HBV with precore and basal core promoter mutations exhibits a high replication phenotype and causes ER stress-mediated cell death in humanized liver chimeric mice.

BACKGROUND AND AIMS: Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . APPROACH AND RESULTS: Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. CONCLUSION: PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.

Comprehensive genomic profiling testing in Japanese castration-resistant prostate cancer patients: results of a single-center retrospective cohort study.

OBJECTIVE: Comprehensive genomic profiling testing using a hybrid-capture next-generation sequencing is commonly used in clinical practice to employ precision medicine in cancer treatment worldwide. In this study, we aimed to analyze the profiles obtained using comprehensive genomic profiling testing that was performed in Japanese castration-resistant prostate cancer patients and to discuss the genetic findings in a real-world setting. METHODS: A total of 60 cases and 57 castration-resistant prostate cancer patients underwent comprehensive genomic profiling testing between 1 January 2021 and 31 December 2022. Four types of comprehensive genomic profiling testing were selected, and clinically significant cancer-specific gene alterations were identified. RESULTS: The median age of patients was 74 years, and the median prostate-specific antigen value at the time of submission was 18.6 ng/ml. Fifty-seven (95%) of 60 cases were metastatic castration-resistant prostate cancers, and 3 cases (5%) were non-metastatic. Among all genetic alterations, androgen-receptor alteration was the most frequently detected in 17 cases (28.3%), followed by 15 cases of TP53 (25.0%), 14 cases of CDK12 (23.3%), 10 cases of phosphatase and tensin homolog (16.7%) and 9 cases of ATM (15.0%) mutations. A total of 13 patients (21.7%) received systemic therapy according to the comprehensive genomic profiling testing results. Overall, the survival rate was significantly greater in the group treated through systemic therapy based on comprehensive genomic profiling testing compared with the group without new therapeutic treatment (P = 0.041). CONCLUSIONS: Comprehensive genomic profiling testing is recommended in castration-resistant prostate cancer patients identified as resistant to standard therapy as this can provide a new therapeutic option.

Hepatitis B Virus (HBV) Upregulates TRAIL-R3 Expression in Hepatocytes Resulting in Escape From Both Cell Apoptosis and Suppression of HBV Replication by TRAIL.

BACKGROUND: Hepatitis B virus (HBV) evades host immunity by regulating intracellular signals. To clarify this immune tolerance mechanism, we performed gene expression analysis using HBV-infected humanized mouse livers. METHODS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 3 (TRAIL-R3) was significantly upregulated in livers of HBV-infected human hepatocyte transplanted mice by cDNA microarray and next-generation sequencing. We analyzed the significance of TRAIL-R3 upregulation in HBV infection using human hepatocyte transplanted mice and HepG2 cell lines. RESULTS: TRAIL-R3 induction by HBV infection was verified by in vitro and in vivo HBV replication models, and induction was inhibited by antiviral nucleot(s)ide analogue treatment. TRAIL-R3 transcription was regulated by the TRAIL-R3 promoter at -969 to -479 nucleotides upstream from the transcription start site, and by hepatitis B x (HBx) via activation of nuclear factor-κB (NF-κB) signal. TRAIL not only induced cell apoptosis but also inhibited HBV replication. TRAIL-R3 upregulation could inhibit both TRAIL-dependent apoptosis in HBV-infected hepatocytes and TRAIL-mediated suppression of HBV replication. CONCLUSIONS: These results suggest a mechanism by which HBV persists by escaping host immunity through upregulation of TRAIL-R3. Development of novel drugs to inhibit this escape system might lead to complete HBV elimination from human hepatocytes.

