Significant association between HLA-B*35:01 and onset of drug-induced liver injury caused by Kampo medicines in Japanese patients.

AIM: Drug-induced liver injury (DILI) is a severe and life-threatening immune-mediated adverse effect, occurring rarely among treated patients. We examined genomic biomarkers in the Japanese population that predict the onset of DILI after using a certain class of drugs, such as Kampo products (Japanese traditional medicines). METHODS: A total of 287 patients diagnosed as DILI by hepatology specialists were recruited after written informed consent was obtained. A genome-wide association analysis and human leukocyte antigen (HLA) typing in four digits were performed. RESULTS: We found a significant association (p = 9.41 × 10-10 ) of rs146644517 (G > A) with Kampo product-related DILI. As this polymorphism is located in the HLA region, we evaluated the association of HLA types and found that 12 (63.2%) of 19 Kampo-DILI patients contained HLA-B*35:01, whereas only 15.2% were positive for this HLA among healthy volunteers. The odds ratio was 9.56 (95% confidence interval 3.75-24.46; p = 2.98 × 10-6 , corrected p = 4.17 × 10-5 ), and it increased to 13.55 compared with the DILI patients not exposed to Kampo products. The individual crude drug components in the Kampo products, including Scutellaria root (ougon in Japanese), rhubarb (daiou), Gardenia fruit (sanshishi), and Glycyrrhiza (kanzou), were significantly associated with HLA-B*35:01. CONCLUSIONS: HLA-B*35:01 is a genetic risk factor and a potential predictive biomarker for Kampo-induced DILI in the Japanese population.

Frequency of null genotypes of glutathione S-transferase M1 and T1 in Japanese patients with drug-induced liver injury.

AIM: Previous reports suggest that the null genotype (*0/*0) of glutathione S-transferase (GST) M1 and/or GSTT1 could be risk factors for drug-induced liver injury (DILI). However, multi-institutional pharmacogenetic research with various suspected drugs has rarely been performed in Japan. Therefore, the aim of this study was to investigate the role of GSTM1 and GSTT1 null genotype in the occurrence of DILI in Japanese patients. METHODS: Blood samples of 270 DILI patients from 23 hospitals throughout Japan collected between 2010 and 2018 were subjected to genotyping of null genotypes of GSTM1 and GSTT1 using the SmartAmp-2 method. We also collected information on DILI types, time to onset of DILI, pharmacological classification of suspected drugs and Digestive Disease Week-Japan score, as well as genotypes of GSTM1 and GSTT1 in each patient with DILI. RESULTS: The distribution of a combination of null genotypes of GSTM1 and GSTT1 in Japanese patients with DILI was significantly different from that reported in the general Japanese population. Notably, the incidence of the GSTM1 null genotype in patients with DILI was significantly higher than that of the control population. A significant relationship between the frequency of GSTM1 and GSTT1 null genotypes and pharmacological classification of suspected drugs, clinical laboratory data for liver function, time to onset of DILI, and Digestive Disease Week-Japan scores was not observed. CONCLUSIONS: The GSTM1 null genotype was associated with an increased incidence of DILI in Japanese patients.

Low prevalence of colonic mucosal injury and bleeding in patients taking direct oral anticoagulants for non-valvular atrial fibrillation.

OBJECTIVE: Direct oral anticoagulants are frequently used to prevent systemic embolism associated with atrial fibrillation. Gastrointestinal bleeding is a common adverse event of this pharmacotherapy, especially in the lower gastrointestinal tract. However, the prevalence of mucosal injury of the colon in patients taking direct oral anticoagulants has remained unknown. MATERIALS AND METHODS: This was a retrospective study using endoscopic records of the colon from patients taking oral anticoagulants. Records from colonoscopies for 120 patients with non-valvular atrial fibrillation who had been prescribed direct oral anticoagulants between April 2011 and June 2017 were reviewed to determine the prevalence of mucosal injury and other findings, compared with those of 140 patients on warfarin. RESULTS: The prevalence of mucosal injury was 1.6% in patients taking direct oral anticoagulants and 1.4% in those taking warfarin, lower than other findings such as diverticula, hemorrhoids, and polyps. Bleeding was more frequent with direct oral anticoagulants (18 patients; 15%) than with warfarin (9 patients; 6.4%). Colonic diverticulum was the most common cause of bleeding in patients on direct oral anticoagulants. The prevalence of mucosal injury and causes of bleeding did not differ among direct oral anticoagulants. CONCLUSION: Colonic mucosal injury was infrequent in patients on direct oral anticoagulants. Bleeding was more frequent with direct oral anticoagulants than with warfarin. Colonic diverticulum and vascular ectasia were common causes of bleeding in patients on direct oral anticoagulants. Little difference in cause of bleeding was evident among oral anticoagulants.

