Focused panel sequencing points to genetic predisposition in non-cirrhotic intrahepatic portal hypertension patients in India.

OBJECTIVE: Non-cirrhotic intrahepatic portal hypertension (NCIPH), a portal microangiopathy affecting small portal vein radicles, is a disease of Indian sub-continent. NCIPH appears to be a complex disease with interactions between inherited and acquired factors, though the exact pathophysiological mechanism is unknown. We aimed at investigating the genetic variants that might contribute to susceptibility to NCIPH. METHODS: In this case-control study, we analyzed genes associated with microangiopathy-VWF-ADAMTS13 (von Willebrand factor and its cleavase enzyme - a disintegrin and matrix metalloprotease with thrombospondin type-1 motifs member 13) and alternative complement system vitamin B12 metabolism and with familial NCIPH. RESULT: Eighty-four Indian patients with liver biopsy-proven NCIPH (cases) and 103 healthy controls (matched for residential region of India) were included in the study. Targeted next-generation sequencing (NGS) panel, comprising 11 genes of interest, was done on 54 cases. Genotyping of selected variants was performed in 84 cases and 103 healthy controls. We identified variants in MBL2, CD46 and VWF genes either associated or predisposing to NCIPH. We also identified a single case with a novel compound heterozygous mutation in MBL2 gene, possibly contributing to development of NCIPH. CONCLUSION: In this first of a kind comprehensive gene panel study, multiple variants of significance have been noted, especially in ADAMTS13-VWF and complement pathways in NCIPH patients in India. Functional significance of these variants needs to be further studied.

What makes non-cirrhotic portal hypertension a common disease in India? Analysis for environmental factors.

In India, an unexplained enteropathy is present in a majority of non-cirrhotic intrahepatic portal hypertension (NCIPH) patients. Small intestinal bacterial contamination and tropical enteropathy could trigger inflammatory stimuli and activate the endothelium in the portal venous system. Groundwater contaminated with arsenic is an environmental factor of epidemic proportions in large areas of India which has similar consequences. Von Willebrand factor (a sticky protein) expressed by activated endothelium may promote formation of platelet microthrombi and occlusion of intrahepatic portal vein branches leading to NCIPH. Environmental factors linked to suboptimal hygiene and sanitation, which enter through the gastrointestinal (GI) tract, predispose to platelet plugging onto activated endothelium in portal microcirculation. Thus, NCIPH, an example of poverty linked thrombophilia, is a disease mainly affecting the lower socio-economic strata of Indian population. Public health measures to improve sanitation, provide clean drinking water and eliminate arsenic contamination of drinking water are urgently needed. Till such time as these environmental factors are addressed, NCIPH is likely to remain 'an Indian disease'.

Rodenticidal hepatotoxicity: Raised plasma Von Willebrand factor levels predict in-hospital survival and preliminary report of the outcome of Von Willebrand factor reducing management protocol.

BACKGROUND: High Von Willebrand factor (VWF) levels may predispose to multi-organ failure in acute liver failure (ALF). In rodenticide-induced hepatotoxicity patients, we analyzed if plasma VWF levels predicted survival and also the outcome of VWF lowering by N-acetyl cysteine (NAC), fresh frozen plasma (FFP) infusions, and plasma exchange (PLEX). METHODS: We retrospectively analyzed prospectively collected data. Hepatotoxicity was classified as uncomplicated acute hepatitis (UAH), acute liver injury (ALI), and ALF. ALF patients, if not opting for liver transplantation, had PLEX and NAC; ALI patients received NAC ± FFP (PLEX, if worsening); UAH patients had NAC. Plasma VWF antigen was measured (normal, 50% to 150%). In-hospital survival was analyzed as discharged alive or died/discharged in a terminal condition (poor outcome). RESULTS: Twenty-four consecutive rodenticide-induced hepatotoxicity patients (UAH in 1, ALI in 20, ALF in 3) from December 2017 to January 2019 were studied. Baseline VWF levels were 153%, 423 (146-890)% median (range), and 448 (414-555)% in UAH, ALI, ALF patients; model for end-stage liver disease (MELD) scores were 11, 24 (12-38), 36 (32-37) and in-hospital survival rates were 100%, 85%, 67%, respectively. VWF levels were higher in patients with poor outcome (555 [512-890]%) than in those discharged alive (414 [146-617]%) (p-value = 0.04). The area under the receiver operating curve of the VWF level, MELD score, and sequential organ failure assessment score to predict survival was 0.92, 0.84, and 0.66, respectively. Of 4 patients meeting criteria for liver transplantation (none had transplantation), 3 (75%) survived. CONCLUSIONS: High VWF levels predict poor outcome in rodenticide-induced hepatotoxicity. VWF reduction may be useful in such patients.