Impact and usefulness of the transition to the new MAFLD classification for non-B, non-C HCC: a retrospective cohort study.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) represents a new classification system for fatty liver disease. In this study, we investigated the clinical characteristics of patients with MAFLD-hepatocellular carcinoma (HCC) in comparison with those with nonalcoholic fatty liver disease (NAFLD) and considered the validity and challenges of the new criteria. METHODS: This study included 237 untreated non-B, non-C HCC patients with hepatic steatosis. We examined the profile and laboratory findings of patients with MAFLD-HCC and NAFLD-HCC. We also classified MAFLD-HCC patients according to the factors on which the diagnosis was based and compared their clinical characteristics. RESULTS: A total of 222 (94%) and 101 (43%) patients were diagnosed with MAFLD and NAFLD, respectively. MAFLD-HCC patients were more likely to be male than NAFLD-HCC, but there were no significant differences in metabolic indices, noninvasive liver fibrosis score or HCC status. In a study of MAFLD-HCC patients by diagnostic factor, those with overweight only were younger and had advanced liver fibrosis histologically, and when limited to patients younger than 70 years, the majority were overweight. Redefinition of overweight as BMI ≥ 25 reduced the number of MAFLD-HCC patients by only 5, from 222 to 217. CONCLUSIONS: MAFLD accounted for the majority of non-B, non-C HCC cases with hepatic steatosis. Examination of additional cases and revision of the detailed criteria is needed so that it can be used to efficiently select patients with fatty liver who are at high risk of developing HCC.

Correlation between serum pro-inflammatory cytokine levels and the prognosis of the patients with acute liver failure.

BACKGROUND AND AIM: The prognosis of acute liver failure (ALF) remains poor, and liver transplantation is an alternative treatment option. Assessing the prognosis of ALF is important in determining treatment strategies. Here, we investigated clinical factors including serum pro-inflammatory cytokine levels that are associated with the prognosis of ALF. METHODS: Sixty-six patients who developed ALF were enrolled in this study. Serum concentrations of 12 pro-inflammatory cytokines were measured on admission. The prognosis and factors associated with survival and development of hepatic coma were analyzed. RESULTS: Of 66 patients, 4 patients underwent liver transplantation, and 49 patients were rescued without liver transplantation, while the remaining 13 patients died. Serum concentrations of interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-13, TNF, IFN -γ, IP-10, and G-CSF were significantly elevated in ALF patients. IL-4 and IL-8 levels were higher in patients who underwent liver transplantation or died than in rescued patients. Multivariable analysis identified age ≥ 55 years and IL-4 ≥ 1.2 pg/mL on admission as independent factors for mortality. Serum IL-8 levels were higher in patients with hepatic coma, and prothrombin-international normalized ratio ≥ 3.5 and IL-8 ≥ 77.2 pg/mL on admission were associated with development of hepatic coma after admission. CONCLUSION: Serum levels of several pro-inflammatory cytokines were elevated in ALF patients. IL-4 and IL-8 were correlated with survival and development of hepatic coma after admission, respectively. Measurement of serum pro-inflammatory cytokines seems to be useful for the management of ALF.

Untying relaxed circular DNA of hepatitis B virus by polymerase reaction provides a new option for accurate quantification and visualization of covalently closed circular DNA.

Hepatitis B virus (HBV) is a small hepatotropic DNA virus that replicates via an RNA intermediate. After entry, the virus capsid carries relaxed circular DNA (rcDNA) into the nucleus where the viral genome is converted into covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. To monitor cccDNA levels, preprocessing methods to eliminate rcDNA have emerged for quantitative PCR, although Southern blotting is still the only method to discriminate cccDNA from other DNA intermediates. In this study, we have established a robust method for untying mature rcDNA into double stranded linear DNA using specific polymerases. Untying rcDNA provides not only an alternative method for cccDNA quantification but also a sensitive method for visualizing cccDNA. We combined this method with plasmid-safe DNase and T5 exonuclease preprocessing and revealed that accurate quantification requires cccDNA digestion by a restriction enzyme because heat stability of cccDNA increases after T5 exonuclease treatment. In digital PCR using duplex TaqMan probes, fewer than 1000 copies of cccDNA were successfully visualized as double positive spots that were distinct from single positives derived from untied rcDNA. This method was further applied to the infection model of primary hepatocytes treated with nucleoside analogues and a core protein allosteric modulator to monitor cccDNA levels. Relative quantification of cccDNA by human genome copy demonstrated the possibility of precise evaluation of cccDNA level per nucleus. These results clearly indicate that the sequential reaction from untying rcDNA is useful to investigate cccDNA fates in a small fraction of nuclei.

Prevalence of immune-related adverse events and anti-tumor efficacy following immune checkpoint inhibitor therapy in Japanese patients with various solid tumors.

