 interferes with recruitment of transcriptional coactivators to RXR, preventing transcription of target genes. In tumor cells and AEG-1 transgenic hepatocytes, overexpressed AEG-1 entraps RXR in cytoplasm, pre...
The characteristics of 7a-deh~droxylase, a bile zvmes and intestinal bacterial transformation re-... more The characteristics of 7a-deh~droxylase, a bile zvmes and intestinal bacterial transformation re- acid-biotransforming enzyme, were determined using dia- lyzed cell extracts of Eubacterium sp. V.P.I. 12708. 7a-De- hydroxylase was induced by cholic acid in this organism. Induction by cholic acid resulted in the differential syn- thesis of at least five new polypeptides with molecular weights of 77,000, two at 56,000, 27,000 and 23,500, as de- termined by both one and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. The relative molecular weight of 7a-dehydroxylase activity was esti- mated by anaerobic Bio-Gel .4 1.5 M gel filtration chro- matography to be 114,000. NAD+ was the only cofactor found to consistently stimulate 7a-dehydroxylase activity in dialyzed cell extracts. The specific activity increased 4- to 6-fold with either cholic or chenodeoxycholic acid as a substrate in the presence of NAD+. NAD+ was also re- quired for the reduction of the A6-inter...
The gut microbiome is altered in cirrhosis; however its evolution with disease progression is onl... more The gut microbiome is altered in cirrhosis; however its evolution with disease progression is only partly understood. We aimed to study changes in the microbiome over cirrhosis severity, its stability over time and its longitudinal alterations with decompensation. Controls and age-matched cirrhotics (compensated/decompensated/hospitalized) were included. Their stool microbiota was quantified using multi-tagged pyrosequencing. The ratio of autochthonous to non-autochthonous taxa was calculated as the cirrhosis dysbiosis ratio (CDR); a low number indicating dysbiosis. Firstly, the microbiome was compared between controls and cirrhotic sub-groups. Secondly, for stability assessment, stool collected twice within 6months in compensated outpatients was analyzed. Thirdly, changes after decompensation were assessed using (a) longitudinal comparison in patients before/after hepatic encephalopathy development (HE), (b) longitudinal cohort of hospitalized infected cirrhotics MELD-matched to uninfected cirrhotics followed for 30days. 244 subjects [219 cirrhotics (121 compensated outpatients, 54 decompensated outpatients, 44 inpatients) and 25 age-matched controls] were included. CDR was highest in controls (2.05) followed by compensated (0.89), decompensated (0.66), and inpatients (0.32, p<0.0001) and negatively correlated with endotoxin. Microbiota and CDR remained unchanged in stable outpatient cirrhotics (0.91 vs. 0.86, p=0.45). In patients studied before/after HE development, dysbiosis occurred post-HE (CDR: 1.2 to 0.42, p=0.03). In the longitudinal matched-cohort, microbiota were significantly different between infected/uninfected cirrhotics at baseline and a low CDR was associated with death and organ failures within 30days. Progressive changes in the gut microbiome accompany cirrhosis and become more severe in the setting of decompensation. The cirrhosis dysbiosis ratio may be a useful quantitative index to describe microbiome alterations accompanying cirrhosis progression.
Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression... more Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21Cip-1/WAF1/mda6 (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27(Kip-1) (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2alpha phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes.