Raised plasma levels of H2S and nitrate predict intrapulmonary vascular dilations: A preliminary report in patients with cryptogenic cirrhosis.

BACKGROUND AND AIMS: The role of vasoactive chemicals in the pathogenesis of hepatopulmonary syndrome (HPS), a disorder characterized by intrapulmonary vascular dilation (IPVD), is only vaguely elucidated. We aimed to study the association between plasma H2S, nitrate levels, and presence and severity of IPVD and HPS. METHODS: Consecutive adult patients with cryptogenic cirrhosis were evaluated for IPVD (by contrast echocardiography) and for hypoxemia (by arterial blood gas analysis). Plasma H2S and nitrate levels were measured in these patients. RESULTS: Fifty-eight patients with cryptogenic cirrhosis (male, 45; median age, range, 45, 16-74 years; Child's class; A, 30; B, 18; C, 10) were enrolled in this study. Thirty-four of the 58 (59%) patients had IPVD and 13 (22%) had HPS (mild, 4; moderate, 5; severe, 2; very severe, 2). Plasma H2S levels were significantly higher in patients with IPVD (19.6, 5.7-83 µmol/L) as compared to patients who had no IPVD (12.3, 0-47 µmol/L; p-value 0.03) with an area under receiver operating characteristic curve of 0.68 (95% CI 0.53-0.84). Plasma H2S levels were higher in patients with IPVD irrespective of liver disease severity. There was a trend for higher plasma nitrate levels in patients with IPVD (47, 15.8-126.4 nmol/mL) as compared to patients who had no IPVD (32.3, 6.9-51.4 nmol/mL; p-value 0.1). Raised plasma H2S and nitrate levels had an additive effect on the presence of IPVD. Neither plasma H2S nor plasma nitrate levels correlated with the degree of hypoxemia. CONCLUSION: Raised plasma H2S and nitrate levels predict the presence of IPVD in patients with cryptogenic cirrhosis.

ADAMTS13 missense variants associated with defective activity and secretion of ADAMTS13 in a patient with non-cirrhotic portal hypertension.

BACKGROUND: Non-cirrhotic intrahepatic portal hypertension (NCIPH) is characterized by thrombotic microangiopathy of the portal venous system, low ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-13), and high vWF (von Willebrand factor) levels. This study aimed to screen for ADAMTS13 mutations, focusing on the CUB domain, in these patients. METHODS: Prospectively recruited NCIPH patients and healthy volunteers underwent tests for plasma vWF-ADAMTS13 balance. Sanger sequencing of the CUB domain of ADAMTS13 was done in a subset of the NCIPH patients, and the detected mutation was screened for in all the study participants. Next-generation sequencing of clinically relevant exome and liver immunostaining for ADAMTS13 was done in patients with detected ADAMTS13 mutation. RESULTS: Plasma vWF-ADAMTS13 balance was significantly altered in 24 NCIPH patients (Child's class A:23, B:1) as compared to 22 controls. On initial sequencing of the CUB domain (17 cases and 3 controls), one NCIPH patient showed a rare missense variant (SNV) at position c.3829C >T resulting in p.R1277W (rs14045669). Subsequent RFLP analysis targeted to the R1277W variant did not detect this in any other NCIPH patient, nor in any of the 22 controls. The NCIPH patient with the R1277W variant had severe ADAMTS13 deficiency, consistently high vWF, other missense SNVs in ADAMTS13, vWF, and complement genes. Immunostaining of his liver biopsy revealed globules of ADAMTS13 within stellate cells. CONCLUSIONS: We report missense variants in ADAMTS13, vWF, and complement genes in a patient with NCIPH who had decreased secretion and activity of ADAMTS13 protein. Further studies are needed in NCIPH patients in this regard.