BACKGROUND: While immune checkpoint inhibitors (ICIs) occasionally cause immune-related adverse events (irAEs) in various organs, the prevalence of irAEs and potential risk factors have not been clarified. We identified irAE predictive factors and examined the relationship between the effect of ICIs and irAEs for patients with malignancies. METHODS: A total of 533 cases treated with ICIs, including programmed death 1 (PD-1), PD-ligand 1 (PD-L1), and cytotoxic T-lymphocyte antigen 4 (CTLA-4), for various malignancies were included retrospectively. We recorded irAEs from medical records and graded them using the Common Terminology Criteria for Adverse Events version 5. Prevalence and predictive factors associated with immune-related liver injury and the relationship between irAE and treatment response were analyzed. RESULTS: During a median of 10 (1-103) cycles with a median follow-up after several ICI initiations of 384 (21-1715) days, irAEs with all grades and with grade ≥ 3 developed in 144 (27.0%) and 57 (10.7%) cases. Cumulative irAE development rates were 21.9, 33.5, and 43.0% in all grades and 8.8, 14.9, and 20.7% in grade ≥ 3 at 5, 10, and 20 cycles, respectively. Patients who received anti-CTLA4 therapy were more likely to develop irAEs compared to those who received anti-PD-1 or anti-PD-L1 monotherapy. Liver injury was the most common irAE. Multivariate analysis identified the combination of PD-1 and anti-CTL-4 antibodies (hazard ratio [HR], 17.04; P < 0.0001) and baseline eosinophil count ≥130/µL (HR, 3.01 for < 130; P = 0.012) as independent risk factors for the incidence of immune-related liver injury with grade ≥ 2. Patients who developed irAEs had a higher disease control rate (P < 0.0001) and an increased overall survival rate compared to those without irAEs (P < 0.0001). CONCLUSION: Combination therapy with anti-PD-1 and anti-CTL-4 antibodies resulted in higher a frequency of irAEs. Baseline absolute eosinophil count was found to be a predictive factor for immune-related liver injury. Occurrence of irAEs may be associated with higher efficacy of ICI treatment and longer survival among patients who receive ICI therapy.

Road to elimination of HCV: Clinical challenges in HCV management.

Since its discovery in 1989, the road to a cure for hepatitis C virus (HCV) has been slow, but most patients can now expect to achieve a sustained virological response (SVR). With direct-acting antiviral (DAA) combination therapies such as glecaprevir/pibrentasvir and velpatasvir/sofosbuvir, 98% of patients successfully eradicate the virus, even if previous treatments failed or if resistance-associated substitutions (RASs) are present. Adverse events are rare or mild, and patients with compensated cirrhosis and other co-morbidities are often eligible for treatment. However, a small number of patients fail to eradicate the virus even after retreatment. The cause of failure is mainly due to emergence of NS5A P32 deletion mutants after initial DAA therapy in genotype 1b patients, although the reason is unknown for some patients. Alternative therapies that do not rely on NS5A inhibitors, such as sofosbuvir plus ribavirin, can be attempted in these patients. While scaled-up treatment efforts present a challenge, another problem is that many carriers are unaware of their infection. Long-term damage to the liver becomes irreversible, and patients who are not diagnosed in time can develop liver cancer or liver failure even after eliminating the virus. The long-term costs of treatment of advanced liver disease in undiagnosed patients relative to the immediate costs of DAA therapy should be considered. As no vaccine is yet available, eventual elimination of the virus requires identifying and treating undiagnosed cases and screening of high-risk populations such as injection drug users and men who have sex with men and female sex workers.

Underestimation of impaired glucose tolerance and usefulness of a continuous glucose monitoring system in chronic liver disease.

