Antiviral Therapy for Patients Chronically Infected with Hepatis C Virus at Tokai University Hospital.

OBJECTIVE: Hepatitis C virus (HCV) was identified in 1989. In 2020, three decades after HCV identification, three researchers won the Nobel Prize in Physiology or Medicine for the discovery of this virus. In 1992, three years after the discovery, interferon (IFN) was launched as the first anti-HCV therapy in Japan; however, the efficacy of IFN therapy was far from acceptable due to severe adverse effects. The advent of IFN-free direct-acting antivirals (DAAs) in 2014 dramatically improved the outcomes of antiviral treatment without serious adverse effects. In this study, we aimed to summarize anti-HCV therapy at the Tokai University Hospital. METHODS: We identified patients who underwent anti-HCV therapy by searching medical records from January 1992 to December 2020, analyzed their background, and compared safety and efficacy among treatments. RESULTS: A total of 1777 treatments were given to 1299 patients. The sustained virologic response rate has dramatically increased over the past 30 years, with only 7% for IFN monotherapy and 95% or higher for recent IFN-free DAA therapies. CONCLUSIONS: We documented the results of anti-HCV therapy at the Tokai University Hospital. In the 30 years since the discovery of HCV, surprisingly successful progress has been accomplished in the anti-HCV treatment.

Mutations in a ß-group of solute carrier gene are responsible for egg and eye coloration of the brown egg 4 (b-4) mutant in the silkworm, Bombyx mori.

The brown egg 4 (b-4) is a recessive mutant in the silkworm (Bombyx mori), whose egg and adult compound eyes exhibit a reddish-brown color instead of normal purple and black, respectively. By double digest restriction-site associated DNA sequencing (ddRAD-seq) analysis, we narrowed down a region linked to the b-4 phenotype to approximately 1.1 Mb that contains 69 predicted gene models. RNA-seq analysis in a b-4 strain indicated that one of the candidate genes had a different transcription start site, which generates a short open reading frame. We also found that exon skipping was induced in the same gene due to an insertion of a transposable element in other two b-4 mutant strains. This gene encoded a putative amino acid transporter that belongs to the ß-group of solute carrier (SLC) family and is orthologous to Drosophila eye color mutant gene, mahogany (mah). Accordingly, we named this gene Bmmah. We performed CRISPR/Cas9-mediated gene knockout targeting Bmmah. Several adult moths in generation 0 (G0) had totally or partially reddish-brown compound eyes. We also established three Bmmah knockout strains, all of which exhibit reddish-brown eggs and adult compound eyes. Furthermore, eggs from complementation crosses between the b-4 mutants and the Bmmah knockout mutants also exhibited reddish-brown color, which was similar to the b-4 mutant eggs, indicating that Bmmah is responsible for the b-4 phenotypes.

Live-bearing without placenta: Physical estimation indicates the high oxygen-supplying ability of white shark uterus to the embryo.

One of the mysteries of shark aplacental viviparity is the ability of the embryos to acquire oxygen from their mothers without a placental connection. It has been assumed that embryonic respiration in aplacental viviparous shark depends on oxygen from the uterine wall, although this hypothesis has not been confirmed quantitatively. Morphological observations of the uterine wall of white shark (Carcharodon carcharias) provided the first quantitative evidence to support the ability of the uterus to supply ample oxygen to the embryo of viviparous elasmobranchs. The uterine surface of the white shark is characterized by (1) uterine lamellae that develop perpendicular to the uterine wall, (2) uterine lamellae folded in an accordion-like fashion, and (3) numerous micro-ridges on the lamellar surface. These modifications result in increased uterine surface are to up to 56 folds compared to the uterus with a smooth surface. Histological observations revealed that the diffusion barrier of the uterine wall is approximately 12 µm. By using these values, the oxygen-diffusion capacity of 1 cm2 of the uterine wall of white shark was estimated to be 63.6 nmol·min-1·torr-1. This value is 250-400 times greater than that observed in other aplacental viviparous sharks (Squalus spp.) and is comparable with that of fish gills.

Effects of ethanol and acetaldehyde load on erythrocyte deformability in healthy subjects and patients with liver cirrhosis.