[Second-line therapy with mycophenolate mofetil in patients with autoimmune hepatitis who were intolerant or failed to respond to standard treatment].

Although standard treatment for autoimmune hepatitis (AIH) comprises prednisolone (PSL) and azathioprine (AZA), some patients are intolerant to or do not respond to PSL and/or AZA. The clinical practice guidelines of AIH in Europe and North America recommend mycophenolate mofetil (MMF) as second-line treatment in these patients. We administered MMF as second-line therapy to 7 patients with AIH (male/female 1/6, age range 27-79 years) who were intolerant to or failed to respond to standard treatment. At the commencement of MMF, the median ALT value was 84U/L (28-254U/L), and the PSL dose was 15.0mg/day (0-45mg/day). In terms of adverse effects of PSL, diabetes mellitus was observed in 4 patients (insulin injection in 2) and femoral head necrolysis in 2. Adverse effects of AZA were present in 2, and 5 patients were not treated with AZA. At 24 weeks of MMF treatment, the median ALT and daily PSL dose were decreased to 16U/L (6-41U/L) and 7.0mg, respectively. Blood sugar control improved, and insulin injection was discontinued in both the patients. While intractable diarrhea developed in 1 patient with cirrhosis, no adverse effect was observed in other 6 patients. In conclusion, MMF appeared effective and safe in at least non-cirrhotic patients with AIH who were intolerant or failed to respond to standard treatment with PSL and AZA in Japanese clinical practice.

A validation study of the Ursodeoxycholic Acid Response Score in Japanese patients with primary biliary cholangitis.

BACKGROUND/PURPOSE: Although ursodeoxycholic acid (UDCA) is a first-line treatment for primary biliary cholangitis (PBC), 20%-30% of patients with PBC exhibit an incomplete response to UDCA. Recently, the UDCA Response Score was proposed for predicting response to UDCA using pretreatment parameters in patients with PBC. We aimed to validate the UDCA Response Score in Japanese patients with PBC. METHODS: Registry data of Japanese patients (n = 873) were collected. Patients with data on all clinical parameters required for calculating the UDCA Response Score were selected. The endpoint was UDCA response, defined as alkaline phosphatase <1.67 times the upper limit of the normal value after 12 months of UDCA treatment. RESULTS: All parameters were available in 804 patients (male/female = 120/684, age 58.9 [interquartile range 51.1-66.9] years). Bezafibrate was commenced within 12 months of UDCA in 78 patients (9.7%) because of the lack of an early response. We found that the endpoint was not reached in these 78 patients, and the area under the receiver operating characteristic curve (AUROC) of the score was 0.74 (95% confidence interval [CI] 0.70-0.79). The AUROC was 0.77 (95% CI 0.70-0.83) in patients undergoing UDCA monotherapy (n = 726). Finally, the AUROC of the modified UDCA Response Score using only data from the treatment start date was 0.80 (95% CI 0.70-0.90) in patients receiving a combination therapy of UDCA and bezafibrate (n = 160). CONCLUSION: The validity of the UDCA Response Score was acceptable in Japanese patients; this score will be informative in patients treated with a combination therapy of UDCA and bezafibrate.

Analysis of 307 cases with drug-induced liver injury between 2010 and 2018 in Japan.