BACKGROUND AND AIM: The prevalence of glucose intolerance in chronic liver disease patients is high, but glucose intolerance may be overlooked in a single blood test. The purpose of this study is to evaluate blood glucose variability in patients with chronic liver disease by a continuous glucose monitoring system (CGMS) and to examine the discrepancy between hemoglobin A1c (HbA1c) levels estimated from average blood glucose levels and HbA1c. METHODS: This study included 335 patients with chronic liver disease associated with glucose intolerance. A fasting blood test and 72-h CGMS were performed. The estimated HbA1c was calculated from the average blood glucose level, and the correlation between hepatic functional reserve and blood glucose-related parameters was analyzed. From the obtained data, we created a new formula to calculate HbA1c without using CGMS. RESULTS: As hepatic functional reserve decreased, average blood glucose and insulin resistance increased while HbA1c decreased (P < 0.0001). The discrepancy between the estimated HbA1c calculated from the mean blood glucose level and the serum HbA1c (ΔHbA1c) increased as the liver reserve decreased. Using multiple regression analysis, a formula based on fasting blood glucose, HbA1c, body mass index, albumin, and liver function was constructed, and its validity was demonstrated in a study using a different control group. CONCLUSIONS: Hemoglobin A1c may be underestimated because of decreased hepatic functional reserve. CGMS was useful in assessing accurate glycemic control of blood glucose and in detecting postprandial hyperglycemia and nocturnal hypoglycemia. Patients with chronic hepatic impairment should be corrected for hepatic functional reserve before glycemic control.

Investigating a psychological model of mental conditions and coping during the COVID-19 pandemic driven by participatory methods.

BACKGROUND: There is evidence of increased mental health problems during the early stages of the COVID-19 pandemic. We aimed to identify the factors that put certain groups of people at greater risk of mental health problems. METHODS: We took a participatory approach, involving people with lived experience of mental health problems and/or carers, to generate a set of risk factors and potential moderators of the effects of COVID on mental health. An online cross-sectional survey was completed by 1464 United Kingdom residents between 24th April and 27th June 2020. The survey had questions on whether respondents were existing mental health service users and or carers, level of depression (PHQ9) and anxiety (GAD7), demographics, threat and coping appraisals, perceived resilience (BRS), and specific coping behaviours (validated as part of this study). The relationship between responses and coping strategies was measured using tetrachoric correlations. Structural equation modelling was used to test the model. RESULTS: A model significantly fit our data (rel χ2 = 2.05, RMSEA = 0.029 95%, CI (0.016, 0.042), CFI = 0.99, TLI = 0.98, SRMR = 0.014). Age and coping appraisal predicted anxiety and depression. Whereas, threat appraisal and ethnicity only predicted anxiety, and resilience only predicted depression. Additionally, specific coping behaviours predicted anxiety and depression, with overlap on distraction. CONCLUSIONS: Some, but not all, risk factors significantly predict anxiety and depression. While there is a relationship between anxiety and depression, different factors may put people at greater risk of one or the other during the pandemic.

The expression of pregnancy-associated plasma protein-A (PAPP-A) in human blastocoel fluid-conditioned media: a proof of concept study.

PURPOSE: The aim of this study was to determine if pregnancy-associated plasma protein-A (PAPP-A), typically measured in maternal serum and a potential predictor of adverse maternal and fetal outcomes such as spontaneous miscarriage, pre-eclampsia, and stillbirth, is expressed in blastocoel fluid-conditioned media (BFCM) at the embryonic blastocyst stage. DESIGN: This is an in vitro study. METHODS: BFCM samples from trophectoderm-tested euploid blastocysts (n = 80) from in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) patients were analyzed for PAPP-A mRNA. BFCM was obtained from blastocyst stage embryos in 20 uL drops. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy prior to blastocyst vitrification and BFCM collection for snap freezing. cfDNA was synthesized using BFCM collected from 80 individual euploid blastocysts. Next, real-time qPCR was performed to detect expression of PAPP-A with GAPDH for normalization of expression in each sample. RESULTS: PAPP-A mRNA was detected in 45 of 80 BFCM samples (56.3%), with varying levels of expression across samples. CONCLUSION: Our study demonstrates the expression of PAPP-A in BFCM. To our knowledge, this is the first study to report detection of PAPP-A mRNA in BFCM. Further studies are required and underway to investigate a greater number of BFCM samples as well as the possible correlation of PAPP-A expression with pregnancy outcomes of transferred euploid blastocysts. If found to predict IVF and obstetric outcomes, PAPP-A may provide additional information along with embryonic euploidy for the selection of the optimal blastocyst for embryo transfer.