Role of Oxygen Free Radicals, Nitric Oxide and Mitochondria in Mediating Cardiac Alterations During Liver Cirrhosis Induced by Thioacetamide.

Thioacetamide (TAA) administration is widely used for induction of liver cirrhosis in rats, where reactive oxygen radicals (ROS) and nitric oxide (NO) participate in development of liver damage. Cardiac dysfunction is an important complication of liver cirrhosis, but the role of ROS or NO in cardiac abnormalities during liver cirrhosis is not well understood. This was investigated in animals after TAA-induced liver cirrhosis and temporal changes in oxidative stress, NO and mitochondrial function in the heart evaluated. TAA induced elevation in cardiac levels of nitrate before development of frank liver cirrhosis, without gross histological alterations. This was accompanied by an early induction of P38 MAP kinase, which is influenced by ROS and plays an important signaling role for induction of iNOS. Increased nitrotyrosine, protein oxidation and lipid peroxidation in the heart and cardiac mitochondria, suggestive of oxidative stress, also preceded frank liver cirrhosis. However, compromised cardiac mitochondrial function with a decrease in respiratory control ratio and increased mitochondrial swelling was seen later, when cirrhosis was evident. In conclusion, TAA induces elevations in ROS and NO in the heart in parallel to early liver damage. This leads to later development of functional deficits in cardiac mitochondria after development of liver cirrhosis.

Plasma von Willebrand factor levels predict in-hospital survival in patients with acute-on-chronic liver failure.

BACKGROUND AND AIMS: Circulating levels of von Willebrand factor (vWF) predict mortality in patients with cirrhosis. We hypothesized that systemic inflammation in acute-on-chronic liver failure (ACLF) will stimulate endothelium, increase vWF levels, and promote platelet microthrombi causing organ failure. METHODS: In this prospective study, we correlated plasma vWF levels with organ failure, liver disease severity, sepsis, and systemic inflammatory response syndrome (SIRS) and also analyzed if vWF levels predicted in-hospital composite poor outcome (i.e. death/discharged in terminal condition/liver transplantation) in consecutive ACLF patients. RESULTS: Twenty-one of the 50 ACLF patients studied had composite poor outcome. ACLF patients had markedly elevated vWF antigen and activity (sevenfold and fivefold median increase, respectively) on days 1 and 3. Median ratio of vWF to a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS13) activity on day 1 was significantly higher in ACLF patients (11.2) compared to 20 compensated cirrhosis patients (3.3) and healthy volunteers (0.9). On day 1, area under ROC curve (AUROC) to predict composite poor outcome of hospital stay for ACLF patients for vWF antigen, vWF activity, and model for end-stage liver disease (MELD) score were 0.63, 0.68, and 0.74, respectively. vWF activity correlated better with liver disease severity (MELD score, ACLF grade) and organ failure (Sequential Organ Failure Assessment [SOFA] score) than vWF antigen; in contrast, neither vWF antigen nor activity correlated with platelet count, sepsis, or SIRS. CONCLUSIONS: vWF levels are markedly elevated, correlate with organ failure, and predict in-hospital survival in ACLF patients. This data provides a mechanistic basis for postulating that vWF-reducing treatments such as plasma exchange may benefit ACLF patients.

Arsenicosis, possibly from contaminated groundwater, associated with noncirrhotic intrahepatic portal hypertension.