AIM: In order to know the present status of drug-induced liver injury (DILI) in Japan, we present the data of prospectively collected DILI cases between 2010 and 2018 from 27 hospitals. METHODS: Drug-induced liver injury cases diagnosed by DILI experts from 27 hospitals all over Japan have been prospectively collected since 2010. Alanine aminotransferase level ≥150 U/L and/or alkaline phosphatase ≥2× upper limit of normal were inclusion criteria. RESULTS: In total, data of 307 cases (125 male and 182 female individuals) aged between 17 and 86 years old were collected. The types of liver injury were as follows: 64% hepatocellular type, 20% mixed type, and 16% cholestatic type. A drug-induced lymphocyte stimulation test was carried out in 59% of cases, and was positive in 48% and semipositive in 3% of cases. Eosinophilia ≥6% was observed in 27% of cases. Fifty-three percent of DILI cases occurred within 30 days and 79% of DILI cases occurred within 90 days after starting drug administration. By the diagnostic scale of the Digestive Disease Week (DDW)-Japan 2004 workshop, 93.8% of cases were diagnosed as "highly probable", and 5.9% as "possible". CONCLUSIONS: Japanese DILI patients are somewhat different from those of Europe and North America. The diagnostic scale of the DDW-Japan 2004 workshop has been used in Japan. However, there are many issues to improve the causality assessment of DILI that we must investigate in the future. It is critical to elucidate the mechanisms of drug metabolism and the pathophysiology of liver injury by various drugs to prevent DILI.

Validation of the Japanese version of the Chronic Liver Disease Questionnaire for the assessment of health-related quality of life in patients with chronic viral hepatitis.

AIM: Patients with chronic liver diseases (CLD) suffer from a variety of subjective symptoms, and the assessment of health-related quality of life (HRQOL) is crucial. The Chronic Liver Disease Questionnaire (CLDQ) is the first liver disease-specific instrument for this purpose. In this study we aimed to develop the Japanese version of CLDQ and to assess its validity and reliability in Japanese patients with chronic viral hepatitis. METHODS: The participants included 135 Japanese patients chronically infected with hepatitis B or C virus. The Japanese version of the CLDQ was developed according to the standard "back-translation" method. In addition to the Japanese version of the CLDQ, we asked the patients to fill out two other self-report questionnaires: the Japanese versions of the 36-Item Short Form Survey (SF-36) and Hospital Anxiety and Depression Scale (HADS). Then, the internal consistency, convergent and discriminant validity of the Japanese version of CLDQ were statistically examined. RESULTS: Cronbach's alpha of the Japanese version of the CLDQ was acceptable. The mean score was lower in emotional domains of the CLDQ, compared with those in somatic domains. Pearson correlations between Japanese CLDQ and SF-36 and HADS were significant. The mean of the CLDQ scores decreased in all domains in patients with liver cirrhosis compared with those in patients with chronic hepatitis. CONCLUSION: The Japanese version of the CLDQ is a reliable and valid instrument for assessment of the HRQOL of Japanese patients with chronic viral hepatitis. The results also suggest that the HRQOL of Japanese patients is mainly impaired by emotional factors rather than somatic symptoms, and significantly worsened by progression of the disease.

Benefit of cystatin C in evaluation of renal function and prediction of survival in patients with cirrhosis.

AIM: The assessment of renal function is of vital importance in management of patients with cirrhosis. While serum creatinine (Cr) is routinely used for this purpose, Cr-based estimated glomerular filtration rate (eGFR) does not reflect true renal function because of muscle wasting and impaired liver function. By contrast, cystatin C (CysC) is unrelated to muscle volume and liver function. In this study, we examined whether CysC-based GFR estimation is beneficial in assessment of renal function in patients with cirrhosis. METHODS: First, we assessed the performance of GFR-predicting equations based on serum Cr or CysC in 14 patients with cirrhosis, by comparison with inulin clearance as a gold standard of GFR (measured GFR [mGFR]). Next, in 49 patients with cirrhosis, we examined serum Cr and CysC at baseline, and examined which GFR-predicting equations were more suitable for predicting the outcome. RESULTS: In the first experiment, mGFR was 54.3 ± 23.0 mL/min, and CysC-based GFR-estimating equations had better performances compared with Cr-based equations in terms of bias, precision and accuracy. Cr-based estimated GFR (eGFRcreat) was significantly different from mGFR (P < 0.05). In the follow-up study of 49 patients (observational period, 30.7 ± 32.0 months), multivariate analysis demonstrated that CysC-based estimated GFR (eGFRcys), along with albumin, Child-Pugh grade and presence of hepatocellular carcinoma, was independently associated with overall survival (odds ratio, 4.19; 95% confidence interval, 1.44-12.2, P = 0.009). CONCLUSION: These results suggest that eGFRcys could estimate renal function and predict outcome more accurately compared with Cr-based eGFR in cirrhotic patients.