Novel drug resistance-associated substitutions against pibrentasvir emerged in genotype 1b hepatitis C virus-infected human hepatocyte transplanted mice.

Combination therapy with glecaprevir and pibrentasvir (PIB) has high efficacy for patients with hepatitis C virus (HCV) infection except among those who experienced NS5A-P32 deletion (del) mutation during prior DAA treatment failure. However, some patients fail to achieve SVR through combination treatment even in the absence of NS5A-P32del. We analyzed emergence of NS5A resistance-associated substitutions (RASs) against PIB using HCV-infected mice. Male human hepatocyte transplanted mice were infected with genotype 1b wild-type HCV. Mice were treated with PIB, resulting in a transient decrease in serum HCV RNA levels but followed by relapse during the treatment. Direct sequence analysis showed emergences of various mutations in the NS5A region, including L31V/P32del, L31F/P32del/Y93H, NS5A-P29del/Y85C, and NS5A-F37Y. PIB was less effective in mice with NS5A-F37Y mutations compared to mice with wild-type HCV. NS5A-F37Y showed 5.4-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. The present in vivo and in vitro studies identified NS5A-F37Y as a novel RAS against PIB and showed the possibility of emergence of various NS5A RASs including P29del, P32del and F37Y following PIB treatment. These mutations might emerge and lead to failure to respond to DAA therapies including PIB-based regimens in chronic hepatitis C patients.

Serum PreS1 and HBsAg ratio reflects liver fibrosis and predicts the development of hepatocellular carcinoma in chronic hepatitis B patients.

While the preS1 region of the large hepatitis B surface protein plays an essential role in hepatitis B virus (HBV) infection, the effect of preS1 on liver fibrosis and hepatocarcinogenesis in chronic hepatitis B (CHB) patients is not well known. In this study, we measured serum preS1 levels by chemiluminescent immunoassay technology in 690 CHB patients and evaluated the correlation between serum preS1 levels and HBV, liver function markers and liver inflammation, fibrosis assessed by histological findings. Predictive factors for hepatocellular carcinoma (HCC) development in patients who had no previous history of HCC at the time of preS1 level measurement were also analysed. Median hepatitis B surface antigen (HBsAg) and preS1 levels were 3.08 log IU/mL and 98 ng/mL, respectively. PreS1 values were significantly correlated with serum HBsAg (p <0.001), hepatitis B core-related antigen (HBcrAg) (p <0.001) and HBV DNA levels (p <0.01). PreS1 values were also significantly correlated with serum alanine aminotransferase levels (p <0.001) and were significantly higher in patients who had higher grading of liver inflammatory activity (p <0.05). HBsAg level was correlated, but preS1/HBsAg ratio reflected liver fibrosis staging more directly than HBsAg alone. Multivariate analysis identified age ≥53 years (hazard ratio [HR], 18.360 for <53 years; p = 0.021) and preS1/HBsAg ratio ≥0.12 (HR, 6.205 for <0.12; p = 0.040) as significant and independent factors for HCC development in CHB patients. The preS1/HBsAg ratio directly reflects liver fibrosis, and the ratio might be a predictive marker for HCC development in CHB patients.

Abatacept treatment for patients with severe acute hepatitis caused by hepatitis B virus infection-Pilot study.

Although glucocorticoids have been used for immunosuppression of patients with primary hepatitis B virus (HBV) infection-induced severe hepatitis, the treatment is associated with a high frequency of adverse events. We conducted a pilot study for evaluating the efficacy and safety of abatacept, a cytotoxic T lymphocyte antigen-4 immunoglobulin (CTLA4), for acute hepatitis B. Five patients with severe acute hepatitis B (prothrombin activity ≤ 60%) were treated for immunosuppression by abatacept. Four patients received abatacept concurrently with methylprednisolone, and another patient was treated with abatacept alone. Rapid decrease in serum alanine aminotransferase levels, increase in prothrombin activity and improvement of general condition were obtained in four out of five patients. The patient with the most severe hepatitis underwent liver transplantation due to exacerbation of hepatitis in spite of treatment with both abatacept and methylprednisolone. None of the patients developed significant adverse events associated with the use of abatacept. Hepatitis B surface antigen (HBsAg) became negative in all five patients. The effect of abatacept and methylprednisolone for severe hepatitis B was compared using a mouse model. Rapid reduction in mouse serum HBV DNA and human albumin levels and elevation of serum interferon-gamma and granzyme A levels were observed in HBV-infected human hepatocyte-transplanted immunodeficient mice that were administered human peripheral blood mononuclear cells. These hepatocyte injuries were inhibited to a greater extent by abatacept compared to methylprednisolone. Abatacept might be an effective therapy alternative to methylprednisolone to reduce acute massive liver damage for patients with severe acute hepatitis caused by HBV infection.

