The thyroid target Ag for disease-inducing autoantibodies in Graves’ disease is the receptor for thyroid-stimulating hormone (TSH), but little is known about the molecular basis of this pathogenic Ab response. We describe the characteristics of two high- affinity mAbs developed from an experimental murine model of hyperthyroid Graves’ disease that exhibit potent thyroid-stimulating activity. Nanogram concentrations of the IgG mAbs KSAb1 and KSAb2 and their Fab induce full stimulation of the TSH receptor that is matched by the ligand TSH and, thus, act as full agonists for the receptor. However, KSAb1 and KSAb2 display differential activities in their ability to block TSH-mediated stimulation of the receptor, indicating subtle differences in their biological properties. In displacement studies, IgG and Fabs of KSAb1 and KSAb2 compete with Graves’ disease autoantibodies as well as thyroid-blocking Abs present in some hypothyroid patients, indicating a close relationship between these autoimmune determinants on the receptor. In passive transfer studies, single injections of microgram quantities of KSAb1 or KSAb2 IgG led to rapid elevation of serum thyroxine and a hyperthyroid state that was maintained for a number of days. The thyroid glands showed evidence of cell necrosis, but there was no accompanying mononuclear cell infiltrate. In studying their receptor activation pathways, both KSAb1 and KSAb2 provoked phosphorylation of the intracellular ERK1/2 pathway in primary thyrocytes, indicating that multiple signaling pathways may participate in the pathogenesis of Graves’ disease. In summary, our findings emphasize the similarities of the experimental mouse model in reproducing the human disorder and provide improved means for characterizing the molecular basis of this pathogenic response.

SummaryBackgroundRifaximin reduces the risk of overt hepatic encephalopathy (HE) and is associated with significant reductions in hospitalisations and 30‐day readmissions.AimTo examine the outcomes of patients listed for liver transplantation with a diagnosis of HE on rifaximin compared to those naïve to the drug.MethodsPatient records of those listed for liver transplantation over a 2‐year period were retrospectively reviewed. Patients were included if they had at least two episodes of overt HE resulting in hospitalisation or were encephalopathic at the time of assessment.ResultsOf the 622 patients listed for transplantation, 101 had HE. Sixty‐six patients were treated with rifaximin and 35 were naïve at listing. The use of concurrent lactulose was not significantly different between groups. Median MELD score was similar (15 [14‐16)] rifaximin‐treated and 16 [14‐18] rifaximin‐naïve). Patients on the waiting list treated with rifaximin had reduced all‐cause admissions, episodes of spontaneous bacterial peritonitis and variceal bleeding. Mean length of stay was 9 days (95% CI 6‐12) in the rifaximin‐treated group vs 14 (95% CI 7‐21) in the rifaximin‐naïve group. Multivariate regression analysis demonstrated that rifaximin was independently associated with an increase in average days to readmission (adjusted effect estimate 71, 95% CI 3‐140 days) and reduced likelihood of requirement for prioritisation on the waiting list (odds ratio 0.29; 95% CI 0.89‐0.93).ConclusionRifaximin prescribed for HE in patients listed for liver transplantation improved outcomes with significant reduction in admissions related to spontaneous bacterial peritonitis, ascites and variceal bleeding.

In the absence of adequate compensatory regeneration, overwhelming liver damage can cause acute liver failure (ALF) and death without emergent liver transplantation (LT). Auxiliary LT produces satisfactory outcomes in this setting, with the prospect of native liver regeneration sustaining long-term survival. Since animal models only partially recapitulate human liver regeneration, we investigated the molecular mechanisms controlling it in this unique LT setting, as an exemplar of human liver regeneration. We demonstrate coordinated changes in expression of microRNA (miRNA) during regeneration that drive proliferation, innate immunity and angiogenesis. In contrast, failed regeneration in a similar cohort is associated with distinct miRNA enforcing cell cycle inhibition and DNA methylation. The miRNA expression associated with successful or failed regeneration when recapitulated in vitro, triggered expression of cardinal regeneration-linked genes promoting cell cycle entry or inhibition, respectively. Furthermore, inhibition of miRNA 150, 663 and 503, whose downregulation is associated with successful regeneration, induced cell proliferation which a key determinant of successful regeneration. Our data indicate that human liver regeneration may be orchestrated by distinct miRNA controlling key regeneration-linked processes including hepatocyte proliferation. To our knowledge this is the first characterization of molecular processes associated with human liver regeneration.