Although hepatic encephalopathy (HE) is linked to the gut microbiota, stool microbiome analysis h... more Although hepatic encephalopathy (HE) is linked to the gut microbiota, stool microbiome analysis has not found differences between HE and no-HE patients. This study aimed to compare sigmoid mucosal microbiome of cirrhotic patients to controls, between HE vs. no-HE patients, and to study their linkage with cognition and inflammation. Sixty cirrhotic patients (36 HE and 24 no-HE) underwent cognitive testing, stool collection, cytokine (Th1, Th2, Th17, and innate immunity), and endotoxin analysis. Thirty-six patients (19 HE and 17 no-HE) and 17 age-matched controls underwent sigmoid biopsies. Multitag pyrosequencing (including autochthonous genera, i.e., Blautia, Roseburia, Fecalibacterium, Dorea) was performed on stool and mucosa. Stool and mucosal microbiome differences within/between groups and correlation network analyses were performed. Controls had significantly higher autochthonous and lower pathogenic genera compared with cirrhotic patients, especially HE patients. HE patients had worse MELD (model for end-stage liver disease) score and cognition and higher IL-6 and endotoxin than no-HE. Mucosal microbiota was different from stool within both HE/no-HE groups. Between HE/no-HE patients, there was no difference in stool microbiota but mucosal microbiome was different with lower Roseburia and higher Enterococcus, Veillonella, Megasphaera, and Burkholderia abundance in HE. On network analysis, autochthonous genera (Blautia, Fecalibacterium, Roseburia, and Dorea) were associated with good cognition and decreased inflammation in both HE/no-HE, whereas genera overrepresented in HE (Enterococcus, Megasphaera, and Burkholderia) were linked to poor cognition and inflammation. Sigmoid mucosal microbiome differs significantly from stool microbiome in cirrhosis. Cirrhotic, especially HE, patients' mucosal microbiota is significantly different from controls with a lack of potentially beneficial autochthonous and overgrowth of potentially pathogenic genera, which are associated with poor cognition and inflammation.
Hepatic encephalopathy (HE) has been related to gut bacteria and inflammation in the setting of i... more Hepatic encephalopathy (HE) has been related to gut bacteria and inflammation in the setting of intestinal barrier dysfunction. We aimed to link the gut microbiome with cognition and inflammation in HE using a systems biology approach. Multitag pyrosequencing (MTPS) was performed on stool of cirrhotics and age-matched controls. Cirrhotics with/without HE underwent cognitive testing, inflammatory cytokines, and endotoxin analysis. Patients with HE were compared with those without HE using a correlation-network analysis. A select group of patients with HE (n = 7) on lactulose underwent stool MTPS before and after lactulose withdrawal over 14 days. Twenty-five patients [17 HE (all on lactulose, 6 also on rifaximin) and 8 without HE, age 56 ± 6 yr, model for end-stage liver disease score 16 ± 6] and ten controls were included. Fecal microbiota in cirrhotics were significantly different (higher Enterobacteriaceae, Alcaligeneceae, and Fusobacteriaceae and lower Ruminococcaceae and Lachnospiraceae) compared with controls. We found altered flora (higher Veillonellaceae), poor cognition, endotoxemia, and inflammation (IL-6, TNF-α, IL-2, and IL-13) in HE compared with cirrhotics without HE. In the cirrhosis group, Alcaligeneceae and Porphyromonadaceae were positively correlated with cognitive impairment. Fusobacteriaceae, Veillonellaceae, and Enterobacteriaceae were positively and Ruminococcaceae negatively related to inflammation. Network-analysis comparison showed robust correlations (all P < 1E-5) only in the HE group between the microbiome, cognition, and IL-23, IL-2, and IL-13. Lactulose withdrawal did not change the microbiome significantly beyond Fecalibacterium reduction. We concluded that cirrhosis, especially when complicated with HE, is associated with significant alterations in the stool microbiome compared with healthy individuals. Specific bacterial families (Alcaligeneceae, Porphyromonadaceae, Enterobacteriaceae) are strongly associated with cognition and inflammation in HE.