BACKGROUND AND AIMS: Idiopathic noncirrhotic intrahepatic portal hypertension (NCIPH), a chronic microangiopathy of the liver caused by arsenicosis from use of contaminated groundwater, was reported from Asia. This study aimed to see, if in the twenty-first century, arsenicosis was present in NCIPH patients at our hospital and, if present, to look for groundwater contamination by arsenic in their residential locality. METHODS: Twenty-seven liver biopsy proven NCIPH patients, 25 portal hypertensive controls with hepatitis B or C related cirrhosis and 25 healthy controls, matched for residential locality, were studied. Eighty-four percent to 96 % of study subjects belonged to middle or lower socioeconomic category. Arsenicosis was looked for by estimation of arsenic levels in finger/toe nails and by skin examination. Arsenic levels in nails and in ground water (in NCIPH patients with arsenicosis) was estimated by mass spectrometry. RESULTS: Nail arsenic levels were raised in five (10 %) portal hypertensive study subjects [two NCIPH patients (both had skin arsenicosis) and three portal hypertensive controls]. All of these five patients were residents of West Bengal or Bangladesh. Skin arsenicosis was noted in three NCIPH patients (11 %) compared to none of disease/healthy controls. Ground water from residential locality of one NCIPH patient with arsenicosis (from Bangladesh) showed extremely high level of arsenic (79.5 µg/L). CONCLUSIONS: Arsenicosis and microangiopathy of liver, possibly caused by environmental contamination continues in parts of Asia. Further studies are needed to understand the mechanisms of such 'poverty-linked thrombophilia'.

Serum cholinesterase is an excellent biomarker of liver cirrhosis.

BACKGROUND: Serum cholinesterase (ChE) is an enzyme synthesised by hepatocytes and its serum levels reflect the synthetic function of liver. METHODS: In patients with cirrhosis, liver function tests, PT INR and serum ChE levels were done within a week of enrolment. We studied 178 cirrhosis patients and 154 healthy controls prospectively. Receiver operator characteristics (ROC) curve analysis was employed to compute an optimal cut-off level to distinguish these groups. Correlation between ChE activity and serum bilirubin, albumin, PT INR and MELD score (Model for End-Stage Liver Disease) was analysed. RESULTS: Median serum ChE in cirrhotics was 1590 IU/L (110-8143) compared to controls 7886 IU/L (2022- 21673), p < 0.001. Serum ChE levels below 3506 had a 98.7% sensitivity and 80.3% specificity in predicting cirrhosis. Median serum ChE was higher (p < 0.001) in CC (n = 51) 4246 IU/L (680-8143) compared to DC (n = 127) 1324 IU/L (110-4550). ChE level less than 2385 IU/L had 80.1% sensitivity and 88.2% specificity in predicting DC. Follow-up levels in 25 patients showed good correlation with clinical course. The correlation coefficient between ChE and albumin was -0.67, 0.53 with PT INR and 0.59 with MELD score, (p < 0.001). CONCLUSIONS: Serum ChE is an excellent biomarker of cirrhosis with good sensitivity and specificity. It shows good correlation with serum albumin, PT INR and MELD score. Since it distinguishes DC from CC well, low levels in cirrhosis may serve as a useful prognostic marker of advanced liver disease. Long-term follow-up studies are warranted to define its exact role in clinical practice.

Investigation into celiac disease in Indian patients with portal hypertension.

BACKGROUND: There is limited data on celiac disease in patients with cryptogenic cirrhosis or idiopathic noncirrhotic intrahepatic portal hypertension (NCIPH). Our objective was to evaluate for celiac disease in patients with portal hypertension in India. METHODS: Consecutive patients with portal hypertension having cryptogenic chronic liver disease (cases) and hepatitis B- or C-related cirrhosis (controls) were prospectively enrolled. We studied tissue transglutaminase (tTG) antibody and duodenal histology in study patients. RESULT: Sixty-one cases (including 14 NCIPH patients) and 59 controls were enrolled. Celiac disease was noted in six cases (including two NCIPH patients) as compared to none in controls. In a significant proportion of the remaining study subjects, duodenal biopsy showed villous atrophy, crypt hyperplasia, and lamina propria inflammation, not accompanied by raised intraepithelial lymphocytes (IELs); this was seen more commonly in cases as compared to controls. An unexpectedly high rate of tTG antibody positivity was seen in study subjects (66 %) of cases as compared to 29 % in controls (p-value < 0.001), which could indicate false-positive test result. CONCLUSION: In this study, 10 % of patients with unexplained portal hypertension (cryptogenic chronic liver disease) had associated celiac disease. In addition, an unexplained enteropathy was seen in a significant proportion of study patients, more so in patients with cryptogenic chronic liver disease. This finding warrants further investigation.