Clinical Usefulness of Serum Autotaxin for Early Prediction of Relapse in Male Patients with Type 1 Autoimmune Pancreatitis.

BACKGROUND: Serum IgG4 level is a useful diagnostic marker for autoimmune pancreatitis (AIP), but it is difficult to use to predict relapse. AIMS: We investigated whether serum autotaxin (ATX) level is predictive of AIP relapse after steroid therapy. METHODS: Fifty-six patients with type 1 AIP were investigated. We measured serum ATX at the time of diagnosis. We selected 24 males for whom serum samples during steroid therapy had been obtained and measured serum ATX at steroid therapy for induction of remission and at maintenance therapy. In the relapse group, we also measured ATX at the time of relapse. RESULTS: ATX was significantly higher in female patients than in male patients. In order to clarify changes in ATX during steroid therapy, we focused on 24 male patients. We found that ATX decreased significantly during steroid therapy for induction of remission and at the time of maintenance therapy. In half of all patients who relapsed during maintenance therapy, ATX was significantly elevated at the time of relapse compared with that of induction therapy (P = 0.039). When we compared ATX at the time of maintenance therapy between patients with relapse and without, we observed significantly higher ATX in the former (P = 0.024). We found that the combination of ATX and elastase-1 could predict relapse with high accuracy (95%). CONCLUSIONS: Preliminary evidence suggests that serum ATX might serve as a candidate biomarker to predict relapse of AIP as well as to monitor the effect of steroid therapy.

Acute fasciolosis in an alpaca: a case report.

BACKGROUND: The popularity of new world camelids, particularly alpacas, is growing rapidly in Ireland, presenting a clinical challenge to veterinary practitioners who may not have worked with these species previously. To the authors' knowledge, the clinical course of a case of acute fasciolosis in an alpaca has not previously been reported, and fasciolosis has not been reported at all in alpacas in Ireland, making this case report a valuable addition to the current literature. CASE PRESENTATION: A three-year-old male castrated huacaya alpaca was admitted to UCD Veterinary Hospital with a two-day history of colic and tenesmus. He had been treated with albendazole, dexamethasone and potentiated amoxycillin by the referring veterinary practitioner with no response. On initial clinical exam, sensitivity to abdominal palpation was the only abnormality. However, the alpaca proceeded to show abnormal lying positions, tenesmus and reduced faecal output over the next 24 h. A general blood panel demonstrated moderate anaemia, marked hyperglobulinaemia and moderately increased hepatocellular and hepatobiliary enzyme activity. Abdominal radiography revealed enlargement of the first forestomach compartment without evidence of gastrointestinal obstruction or peritonitis. An abdominal ultrasound exam revealed an elongated, heterogenous mass in the caudoventral abdomen that appeared to be contiguous with the liver. FNA of this mass revealed that it was in fact a liver lobe with biliary stasis and inflammation. Faecal sedimentation demonstrated Fasciola hepatica eggs. In spite of treatment with triclabendazole and supportive treatment including blood transfusion, the alpaca's condition continued to deteriorate and he was euthanised. On post-mortem exam, acute fasciolosis was diagnosed. CONCLUSIONS: The clinical presentation and course of a case of acute fasciolosis in an individual alpaca is described, including the results of a range of diagnostic tests that were carried out. The final diagnosis is supported by a description of post-mortem findings. This information will serve as a resource for veterinary practitioners involved in the diagnosis and treatment of similar cases.

Nutrient digestibility and endogenous protein losses in the foregut and small intestine of weaned dairy calves fed calf starters with conventional or enzyme-treated soybean meal.