Alcohol intake leads to the distribution of alcohol and its metabolite, acetaldehyde throughout the blood and organs. Hepatic cirrhosis is associated with abnormal red blood cell morphology and function, particularly impaired red blood cell deformability. To investigate the effect of drinking on red blood cells in patients with hepatic cirrhosis, erythrocyte deformability was evaluated in response to alcohol and acetaldehyde tolerance. Erythrocyte deformability in 10 healthy and 15 cirrhotic subjects was examined by filterability of the red blood cells. Erythrocyte deformability decreased markedly in the cirrhosis group compared with the healthy group (p < 0.05). No significant change in erythrocyte deformability was observed in healthy or cirrhotic subjects due to ethanol 100 mM tolerance. Acetaldehyde tolerance elicited a significant decrease in erythrocyte deformability at 2 mM in the cirrhosis group (p < 0.05). Alcohol consumption in cirrhotic patients was suggested to worsen erythrocyte deformability and red blood cell function. Decreased erythrocyte deformability worsens microcirculation in the liver, resulting in more severe hepatic dysfunction.

Growth of the Coconut Crab, Birgus latro, at Its Northernmost Range Estimated from Mark-Recapture Using Individual Identification Based on Carapace Grooving Patterns.

Although populations of the coconut crab, Birgus latro, have declined in the tropical Indo-Pacific region, insufficient knowledge exists for the management of this species. We investigated the growth of the northernmost coconut crab population, located at Ocean Expo Park, Okinawa, southern Japan, using a mark-recapture method based on the identification of individual carapace grooving patterns. Of the 485 crabs photographed (264 males, 221 females; 14.3-68.8 mm thoracic length [ThL]), 64 males and 62 females were recaptured (recapture rate 26%). The liberty period ranged from two to 2384 days. The annual data indicated that most crabs molted during winter, except for juveniles and crabs near the maximum size. Using the GROTAG program, the asymptotic ThL (L∞) was estimated as 80.72 and 49.89 mm for males and females, respectively. The Brody growth coefficient (K) was 0.063 for both sexes. The growth curves from these parameters showed that males grew larger than females because of a difference in growth speed. Longevity was estimated at approximately 50 years for both sexes. The growth data obtained in the present study, which are the most precise gathered for the coconut crab to date, can be compared with the results of studies performed in other regions.

Networked-cage microcrystals for evaluation of host-guest interactions.

We have developed a new synthetic protocol for the preparation of a microcrystalline powder (median size: X50 = 25 µm) of networked M6L4 cages 1a for the stationary phase of an affinity column on a greater than 50 g scale. Analogously to large single crystals 1b (X50 ≈ 0.5 mm), microcrystals 1a accommodate guest molecules tetrathiafulvalene (TTF) and fullerene (C60) at up to 32 and 35 wt %, respectively. Importantly, the host-guest interactions within networked cages could be evaluated in terms of the retention time from HPLC analysis by using microcrystals 1a as the stationary phase. In this way, favorable guests for networked cages 1 and even solution M6L4 cage 2 could easily be assessed by HPLC.

Eight-week oral administration of meloxicam, a non-steroidal anti-inflammatory drug, prevents dose reduction of pegylated interferon alpha-2a in the treatment of chronic hepatitis C.

AIM: We conducted a trial to evaluate whether eight-week oral administration of meloxicam, a non-steroidal anti-inflammatory drug, would decrease the rate of the patients who required dose reduction of pegylated interferon alpha-2a in the treatment of chronic hepatitis C. METHODS: Sixty patients given weekly subcutaneous administration of pegylated interferon alpha-2a at a dose of 180 mug for 48 weeks were allocated into the meloxicam group (n = 22) and the control group (n = 38) before interferon treatment. Meloxicam was given orally at a dose of 10 mg once a day for eight weeks from the start of interferon treatment. RESULTS: The cumulative rate of dose-reduction-free patients was significantly higher in the meloxicam group (P < 0.05). Until week eight, 44.7% of the control group and 9.1% of the meloxicam group required dose reduction. Dose was modified by neutropenia in 31.6% and 18.2% of the control and meloxicam groups, respectively. Meloxicam relieved a declineof neutrophil count within the first eight weeks from 54.2% to 44.2% (P < 0.05). Multivariate analysis revealed that greater pretreatment neutrophil count and the use of meloxicam were independent factors associated with avoiding dose reduction. Sustained virological response was obtained in 52.6% of the patients. The multivariate logistic analysis revealed that viral serotype and viral load were the only independent factors associated with sustained virological response. CONCLUSION: Eight-week administration of meloxicam prevented dose reduction of pegylated interferon by relieving a decline of neutrophil count in the treatment of chronic hepatitis C.