Plasma Lipid Profiling of Three Types of Drug-Induced Liver Injury in Japanese Patients: A Preliminary Study.

Drug-induced liver injury (DILI) is a major adverse event caused by drug treatment, which can be categorized into three types: hepatocellular, mixed, and cholestatic. Although nearly every class of drugs can cause DILI, an overall understanding of lipid profiles in DILI patients is lacking. We used lipidomics to analyze the plasma lipid profiles of patients to understand their hepatic pathophysiology and identify DILI biomarkers. We identified 463 lipids and compared their levels between the acute and recovery phases of the three types of DILI patients. Mixed and cholestatic types demonstrated specific plasma lipid alterations between the phases, but the hepatocellular type did not. Moreover, as specific indicators of mixed-type DILI, levels of several ceramides increased in the acute phase, while those of arachidonic acid-containing ether-linked phosphoglycerolipids decreased. In contrast, as specific indicators of cholestatic-type DILI, levels of palmitic acid-containing saturated or monounsaturated phosphatidylcholines increased in the acute phase, while those of arachidonic acid- or docosahexaenoic acid-containing ether-linked phosphoglycerolipids and phosphatidylinositols decreased. We also identified lipids with a relatively high capacity to discriminate the acute phase from the recovery phase and healthy subjects. These findings may help with understanding the pathophysiology of different DILI types and identify candidate biomarkers.

The clinical significance of IgA antimitochondrial antibodies in sera and saliva in primary biliary cirrhosis.

It still remains unclear how antimitochondrial autoantibodies (AMA) are involved with immunopathogenesis of primary biliary cirrhosis (PBC). We have suggested the potential role of IgA-AMA in damage to epithelial cells in PBC. In the current study, we investigated whether IgA-AMA were detectable in sera and saliva of PBC patients, to examine the association between detectable IgA-type autoantibodies in sera or saliva and progression of liver diseases. Fifty-three patients with PBC were enrolled, and IgA-AMA in sera and saliva were sought by immunoblotting using pork heart mitochondria as antigens. The progression of PBC was determined as Scheuer's classification consisting of four histological stages. We found IgA-AMA, IgA-anti-PDC-E2, and IgA-anti-E3BP in 43/53 (81%), 37/53 (70%), and 35/53 (66%) sera of patients with PBC, but none of controls. The progression of PBC was statistically associated with presence of IgA-anti-PDC-E2 (P = 0.0124), but neither with IgA-AMA (P = 0.1296) nor anti-IgA-E3BP (P = 0.5973). In saliva, detectable IgA-AMA, IgA-anti-PDC-E2, and IgA-anti-E3BP were noted in 12/26 (46%), 6/26 (23%), and 11/26 (42%), respectively. Detection of IgA-anti-PDC-E2 was strongly associated with progression of PBC (P = 0.0002), whereas detection of IgA-AMA and IgA-anti-E3BP were not associated (P = 0.2145 and P = 0.5118). The current findings suggest that the presence of IgA-anti-PDC-E2 in sera or saliva might be associated with progression of PBC, although a prospective study with PBC patients with detectable IgA-anti-PDC-E2 at early stages will be required to conclude the contribution of IgA-anti-PDC-E2 to the progression of PBC.

Gilbert Syndrome with Concomitant Hereditary Spherocytosis Presenting with Moderate Unconjugated Hyperbilirubinemia.