The aims of this experiment were (1) to compare the effects of a soybean meal with an enzymatic treatment (ESBM) to reduce the concentration of antinutritional factors versus a standard soybean meal (SBM) on foregut and small intestine digestion in weaned dairy calves and (2) to estimate the endogenous losses of crude protein (CP) in the small intestine. Our hypothesis was that a diet containing ESBM instead of SBM would improve ruminal and small intestine digestion and absorption of nutrients. A T-cannula was placed in the duodenum, and a second T-cannula was installed in the distal ileum of 12 Holstein calves at approximately 3 wk of age. Calves were weaned on d 42, and on d 50 they were assigned randomly to a quadruplicated 3 × 3 Latin square with 10-d periods. Digesta samples were collected on d 7 and 8 from the ileum and d 9 and 10 from the duodenum. The diets were fed for ad libitum intake and consisted of a calf starter (CS) of 20% CP with SBM as the main source of protein (CTRL), and an isonitrogenous CS with an ESBM instead of SBM (ENZT). A third diet with a low content of CP (10%) and no soy protein was fed to estimate endogenous N losses and digestibilities of test ingredients. Flows and digestibilities of nutrients were compared between CTRL and ENZT and their test ingredients (SBM vs. ESBM, respectively). Duodenal net flows of CP and total AA as well as ruminal microbial protein synthesis per kilogram of digested CP were greater, and flow of nonprotein N and CP true (corrected by endogenous and microbial flows) foregut digestibility were lower with ENZT than CTRL. The apparent small intestine digestibilities of CP and total AA were greater for ESBM than SBM, but there were no differences between the CTRL and ENZT diets. We observed no differences in digestibilities at the duodenum or ileum of starch or NDF, but true small intestine digestibilities of CP and all AA were greater with ENZT than CTRL. Total endogenous protein losses in the small intestine estimated from calves fed the low-CP with no soy protein diet were 37 ± 1.5 g of CP and 29 ± 1.4 g of AA/kg of DMI. These values may be considered the basal endogenous losses as they are similar to values obtained with the regression method, which estimates N losses when dietary N is null. Our results indicated that the inclusion of an ESBM improved the efficiency of ruminal microbial protein synthesis per digested kilogram of organic matter and CP, and increased CP and AA absorption in the small intestine despite a greater proportion of undigested dietary protein entering the duodenum.

Effects of spray-dried bovine plasma protein in milk replacers fed at a high plane of nutrition on performance, intestinal permeability, and morbidity of Holstein calves.

Spray-dried plasma protein (SDP) has been shown to improve growth and intestinal function in young calves when included in milk replacers (MR) fed at conventional rates. Use of an SDP and wheat protein blend to replace a portion of whey protein has been shown to perform similarly to using an all-whey protein control MR. However, a trend in the dairy industry is to feed calves for greater rates of growth during the preweaning period. The purpose of this study was to determine the effects of increasing amounts of SDP inclusion in MR on growth and health of calves fed at a high plane of nutrition. Young (<7 d) Holstein calves were offered starter and assigned to 1 of 5 MR treatments: an all-milk protein (whey) control MR (0SDP, n = 26) or MR containing 5% SDP (5SDP, n = 20), 7.5% SDP (7.5SDP, n = 14), 10% SDP (10SDP, n = 20), or 12% of an approximate 1:1 SDP plus wheat protein blend (PW, n = 17). All MR were formulated to contain 26% CP and 16% fat and were fed at a maximum rate of 1 kg of powder (as fed) from d 8 to 36. Amounts of MR powder were decreased by 25%/wk from d 37 to weaning at d 57. Thereafter, calves were provided only starter feed until the end of the study at d 63. On d 4, 15, 36, and 57, intestinal permeability was assessed via oral administration of lactulose and d-mannitol followed by analysis of lactulose and mannitol in blood at 60 min after administration. Increasing SDP led to a small linear decrease in MR consumed. There was a tendency for a positive linear relationship between increasing SDP and average daily gain of body weight, and SDP had mixed effects on body frame variables. Increasing SDP tended to increase fecal scores and increased the amount of fluid therapy given. Diet had no effect on intestinal permeability. Increasing SDP led to an increase in serum total cholesterol and serum urea N and tended to have a quadratic effect on serum glucose concentration on d 36. Calves fed PW tended to have increased withers height, increased intestinal permeability on d 36, and an increased likelihood of being medicated for any reason or being medicated for respiratory illness, but growth and health were not different from the control diet otherwise. Feeding PW led to an increase in serum total cholesterol and tended to lead to increased serum glucose concentration on d 36. Results of this study indicate that SDP can be included at up to 10% as fed in the MR of calves fed at a high plane of nutrition (1 kg/d of MR powder, as fed) with improvements in average daily gain. Additionally, a 1:1 SDP plus wheat protein blend can be used at 12% inclusion with no difference in most health and growth parameters.