An endothelin A receptor antagonist induces dilatation of sinusoidal endothelial fenestrae: implications for endothelin-1 in hepatic microcirculation.

BACKGROUND: Sinusoidal endothelial fenestrae (SEF) regulate the sinusoidal circulation by altering their diameter and number. This study documented the effects of endothelin (ET) receptor antagonists on SEF and hepatic microcirculation. METHODS: The portal pressure and hepatic tissue blood flow were measured with a hydromanometer and a laser Doppler blood flow meter, respectively. BQ-123 (ET(A) receptor antagonist) or BQ-788 (ET(B) receptor antagonist) was continuously infused into normal rats at the rate of 10 nmol/min for 10 min. The sinusoids were observed at 60 min after the infusion by scanning electron microscopy. The localization of ET-1 and ET(A) and ET(B) receptors was examined by the indirect immunoperoxidase method. RESULTS: When BQ-123 was infused, the portal pressure gradually decreased with time, and it showed a significant reduction compared with the control groups. On the other hand, a decrease in portal pressure was not evident in the BQ-788-infused groups. Hepatic tissue blood flow was maintained at the value prior to the infusion in both groups. BQ-123 also caused a marked dilatation of the SEF. The diameters of the SEF after BQ-123 infusion were almost three times those of normal SEF. ET-1 was evenly present along the sinusoidal walls, and the reaction products of the ET(A) receptors were recognized along the portal vein and in the sinusoidal cells, that is, the hepatic stellate cells and endothelial cells. CONCLUSIONS: Action of ET-1 via the ET(A) receptors may regulate the size of SEF in addition to hepatic microcirculation.

Clinical and pathological features of a prolonged type of acute intrahepatic cholestasis.

AIM: We examined the clinical and pathological features of drug-induced acute intrahepatic cholestasis (AIC) to elucidate the pathogenesis of prolonged cases. METHODS: Twenty-six cases of drug-induced AIC were divided into prolonged and non-prolonged groups. Serum bilirubin levels and other biochemical data were compared between the two groups. Biopsy liver specimens were examined by light and electron microscopy. The localization of multidrug resistance protein 2 (MRP2) was immunohistochemically assessed by the Envision technique. RESULTS: The causative drugs of four prolonged cases were found to be tiopronin, chlorpromazine and diclofenac. Two of the patients either died or underwent liver transplantation. The maximal total bilirubin levels (35.2 +/-> 13.8 mg/dL) were significantly higher and a half-life of total bilirubin (78.8 +/-> 69.6 days) was markedly longer in the prolonged cases, in comparison to the non-prolonged cases (16.8 +/-> 8.1 mg/dL, 22.1 +/-> 12.7 days, respectively). The liverbiopsy specimens revealed canalicular cholestasis and a slight degree of lobular inflammation. In the prolonged cases, liver cell injury and cholestasis was marked, and the interlobular bile ducts disappeared in the portal triads. The reaction products of MRP2, recognized on the bile canaliculi in a control liver, were weakened and found in the pericanalicular vesicles in AIC. CONCLUSION: These results indicated disturbances in the canalicular bilirubin transport through MRP2 in the prolonged cases, resulting from severe cholestasis, liver cell injury and vanishing bile ducts. The histological findings of the liver at the acute icteric phase may be important to understand the pathogenesis and to predict the prognosis in AIC.

Clinical significance of serum matrix metalloproteinases and tissue inhibitors of metalloproteinases in chronic liver disease.