We experienced a case of a 19-year-old man with Gilbert syndrome with concomitant hereditary spherocytosis. The patient presented with moderate unconjugated hyperbilirubinemia, and inherited etiology was strongly suspected. The diagnosis of Gilbert syndrome was confirmed by the genetic analysis of the UGT1A1 gene, demonstrating UGT1A1*28 and compound heterozygote UGT1A1*6. In addition, since the laboratory findings and imaging studies revealed lysemia as well as gallstone and splenomegaly, a diagnosis of hereditary spherocytosis was made as a comorbidity. Both Gilbert syndrome and hereditary spherocytosis are hereditary diseases with a high frequency, and the hyperbilirubinemia may be exacerbated when these two diseases are concomitant.

Endoscopic removal of a denture with clasps impacted in the ileocecum.

We report a case of endoscopic removal of a denture with clasps impacted in the ileocecum. The patient was a 63-year-old man hospitalized at another center with aspiration pneumonia. He had a history of cerebral bleeding, inflicted permanent damage with left hemiplegia, and dysphagia. Abdominal radiography for localization of a catheter in the femoral vein revealed a denture in the right lower quadrant of the abdomen. He was subclinical and could not recall when he might have swallowed the denture. The patient was brought by ambulance to our institution. Computed tomography showed a foreign body with the density of metal in the ileocecum without any severe complications such as obstruction or perforation. Following intestinal lavage from a nasogastric tube, we performed colonoscopy and successfully retrieved the denture. The patient showed no complications associated with endoscopic therapy and returned to the previous hospital 3 days after endoscopic removal of the denture.

Autoimmune neutropenia due to antineutrophil antibodies in a patient with primary sclerosing cholangitis.

Autoimmune neutropenia (AIN) is defined as a decrease in the circulating absolute neutrophil count (ANC) to less than 1500/µl caused by serum antineutrophil antibodies. Secondary AIN is associated with various autoimmune diseases. Herein we present the case of a patient with primary sclerosing cholangitis (PSC) who developed secondary AIN. A 19-year-old man was admitted due to liver injury, and a diagnosis of PSC was established by cholangiogram and liver biopsy. Severe neutropenia, with the ANC down to 130/µl, developed during his hospital course. No medications had been given at that time and bone marrow aspiration revealed no abnormality. Therefore we suspected secondary AIN as a causative etiology and examined whether antineutrophil antibodies were detectable in the patient's sera by flow cytometric analysis of the granulocyte indirect immunofluorescence test. We found that antineutrophil antibody was strongly positive on admission, and the titer decreased along with recovery from neutropenia. This is the first reported case of a patient with PSC who developed AIN, with detection of serum antineutrophil antibodies.

Carcinosarcoma of the liver.

Herein we present a 73-year-old man with primary carcinosarcoma of the liver, a rare malignant tumor of the liver. The case was followed up due to HBV-related liver cirrhosis. Regular check-up by ultrasound demonstrated a hyperechoic tumor in the left lobe of the liver, and he was referred and admitted to our hospital. Dynamic CT studies revealed a mostly hypoenhancing hepatic mass with a peripheral ring enhancement. Surgical resection was performed, and the resected tumor was macroscopically a simple nodular type, 3 cm in diameter, with a dense fibrous capsule. Microscopically, undifferentiated cells were dominant in the tumor, while moderately differentiated hepatocellular carcinoma (HCC) were also observed. A transitional zone was noted between the undifferentiated tumor and HCC. Tumor tissue with adenocarcinoma, osteosarcoma and chondrosarcoma were also detected. Immunohistochemical studies demonstrated that tumor cells were HepPar 1 positive in hepatocellular carcinoma, and CK19 and partly CK7 positive in adenocarcinoma. Moreover, CD56, chromogranin A and c-kit were occasionally positive in undifferentiated tumor cells. The diagnosis of carcinosarcoma was made based on the concomitant presence of HCC and sarcomatous components, yet it is noteworthy that various types of tumor cells were observed.

Gene expression profiling in whole liver of bile duct ligated rats: VEGF-A expression is up-regulated in hepatocytes adjacent to the portal tracts.