Immune response and intestinal permeability in children with acute gastroenteritis treated with Lactobacillus rhamnosus GG: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Probiotics have a possible role in the treatment of pediatric acute gastroenteritis. We report the effect of the probiotic Lactobacillus rhamnosus GG (LGG) on intestinal function, immune response, and clinical outcomes in Indian children with cryptosporidial or rotavirus diarrhea. METHODS: Children with gastroenteritis aged 6 months to 5 years, testing positive for either rotavirus or Cryptosporidium species in stool (coinfections were excluded), were randomized to LGG (ATCC 53103) or placebo, once daily for 4 weeks. Baseline demographic and clinical details were obtained. Sera were tested for immunoglobulin G (IgG) and immunoglobulin A (IgA) antibodies to Cryptosporidium and rotavirus, and the lactulose to mannitol ratio for intestinal permeability was determined at baseline and at the end of follow-up. RESULTS: Of the 124 children enrolled, 82 and 42 had rotavirus and cryptosporidial diarrhea, respectively. Median diarrheal duration was 4 days; one-third of the children had severe diarrhea. Baseline and clinical parameters were comparable between children receiving LGG and placebo. At the end of follow-up, fewer children with rotavirus diarrhea on LGG had repeated diarrheal episodes (25% vs 46%; P = .048) and impaired intestinal function (48% vs 72%; P = .027). Significant increase in IgG levels postintervention (456 vs 2215 EU; P = .003) was observed in children with rotavirus diarrhea receiving LGG. Among children with cryptosporidial diarrhea, those receiving LGG showed significant improvement in intestinal permeability. CONCLUSIONS: LGG has a positive immunomodulatory effect and may be useful in decreasing repeated episodes of rotavirus diarrhea. Improvement in intestinal function in children with rotavirus and cryptosporidial gastroenteritis emphasizes the role of probiotics in treating intestinal impairment after infection. CLINICAL TRIALS REGISTRATION: CTRI/2010/091/000339.

Acute fatty liver of pregnancy: an update on mechanisms.

Acute fatty liver of pregnancy (AFLP), characterized by hepatic microvesicular steatosis, is a sudden catastrophic illness occurring almost exclusively in the third trimester of pregnancy. Defective fatty acid oxidation in the fetus has been shown to be associated with this disease. Since the placenta has the same genetic makeup as the fetus and as AFLP patients generally recover following delivery, we hypothesized that the placenta might be involved in pathogenesis of this disease. In an animal model of hepatic microvesicular steatosis (using sodium valproate), we found that microvesicular steatosis results in mitochondrial structural alterations and oxidative stress in subcellular organelles of the liver. In placentas from patients with AFLP, we observed placental mitochondrial dysfunction and oxidative stress in subcellular organelles. In addition, defective placental fatty acid oxidation results in accumulation of toxic mediators such as arachidonic acid. Escape of these mediators into the maternal circulation might affect the maternal liver resulting in microvesicular steatosis.

Activation of phospholipase A2 is involved in indomethacin-induced damage in Caco-2 cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs), widely used in clinical practice, cause adverse effects in the gastrointestinal tract. These effects have been attributed to mechanisms such as drug-induced cyclooxygenase inhibition, oxidative stress, mitochondrial dysfunction and changes in cell membrane lipids. Our previous study showed that indomethacin (an NSAID commonly used in toxicity studies) caused activation of cytosolic phospholipase A(2) (cPLA(2)) in the rat small intestine. We hypothesized that activation of cPLA(2) is an important event in the pathogenesis of indomethacin-induced damage in enterocytes. To test this, we incubated enterocyte-like Caco-2 cells with indomethacin, with and without pretreatment with methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of cPLA(2). Cells treated with indomethacin showed decreased viability and evidence of oxidative stress and morphological cell damage. Phospholipids were degraded in these cells, with increases in the levels of lysophospholipids and arachidonic acid. There was no evidence of apoptosis in the cells in response to the drug. Pretreatment of the cells with MAFP attenuated the drug-induced effects seen. This shows that activation of phospholipase A(2) appears to be an important event in the pathogenesis of indomethacin-induced damage in Caco-2 cells. To our knowledge, this is the first report that implicates the involvement of this enzyme in NSAID-induced enteropathy.