OBJECTIVE: We have attempted to determine serum levels of type IV collagen (IV-C), laminin (LM), prolylhydroxylase (PH), metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in chronic liver disease to elucidate the clinical significance of MMPs and TIMPs in the process of hepatic fibrosis. METHODS: Serum samples were collected from 60 patients with chronic liver disease caused by hepatitis B or C. Serum levels of IV-C, LM, PH, MMP-1, 2 and 3, and TIMP-1 and 2 were measured by a one-step sandwich enzyme immunoassay using monoclonal antibodies. The values were correlated with Histology Activity Index (HAI) scores of liver biopsy specimens. RESULTS: LM and IV-C levels markedly increased in parallel with the progression of the chronic liver disease. The MMP-2 and MMP-3 levels tended to increase in chronic active hepatitis (CAH), and significantly elevated in liver cirrhosis (LC). There was a positive correlation between the IV-C and MMP-2 levels, and the ratio of IV-C to MMP-2 levels was significantly elevated in LC. Both TIMP-1 and TIMP-2 levels were markedly increased in LC. The HAI scores were positively correlated with the serum IV-C and MMP-2 levels. CONCLUSIONS: Serum IV-C and MMP-2 levels may be useful diagnostic markers for hepatic fibrosis, since they increased in parallel with the progression of chronic liver disease. In addition, the imbalances between IV-C, LM, and TIMP-1 and 2 as fibrogenic factors and MMP-2 and 3 as fibrolytic factors may lead to fibrosis in chronic viral liver disease, especially in cirrhosis.

Predictive factors for the response to lamivudine in HBV-infected patients with chronic hepatitis and cirrhosis.

OBJECTIVE: To determine predictors for virological response to lamivudine, a retrospective-cohort study was designed. METHODS: Seventy HBV positive patients who received lamivudine were classified according to virological response into responders and non-responders. Background conditions and normalization and flare-up of hepatitis were compared using student-t test and chi-square test. Logistic regression analysis was performed to determine the effect of explanatory variables, age, sex, ALT, HBV-DNA, hepatic fibrosisi, presence of absence of HBeAg, former IF non-response on the response to lamivudine. RESULTS: There were no difference in gender, age, observed period, ALT level, liver fibrosis, former response to Interferon in background but viral titer and rate of HBeAg (+) was higher in non-responders. Hepatitis normalization rates were not different but flare-up rates were significantly higher in non-responders. Multivariate analysis showed HBeAg is the relevant factor for the response to lamivudine. CONCLUSIONS: The presence of HBeAg was a risk for non-response to lamivudine therapy.

Study of energy metabolism of skeletal muscles in alcoholic liver disease--expired gas analysis during exercise.

BACKGROUND: The effects of chronic alcohol intake on skeletal muscle are clinically observed as muscle cramps with decrease in the amount of muscle. It was clarified by expired gas analysis that acute alcohol load affects disturbed energy metabolism of skeletal muscle. We studied abnormal energy metabolism of skeletal muscles in alcoholic liver diseases using expired gas analysis. METHODS: Subjects of the study were five inpatients with alcoholic liver disease (fatty liver: one case, hepatic fibrosis: one case, liver cirrhosis: three case). Expired gas analysis during exercise was performed using AEROMONITOR. Minute ventilation, oxygen consumption and carbon dioxide output were monitored, and anaerobic threshold (AT) and respiratory compensation point (RCP) were calculated. RESULTS: The anaerobic threshold, which is the limit of the aerobic exercise, was significantly reduced in patients with alcoholic disease (p < 0.01). The respiratory compensation point, which is the limit of the metabolic compensation of intracellular lactic acidosis, was decreased (p < 0.01). CONCLUSIONS: The results of expired gas analysis during exercise indicate that the aerobic energy metabolism of skeletal muscle had been disturbed in alcoholic liver disease. The reduced RCP suggests that the lactate metabolism in skeletal muscle is also disturbed in alcoholic liver disease. Expired gas analysis during exercise allows determination of the amount of exercise required to treat liver diseases through analysis of AT.

[Actual condition of alcohol intake and its effects on lifestyle-related disease in health checkup].