American journal of physiology. Gastrointestinal and liver physiology, Jan 15, 2014
Proton pump inhibitors (PPI) have been associated with infectious complications in cirrhosis, but... more Proton pump inhibitors (PPI) have been associated with infectious complications in cirrhosis, but their impact on distal gut microbiota composition and function is unclear. We aimed to evaluate changes in stool microbiota composition and function in patients with cirrhosis and healthy controls after omeprazole therapy. Both 15 compensated cirrhotic patients and 15 age-matched controls underwent serum gastrin measurement, stool microbiota profiling with multitagged pyrosequencing, and urinary metabolic profiling with NMR spectroscopy to assess microbial cometabolites before/after a 14-day course of 40 mg/day omeprazole under constant diet conditions. Results before (pre) and after PPI were compared in both groups, compared with baseline by systems biology techniques. Adherence was >95% without changes in diet or MELD (model for end-stage liver disease) score during the study. Serum gastrin concentrations significantly increased after PPI in cirrhosis (pre 38.3 ± 35.8 vs. 115.6 ± 7...
A picture is now starting to emerge regarding the liver-bile acid-microbiome axis. Increasing lev... more A picture is now starting to emerge regarding the liver-bile acid-microbiome axis. Increasing levels of the primary bile acid cholic acid (CA) causes a dramatic shift toward the Firmicutes, particularly Clostridium cluster XIVa and increasing production of the harmful secondary bile acid deoxycholic acid (DCA). During progression of cirrhosis, the microbiome, both through their metabolism, cell wall components (LPS) and translocation lead to inflammation. Inflammation suppresses synthesis of bile acids in the liver leading to a positive-feedback mechanism. Decrease in bile acids entering the intestines appears to favor overgrowth of pathogenic and pro-inflammatory members of the microbiome including Porphyromonadaceae and Enterobacteriaceae. Decreasing bile acid concentration in the colon in cirrhosis is also associated with decreases in Clostridium cluster XIVa, which includes bile acid 7α-dehydroxylating bacteria which produce DCA. Rifaximin treatment appears to act by suppressing DCA production, reducing endotoxemia and harmful metabolites without significantly altering microbiome structure. Taken together, the bile acid pool size and composition appear to be a major regulator of microbiome structure, which in turn appears to be an important regulator of bile acid pool size and composition. The balance between this equilibrium is critical for human health and disease.
The 7adehydroxylation of primary bile acids by Eubacterium sp- V.P.I. 12708 required a cell extra... more The 7adehydroxylation of primary bile acids by Eubacterium sp- V.P.I. 12708 required a cell extract prepared from a cholic acid-induced culture and NAD+. NADH (0.5 mM) inhibited bile acid 7-dehydroxylase activity more than 50% when added to reaction mixtures containing NAD+ (0.5 mM). Saturation kinetics and double reciprocal plots of NADH inhibition were consistent with negative cooperativity. 7-Dehydroxylase activity was
HIV protease inhibitor (PI), the core component of highly active antiretroviral treatment (HAART)... more HIV protease inhibitor (PI), the core component of highly active antiretroviral treatment (HAART) for HIV infection, has been implicated in HAART-associated cardiovascular complications. Our previous studies have demonstrated that activation of endoplasmic reticulum (ER) stress is linked to HIV PI-induced inflammation and foam cell formation in macrophages. Raltegravir is a first-in-its-class HIV integrase inhibitor, the newest class of anti-HIV agents. We have recently reported that raltegravir has less hepatic toxicity and could prevent HIV PI-induced dysregulation of hepatic lipid metabolism by inhibiting ER stress. However, little information is available as to whether raltegravir would also prevent HIV PI-induced inflammatory response and foam cell formation in macrophages. In this study, we examined the effect of raltegravir on ER stress activation and lipid accumulation in cultured mouse macrophages (J774A.1), primary mouse macrophages, and human THP-1-derived macrophages, an...