Recurrent hepatitis C virus (HCV) infection is associated with accelerated fibrosis rates after liver transplantation (LT) and is the leading cause of graft failure. Furthermore, distinguishing recurrent HCV from acute cellular rejection (ACR) can be problematic, and this can lead to inappropriate treatments and adverse outcomes. We hypothesized that intragraft micro-RNA (miRNA) expression profiles could distinguish the severity of recurrent HCV and differentiate recurrent HCV from ACR. We established meticulously matched post-LT patient cohorts in order to derive robust global miRNA expression profiles and minimize the impact of variables known to influence HCV recurrence. These cohorts consisted of patients with slow HCV fibrosis progression (Ishak stage < F2), fast HCV fibrosis progression (Ishak stage F2), ACR, and nonviral etiologies. We found increased intragraft expression of miRNA-146a, miRNA-19a, miRNA-20a, and miRNA-let7e in slow progressors versus fast progressors, and we validated these findings with quantitative PCR. This miRNA network regulates the expression of cardinal genes implicated in promoting antifibrogenic, antiangiogenic, and anti-inflammatory pathways. miRNA-19a and miRNA-20a were also specifically detected in the serum of slow progressors. Furthermore, intragraft miRNA expression distinguished fast HCV progression from ACR. Here, changes in the expression of key miRNAs regulating fibrogenic and angiogenic pathways were associated with fast HCV progression. We demonstrate specific miRNA expression signatures that discriminate the rates of fibrosis progression in patients with recurrent HCV, and we distinguish recurrent HCV from ACR after LT. A pathway analysis indicates that specific miRNAs may play a regulatory role in these processes. Selected miRNAs may serve as intragraft and serum biomarkers for recurrent HCV after LT and help to distinguish between ACR and recurrent HCV.

Donation after cardiac death (DCD) livers are marginal organs for transplant and their use is associated with a higher risk of primary non function (PNF) or early graft dysfunction (EGD). The aim was to determine if microRNA (miRNA) was able to discriminate between DCD livers of varying clinical outcome. DCD groups were categorized as PNF retransplanted within a week (n=7), good functional outcome (n=7) peak aspartate transaminase (AST) ≤ 1000 IU/L and EGD (n=9) peak AST ≥ 2500 IU/L. miRNA was extracted from archival formalin fixed post-perfusion tru-cut liver biopsies. High throughput expression analysis was performed using miRNA arrays. Bioinformatics for expression data analysis was performed and validated with real time quantitative PCR (RT-qPCR). The function of miRNA of interest was investigated using computational biology prediction algorithms. From the array analysis 16 miRNAs were identified as significantly different (p<0.05). On RT-qPCR miR-155 and miR-940 had the highest expression across all three DCD clinical groups. Only one miRNA, miR-22, was validated with marginal significance, to have differential expression between the three groups (p=0.049). From computational biology miR-22 was predicted to affect signalling pathways that impact protein turnover, metabolism and apoptosis/cell cycle. In conclusion, microRNA expression patterns have a low diagnostic potential clinically in discriminating DCD liver quality and outcome.

No abstract

We previously demonstrated a distinct hepatic microRNA (miRNA) signature (down-regulation of miRNA-23a, -150, -200b, -503, and -663 and up-regulation of miRNA-20a) is associated with successful regeneration in auxiliary liver transplantation (ALT). This study aimed to evaluate whether the serum expression of this regeneration-linked miRNA signature is associated with clinical outcomes in acute and chronic liver disease. These were represented by patients with acetaminophen-induced acute liver failure (ALF; n = 18) and patients with hepatitis C virus (HCV) undergoing treatment with direct-acting antivirals (n = 56), respectively. Patients were grouped depending on their clinical outcome. Global serum miRNA expression was analyzed using polymerase chain reaction (PCR) arrays and selected miRNA expression using targeted PCR. We demonstrate that specific regeneration-linked miRNAs discriminate outcomes in both clinical scenarios. We further show that miRNA-20a, -23a, -150, -200b, -503, and -663 undergo concordant changes in expression in 3 distinct clinical settings: liver regeneration accompanying successful ALT, clinical recovery after ALF, and clinical recompensation after cure of HCV. This miRNA signature represents a potentially novel biomarker to predict outcome and optimize patient selection for liver transplantation in both acute and chronic liver disease.Liver Transplantation 26 811-822 2020 AASLD. devised and planned the experiments and reviewed and revised the manuscript for submission. Siamak Salehi and Oliver D. Tavabie carried out the microRNA analysis and drafted the manuscript. Suman Verma and William Bernal created the patient cohorts, developed clinical data, and reviewed and revised the manuscript for submission.

Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.