Actual condition of alcohol intake was investigated in forty four thousand one hundred and twenty six individuals who visited the Tokai University Hospital Health Checkup Center from 1989 to 2003. Effects of alcohol intake were also examined in relation to several risk factors for lifestyle-related diseases. The male drinkers who took more than 1 gou of sake per a day were recognized in 53.0% from 1989 to 1991, and decreased to 46.3% from 2001 to 2003. The female drinkers were found in approximately 10%, and remained unchanged during the 15-year survey period. When examined by age, the frequency of habitual drinking among males was 34.4% in the age of 20 years, and then increased to 45% in the 30 years, leading to the peak (54.1%) in the 40 years. In females, the frequency was 27.5% in the age of 20 years, but decreased to 10.9% in the 30 years. The prevalence of systolic hypertension, diastolic hypertension, hyperuricemia, high levels of HbAlc, and hypertriglyceridemia was significantly (P < 0.0001) increased with an increase in alcohol intake. The prevalence of obesity, fatty liver and hyperglycemia at fasting was markedly (P < 0.0001) increased in the drinkers whose intake was more than 2 gou per a day. These findings indicate that habitual drinking may be associated with risk factors for lifestyle-related diseases, such as obesity, fatty liver, hypertension, hypertriglyceridemia and hyperuricemia.

Platelet activation in patients with alcoholic liver disease.

OBJECTIVE: To elucidate the mechanisms of thrombocytopenia in alcoholic liver diseases, we investigated activation status of platelets in patients with alcoholic fatty liver (Al-FL), alcoholic liver cirrhosis (Al-LC) or hepatitis-C liver cirrhosis C (C-LC). METHODS: Platelet activation was evaluated by flow cytometry using monoclonal antibodies against P-selectin (CD62P) and the fibrinogen receptor (PAC-1), both specific for platelet activation, and anti-CD61 antibody for the presence of microparticles (PMP) in seven patients with Al-FL, thirteen patients with Al-LC and, as a non-alcoholic liver disease control, nine patients with C-LC. As a normal control, seventeen healthy subjects without liver dysfunction were also evaluated. RESULTS: Compared with the healthy controls, the platelet count was significantly decreased in patients with alcoholic liver diseases or C-LC. Ten days after discontinuation of alcohol intake, the platelet count was significantly higher in both the Al-FL and Al-LC groups than that measured on admission. There was an inverse correlation between the platelet count and PMP, a marker of platelet activation. The Al-FL, Al-LC and C-LC groups showed significantly higher percentages of platelets positive for CD62P than the healthy controls. The PAC-1 positivity was increased only in the C-LC group. PMP were significantly increased in the Al-FL, Al-LC and C-LC groups compared to that in the healthy group. In the Al-LC group, PMP were significantly decreased 10 days after discontinuation of alcohol intake from that measured on admission. CONCLUSION: Patients with alcoholic liver diseases have increased platelet activation, which may contribute to the occurrence of thrombocytopenia. The formation of PMP might be one of the important factors of thrombocytopenia in alcoholic liver diseases.

Development of quantitative neuropsychological tests for diagnosis of subclinical hepatic encephalopathy in liver cirrhosis patients and establishment of diagnostic criteria-multicenter collaborative study in Japanese.

At present, there are no generally accepted diagnostic criteria or methods for subclinical hepatic encephalopathy (SHE) associated with liver cirrhosis. We therefore developed an easily conducted computer-aided quantitative neuropsychiatric function test system for use in routine medical practice. We established normal values in healthy Japanese subjects and determined differences between healthy persons and liver cirrhosis patients without clinical encephalopathy in a multi-center clinical trial. The test system consists of eight tests: number connection tests A and B, a figure position test, a digit symbol test, a block design test, and reaction time tests A, B and C. The test results were affected by age, but not by gender or facility. No learning effect was noted. The results were therefore reported by 5-year quartile ranges and differences were evaluated between 542 healthy subjects and 292 cirrhotic patients. When the cut-off value was set at the 10th/90th percentile of the results in healthy subjects, the results of each of the 8 tests were abnormal in about 25% of cirrhotic patients, and at least 1 of the 8 tests gave values greater than the 10th/90th percentile cut-off value in 58.2% of the 292 liver cirrhosis patients. SHE patients were thought to be included in these 58.2% of patients. The developed test makes it possible to quantitatively assess neuropsychiatric function, and the results obtained can be used as a basis for the diagnosis of SHE.

Fatal liver failure due to reactivation of lamivudine-resistant HBV mutant.