Fatty acids influence binding of cobalt to serum albumin in patients with fatty liver.

Human serum albumin binds ligands such as fatty acids and metals in circulation. Oxidative stress can modify albumin and affect ligand binding. This study examines the role of oxidative stress and fatty acids in modulating cobalt binding to albumin in patients with fatty liver. Elevated levels of malondialdehyde and protein carbonyls, indicative of oxidative stress were evident in serum of patients with fatty liver. A significant decrease in albumin-cobalt binding was also observed. Albumin isolated from patient serum also showed an increase in bound fatty acids. In vitro experiments indicated that while oxidant exposure or removal of fatty acids independently decreased cobalt binding to albumin, removal of fatty acids from the protein prior to oxidant exposure did not influence the oxidant effect on albumin-cobalt binding. These results suggest that oxidative stress and fatty acids on albumin can influence albumin-cobalt binding in patients with fatty liver by independent mechanisms.

Curcumin reduces indomethacin-induced damage in the rat small intestine.

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in clinical medicine. Their utility is, however, often limited by the adverse effects they produce in the gastrointestinal tract. Oxidative stress has been shown to occur in the small intestine in response to the oral administration of indomethacin, an NSAID commonly used in toxicity studies. In view of this, the effect of curcumin, an agent with anti-oxidant properties, was evaluated on indomethacin-induced small intestinal damage in a rat model. Rats were pretreated with various doses of curcumin (20 mg kg(-1), 40 mg kg(-1) and 80 mg kg(-1)) before administering indomethacin at 20 mg kg(-1). Various parameters of oxidative stress and the extent of small intestinal damage produced by indomethacin, with and without pretreatment with curcumin, were measured. Macroscopic ulceration was found to occur in the small intestine in response to indomethacin. The viability of enterocytes from indomethacin-treated animals was significantly lower than those from control animals. Drug-induced oxidative stress was also evident as seen by increases in the levels of malondialdehyde and protein carbonyl and in activities of pro-oxidant enzymes such as myeloperoxidase and xanthine oxidase in indomethacin-treated rats. Concomitant decreases were seen in the activities of the antioxidant enzymes catalase and glutathione peroxidase in these animals. Pretreatment with curcumin was found to ameliorate these drug-induced changes. Thus, curcumin appears to hold promise as an agent that can potentially reduce NSAID-induced small intestinal damage.

Intestinal mucosal alterations in rats with carbon tetrachloride-induced cirrhosis: changes in glycosylation and luminal bacteria.

Spontaneous bacterial peritonitis is a major cause of mortality after liver cirrhosis. Altered permeability of the mucosa and deficiencies in host immune defenses through bacterial translocation from the intestine due to intestinal bacterial overgrowth have been implicated in the development of this complication. Molecular mechanisms underlying the process are not well known. In order to understand mechanisms involved in translocation of bacteria, this study explored the role of oxidative stress in mediating changes in intestinal mucosal glycosylation and luminal bacterial content during cirrhosis. CCl4-induced cirrhosis in rats led to prolonged oxidative stress in the intestine, accompanied by increased sugar content of both intestinal brush border and surfactant layers. This was accompanied by changes in bacterial flora in the gut, which showed increased hydrophobicity and adherence to the mucosa. Inhibition of xanthine oxidase using sodium tungstate or antioxidant supplementation using vitamin E reversed the oxidative stress, changes in brush border membrane sugar content, and bacterial adherence. In conclusion, oxidative stress in the intestine during cirrhosis alters mucosal glycosylation, accompanied by an increased hydrophobicity of luminal bacteria, enabling increased bacterial adherence onto epithelial cells. This might facilitate translocation across the mucosa, resulting in complications such as spontaneous bacterial peritonitis.