Signal Activation of Hepatitis B Virus-Related Hepatocarcinogenesis by Up-regulation of SUV39h1.

BACKGROUND: Hepatitis B virus (HBV) X (HBx) protein is associated with hepatocellular carcinogenesis via the induction of malignant transformation and mitochondrial dysfunction. However, the association between HBx and histone methyltransferase in carcinogenesis has not been fully clarified. In the current study, we analyzed the association between HBx and the histone methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39h1) using HBV replication models. METHODS: We constructed several HBx and SUV39h1 expression plasmids and analyzed the association between HBx and SUV39h1 with respect to HBV replication and hepatocarcinogenesis. RESULTS: SUV39h1 up-regulation was observed in HBV-infected humanized mouse livers and clinical HBV-related hepatocellular carcinoma tissues, indicating that SUV39h1 expression might be regulated by HBV infection. Through in vitro analysis, we determined that the coactivator domain of HBx interacts with the PSET (PostSET) and SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domains of SUV39h1. The expression levels of 4 genes, activating transcription factor 6, α-fetoprotein, growth arrest and DNA damage-inducible 45a, and dual-specificity phosphatase 1, known to induce carcinogenesis via HBx expression, were up-regulated by HBx and further up-regulated in the presence of both HBx and SUV39h1. Furthermore, histone methyltransferase activity, the main function of SUV39h1, was enhanced in the presence of HBx. CONCLUSIONS: We demonstrated that SUV39h1 and HBx enhance each other's activity, leading to HBx-mediated hepatocarcinogenesis. We propose that regulation of this interaction could help suppress development of hepatocellular carcinoma.

Ribavirin induces hepatitis C virus genome mutations in chronic hepatitis patients who failed to respond to prior daclatasvir plus asunaprevir therapy.

Ribavirin (RBV) induces nucleotide (nt) substitutions in hepatitis C virus (HCV) genome nonstructural (NS) regions. Although emergence of drug resistance-associated variants is associated with direct-acting antiviral treatment failure, the effect of RBV on genome substitutions in such patients is unknown. Genotype 1b HCV subgenomic replicon cells were treated with RBV for 120 hours. Six patients with chronic genotype 1b with HCV-infected patients who failed to respond to prior daclatasvir plus asunaprevir (DCV/ASV) therapy were treated with 12 weeks of sofosbuvir and ledipasvir plus RBV after 4 weeks of RBV monotherapy. RBV-induced genome mutations in the HCV NS region (nt3493-9301) in replicon cells and in patients during 4 weeks of RBV monotherapy were analyzed by deep sequencing. RBV-associated G-to-A and C-to-U transitions increased in a dose-dependent manner in HCV replicon cells after the RBV treatment. In patients with prior DCV/ASV treatment failures, the median serum HCV RNA level was 6.25 ± 0.31 log IU/mL at the start of RBV therapy and decreased significantly to 5.95 ± 0.4 log IU/mL (P = .03) after 4 weeks of RBV monotherapy. Although predominant HCV genome substitutions rates were similar between nontreatment and RBV-treatment periods (0.042 and 0.031 per base pair, respectively; P = .248), the frequencies of G-to-A and C-to-U transitions significantly increased after RBV monotherapy. These transitions were enriched, particularly within the HCV NS3 region in all patients. RBV treatment induces G-to-A and C-to-U transitions in the HCV genome even in chronic patients with hepatitis C with prior DCV/ASV treatment failures.