We report a case of fatal liver failure due to reactivation of lamivudine-resistant HBV. A 53-year-old man was followed since 1998 for HBV-related chronic hepatitis. Serum HBV-DNA was 150 MEq/mL (branched DNA signal amplification assay) and ALT levels fluctuated between 50-200 IU/L with no clinical signs of liver cirrhosis. Lamivudine (100 mg/d) was started in May 2001 and serum HBV-DNA subsequently decreased below undetectable levels. In May 2002, serum HBV-DNA had increased to 410 MEq/mL, along with ALT flare (226 IU/L). The YMDD motif in the DNA polymerase gene had been replaced by YIDD. Lamivudine was continued and ALT spontaneously decreased to the former levels. On Oct 3 the patient presenting with general fatigue, nausea and jaundice was admitted to our hospital. The laboratory data revealed HBV reactivation and liver failure (ALT: 1828 IU/L, total bilirubin: 10 mg/dL, and prothrombin INR: 3.24). For religious reasons, the patient and his family refused blood transfusion, plasma exchange and liver transplantation. The patient died 10 d after admission. The autopsy revealed remarkable liver atrophy.

Problems in serum albumin measurement and clinical significance of albumin microheterogeneity in cirrhotics.

OBJECTIVES: To clarify problems with the determination of serum albumin levels, the definition of hypoalbuminemia, and the implications of microheterogeneity of albumin, serum albumin was measured by using dye-binding methods and the authentic method (immunoassay) in patients with liver cirrhosis and healthy subjects. METHODS: We enrolled 103 patients with liver cirrhosis and 36 healthy subjects. Serum albumin levels were analyzed by immunoassay and the bromcresol green and bromcresol purple methods. Oxidized albumin and glycoalbumin were determined by high-performance liquid chromatography. RESULTS: In cirrhotic patients, serum albumin levels measured by the bromcresol green method was about 0.2 g/dL higher than that by immunoassay. Serum albumin levels measured by the bromcresol purple method also was higher in cirrhotic patients than those measured by immunoassay and varied widely. In addition, extensive variation was found across serum albumin levels determined by the bromcresol green method at individual institutions (five university hospitals) and those determined by immunoassay at a contract laboratory. The percentages of oxidized albumin and glycoalbumin within total serum albumin increased with progression of liver disease. Further, an increase in oxidized albumin led to an increase in the albumin level as measured by the bromcresol purple method. CONCLUSION: These results show that adequate assessment of the pathophysiology and prognosis of patients with liver cirrhosis and the efficacy of treatment is not possible with dye-binding methods for determination of serum albumin. Further, the conventional definition of hypoalbuminemia as a serum albumin level of 3.5 g/dL or lower should be reconsidered, and the clinical implications of qualitative changes in albumin should be investigated in consideration of the microheterogeneity of albumin, such as oxidized albumin and glycoalbumin.

Effect of in vitro interferon-beta administration on hepatitis C virus in peripheral blood mononuclear cells as a predictive marker of clinical response to interferon treatment for chronic hepatitis C.

AIM: To test whether in vitro incubation of peripheral blood mononuclear cells (PBMC) with interferon (IFN) could efficiently decrease hepatitis C virus-RNA (HCV-RNA) amount and to analyze whether this effect was associated with clinical response to IFN. METHODS: Twenty-seven patients with histologically proven chronic hepatitis C were given intravenous administration of 6 million units (MU) IFN-beta daily for 6 weeks followed by three times weekly for 20 weeks. PBMC collected before IFN therapy were incubated with IFN-beta and HCV-RNA in PMBC was semi-quantitatively determined. RESULTS: Twenty-five patients completed IFN therapy. Eight patients (32%) had sustained loss of serum HCV-RNA with normal serum ALT levels after IFN therapy (complete responders). HCV-RNA in PBMC was detected in all patients, whereas it was not detected in PBMC from healthy subjects. In vitro administration of IFN-beta decreased the amount of HCV-RNA in PMBC in 18 patients (72%). Eight of these patients obtained complete response. On the other hand, none of the patients whose HCV-RNA in PBMC did not decrease by IFN-beta was complete responders. Multiple logistic regression analysis revealed that the decrease of HCV-RNA amount in PBMC by IFN-beta was the only independent predictor for complete response (P<0.05). CONCLUSION: The effect of in vitro IFN-beta on HCV in PBMC reflects clinical response and would be taken into account as a predictive marker of IFN therapy for chronic hepatitis C.