BACKGROUND AND AIM: It would be of clinical importance to clarify molecular mechanisms of cholangiocytes proliferation for the treatment of intractable cholestatic diseases. The aim of this study was to elucidate gene expression profiling in the whole liver of bile duct ligated (BDL) rats using microarray analysis. In addition, the localization and time course of up-regulated expression of vascular endothelial growth factor (VEGF) was investigated. METHODS: Male Sprague-Dawley rats were used. The whole liver was removed from BDL and sham-operated rats at day 2 after the procedure, and microarray analysis was performed using an array on which 3757 rat cDNA clones spotted. The up-regulation of VEGF expression was investigated by RT-PCR using livers at day 1, 2, 4 and 7, and immunoblotting and immunohistochemistry at day 2. RESULTS: Marked proliferation of bile ducts was observed in livers of BDL rats. By microarray analysis, 38 up-regulated and 17 down-regulated transcripts were detected in whole liver of the BDL rat. The expression of VEGF-A was significantly elevated in the BDL rats at day 2; the VEGF-A/GAPDH ratio was 4.030 +/- 2.493 in BDL rats and 1.159 +/- 0.125 in sham-operated rats (P = 0.0330). The up-regulation of VEGF-A expression was maximal at day 2. Immunoblotting also demonstrated up-regulated expression of VEGF-A at the protein level. Immunostaining of VEGF revealed that the expression was evident in hepatocytes adjacent to the portal tracts, and scarcely observed in hepatocytes at the centrilobular area or cholangiocytes. CONCLUSION: Gene expression profiling in the whole liver of the BDL rats revealed 38 up-regulated and 17 down-regulated transcripts. In addition, the up-regulated expression of VEGF was mainly observed in hepatocytes surrounding to the portal tracts.

Liver injury induced by levothyroxine in a patient with primary hypothyroidism.

We report a patient with primary hypothyroidism, who developed hepatocellular injury due to levothyroxine, synthetic thyroxine. A 63-year-old male was admitted to our hospital due to elevation of liver enzymes. The patient was diagnosed as having hypothyroidism and had been treated with levothyroxine for almost two months until admission. Drug-induced liver injury induced due to levothyroxine was suspected and liver enzymes were rapidly decreased after discontinuation of levothyroxine and dried thyroid powder, also containing thyroxine. Synthetic triiodothyronine, the deiodinated form of levothyroxine was administered instead, and was well tolerated by the patient. The drug-induced lymphocyte stimulation test (DLST) using levothyroxine was negative. Since triiodothyronine which structurally resembles levothyroxine did not cause liver injury, and DLST using levothyroxine was negative, it is unlikely that levothyroxine itself was targeted by the immune system. Rather, we assume that the complex of levothyroxine as the hapten and liver-related macromolecules in the body as the carrier might have acquired antigenicity in this patient and subsequently resulted in liver injury.

Comparison of urinary excretion of pravastatin and temocapril in bile duct-ligated rats and Eisai hyperbilirubinemic rats (EHBR).

BACKGROUND/PURPOSE: In patients with complete bile duct obstruction, the only pathway for the elimination of cholephilic compounds is through the urine. Although changes in various transporters in the liver and kidney in cholestasis have been elucidated, little is known about how effectively the elimination of these compounds is compensated for by urinary excretion. METHODS: In the present study, the urinary excretion of pravastatin and temocapril was studied in bile-duct-ligated rats (BDLR) for 3 days and in Eisai hyperbilirubinemic rats (EHBR). After urinary bladder cannulation, radiolabeled pravastatin and temocapril were injected intravenously. Urine samples were collected every 1 h for 4 h, and the radioactivity was counted. RESULTS: Urinary excretion of pravastatin was markedly increased in BDLR (85.9% of the dose after 4 h) and moderately increased in EHBR (35.9% of the dose after 4 h) compared with that in control rats (5.5% of the dose after 4 h). Similar but less prominent differences were observed with temocapril after it was administered (50.7%, 38.2%, and 22.0% of the dose after 4 h in BDLR, EHBR, and the controls, respectively). CONCLUSIONS: The absence of biliary excretion of anionic drugs was compensated for by urinary excretion in BDLR and EHBR, and the compensation was more efficient with pravastatin than with temocapril. In patients with complete bile duct obstruction, the only pathway for the elimination of cholephilic compounds is through the urine. Although changes in various transporters in the liver and kidney in cholestasis have been elucidated, little is known about how effectively the elimination of these compounds is compensated for by urinary excretion.