Retinoid X receptor (RXR) regulates key cellular responses such as cell growth and development, a... more Retinoid X receptor (RXR) regulates key cellular responses such as cell growth and development, and this regulation is frequently perturbed in various malignancies, including hepatocellular carcinoma (HCC). However, the molecule(s) that physically govern this deregulation are mostly unknown. Here, we identified RXR as an interacting partner of astrocyte-elevated gene-1 (AEG-1)/metadherin (MTDH), an oncogene upregulated in all cancers. Upon interaction, AEG-1 profoundly inhibited RXR/retinoic acid receptor (RAR)-mediated transcriptional activation. Consequently, AEG-1 markedly protected HCC and acute myelogenous leukemia (AML) cells from retinoid- and rexinoid-induced cell death. In nontumorigenic cells and primary hepatocytes, AEG-1/RXR colocalizes in the nucleus in which AEG-1 interferes with recruitment of transcriptional coactivators to RXR, preventing transcription of target genes. In tumor cells and AEG-1 transgenic hepatocytes, overexpressed AEG-1 entraps RXR in cytoplasm, pre...
The characteristics of 7a-deh~droxylase, a bile zvmes and intestinal bacterial transformation re-... more The characteristics of 7a-deh~droxylase, a bile zvmes and intestinal bacterial transformation re- acid-biotransforming enzyme, were determined using dia- lyzed cell extracts of Eubacterium sp. V.P.I. 12708. 7a-De- hydroxylase was induced by cholic acid in this organism. Induction by cholic acid resulted in the differential syn- thesis of at least five new polypeptides with molecular weights of 77,000, two at 56,000, 27,000 and 23,500, as de- termined by both one and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. The relative molecular weight of 7a-dehydroxylase activity was esti- mated by anaerobic Bio-Gel .4 1.5 M gel filtration chro- matography to be 114,000. NAD+ was the only cofactor found to consistently stimulate 7a-dehydroxylase activity in dialyzed cell extracts. The specific activity increased 4- to 6-fold with either cholic or chenodeoxycholic acid as a substrate in the presence of NAD+. NAD+ was also re- quired for the reduction of the A6-inter...
The gut microbiome is altered in cirrhosis; however its evolution with disease progression is onl... more The gut microbiome is altered in cirrhosis; however its evolution with disease progression is only partly understood. We aimed to study changes in the microbiome over cirrhosis severity, its stability over time and its longitudinal alterations with decompensation. Controls and age-matched cirrhotics (compensated/decompensated/hospitalized) were included. Their stool microbiota was quantified using multi-tagged pyrosequencing. The ratio of autochthonous to non-autochthonous taxa was calculated as the cirrhosis dysbiosis ratio (CDR); a low number indicating dysbiosis. Firstly, the microbiome was compared between controls and cirrhotic sub-groups. Secondly, for stability assessment, stool collected twice within 6months in compensated outpatients was analyzed. Thirdly, changes after decompensation were assessed using (a) longitudinal comparison in patients before/after hepatic encephalopathy development (HE), (b) longitudinal cohort of hospitalized infected cirrhotics MELD-matched to uninfected cirrhotics followed for 30days. 244 subjects [219 cirrhotics (121 compensated outpatients, 54 decompensated outpatients, 44 inpatients) and 25 age-matched controls] were included. CDR was highest in controls (2.05) followed by compensated (0.89), decompensated (0.66), and inpatients (0.32, p<0.0001) and negatively correlated with endotoxin. Microbiota and CDR remained unchanged in stable outpatient cirrhotics (0.91 vs. 0.86, p=0.45). In patients studied before/after HE development, dysbiosis occurred post-HE (CDR: 1.2 to 0.42, p=0.03). In the longitudinal matched-cohort, microbiota were significantly different between infected/uninfected cirrhotics at baseline and a low CDR was associated with death and organ failures within 30days. Progressive changes in the gut microbiome accompany cirrhosis and become more severe in the setting of decompensation. The cirrhosis dysbiosis ratio may be a useful quantitative index to describe microbiome alterations accompanying cirrhosis progression.
Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression... more Previously, using primary hepatocytes residing in early G1 phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21Cip-1/WAF1/mda6 (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27(Kip-1) (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2alpha phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes.
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Papers by Phillip Hylemon