Elevated liver enzymes in a German tertiary-care hospital: Distribution, diagnostic steps and diagnosis groups.

BACKGROUND: The determination of liver blood tests is frequently performed in hospitalized patients, and abnormal values require further diagnostics. Yet, analyses considering the management of elevated liver enzymes are missing. Therefore, this study aimed to analyze the distribution of abnormal liver function tests and the subsequent diagnostic steps across different medical specialties. METHODS: From our Hannover liver-injury database, we identified 63,300 cases of patients who were hospitalized between January 2008 and July 2021 with AST or ALT > 3 ULN or AP or TBI > 2 ULN at any time point during hospitalization. Of these, 29,547 cases fulfilled the inclusion criteria and were subjected to further analysis. Cases were analyzed according to the three groups: internal medicine, surgery and others. Analyses were performed regarding baseline characteristics, liver-related diagnostics and factors influencing hospital mortality. RESULTS: Elevated liver blood tests were mainly observed in internal medicine (n=17,762, 60.1%), followed by the surgery department 34.2% (n=10,105). Notably, 40.2% (n=11,896) developed liver enzyme elevation above the cut-offs during the hospital stay. Testing for hepatitis B and C was more often performed in the surgery department compared to in internal medicine. In total, 5.6% of the cases (n=1,640) had a liver biopsy. Hyperbilirubinemia (total bilirubine ≥ 2ULN) and AST/ALT ratios >2 were associated with in-hospital mortality. CONCLUSION: Clinicians are often faced with elevated liver enzymes. However, diagnostic steps differ between different specialties. Physicians should be aware of the increased in-hospital mortality in cases with hyperbilirubinemia or elevated AST/ALT ratios.

Out of sight for the endoscopist? Gastrointestinal bleeding after aortic repair.

BACKGROUND AND STUDY AIM: Secondary Aortoenteric Fistulas (sAEF) are difficult to diagnose and usually result in fatal gastrointestinal (GI) bleeding following aortic repair. Outcomes are largely dependent on a timely diagnosis, but AEFs remain challenging to identify endoscopically and are usually diagnosed on computed tomography (CT) scans. The aim of our study was optimize diagnosis of AEF by identifying patients developing GI bleeding after aortic repair, investigate their clinical course and identify factors specific to different bleeding sources. METHODS: A retrospective, single-center study capturing all patients developing upper or lower GI bleeding after aortic surgery between January 2009 and March 2020 was performed. Electronic health records were screened for diagnostic codes of the relevant procedures. Bleeding was classified into three groups: AEF with demonstrable fistula, ischemic - macroscopic ulceration plus histological confirmation or imaging and "other" due to other recognized conventional cause, such as peptic ulcer disease. RESULTS: 47 GI bleeding episodes in 39 patients were identified. Of these, 10 episodes (21%) were caused by AEF, 16 (34%) by ischemic ulceration and 21 (45%) due to other causes. Patients with AEF exhibited more frequent hemodynamic instability requiring vasopressors and had higher mortality, while ischemic ulcerations were associated with more recent operation or hypotensive episode. CONCLUSIONS: GI bleeding complications are uncommon following aortic surgery. AEF and ischemic ulceration are however frequent bleeding causes in this cohort. In patients presenting with fulminant bleeding, primary CT-scanning should be considered.

T cell receptor repertoires within liver allografts are different to those in the peripheral blood.

BACKGROUND & AIMS: T cells are the main mediators of allogeneic immune responses. Specific T cell clones can be tracked by their unique T cell receptor (TCR), but specificity and function remain elusive and have not been investigated in human liver biopsies thus far. METHODS: TCR repertoire analysis of CD4+, CD8+, and regulatory T cells of the peripheral blood and liver graft was performed in 7 liver transplant recipients with either stable course (non-rejector, NR), subclinical cellular rejection (SCR), or acute cellular rejection (ACR) during an observation period from pre-transplant to 6 years post-transplant. Furthermore, donor-reactive T cells, identified by their expression of CD154 and glycoprotein A repetitions predominant (GARP) after allogeneic activation, were tracked longitudinally in peripheral blood and within the liver allograft. RESULTS: Although overall clonality of the TCR repertoire did not increase in peripheral blood after liver transplantation, clonality of donor-reactive CD4+ and regulatory T cells increased and these clones accumulated within the liver graft. Surprisingly, the TCR repertoires between the liver graft and the periphery were distinct and showed only limited overlap. Notably, during ACR, TCR repertoires aligned suggesting either graft-specific homing or release of activated T cells from the graft. CONCLUSIONS: This is the first study comparing TCR repertoires between liver grafts and blood in patients with NR, SCR, and ACR. Moreover, we attribute specificity and function to a subgroup of intragraft T cell populations. Given the limited overlap between peripheral blood and intragraft repertoires, future studies investigating function and specificities of T cells after liver transplantation should focus on the intragraft immune response. LAY SUMMARY: In solid organ transplantation, T cells are key mediators of the recipient's immune response directed at the transplanted organ. In our study, we characterised the T cell repertoire in a cohort of 7 liver transplant recipients. We demonstrate that donor-specific T cells expand clonally and accumulate in the transplanted liver. Moreover, we show that the composition of T cells in peripheral blood differs from the T cells in the liver allograft, only aligning in the context of acute cellular rejection but not in normal graft or subclinical cellular rejection. This indicates that the intragraft immune response is not mirrored in the peripheral blood. Our findings clarify the importance of protocol liver biopsies in identifying intragraft immune responses for future investigations of allo-directed immune responses.

Cholemic Nephropathy Causes Acute Kidney Injury and Is Accompanied by Loss of Aquaporin 2 in Collecting Ducts.

Impairment of renal function often occurs in patients with liver disease. Hepatorenal syndrome is a significant cause of acute kidney injury (AKI) in patients with cirrhosis (HRS-AKI, type 1). Causes of non-HRS-AKI include cholemic nephropathy (CN), a disease that is characterized by intratubular bile casts and tubular injury. As data on patients with CN are obtained primarily from case reports or autopsy studies, we aimed to investigate the frequency and clinical course of CN. We identified 149 patients who underwent kidney biopsy between 2000 and 2016 at the Department of Gastroenterology, Hepatology and Endocrinology at Hannover Medical School. Of these, 79 had a history of liver disease and deterioration of renal function. When applying recent European Association for the Study of the Liver criteria, 45 of 79 patients (57%) presented with AKI, whereas 34 patients (43%) had chronic kidney disease (CKD). Renal biopsy revealed the diagnosis of CN in 8 of 45 patients with AKI (17.8%), whereas none of the patients with CKD was diagnosed with CN. Univariate analysis identified serum bilirubin, alkaline phosphatase, and urinary bilirubin and urobilinogen as predictive factors for the diagnosis of CN. Histological analysis of AKI patients with normal bilirubin, elevated bilirubin, and the diagnosis of CN revealed loss of aquaporin 2 (AQP2) expression in collecting ducts in patients with elevated bilirubin and CN. Biopsy-related complications requiring medical intervention occurred in 4 of 79 patients (5.1%). Conclusion: CN is a common finding in patients with liver disease, AKI, and highly elevated bilirubin. Loss of AQP2 in AKI patients with elevated bilirubin and CN might be the result of toxic effects of cholestasis and in part be responsible for the impairment of renal function.

Second-line chemotherapy in biliary tract cancer: Outcome and prognostic factors.

BACKGROUND & AIMS: The prognosis of biliary tract cancer (BTC) is poor. Standard treatment for advanced BTC is a chemotherapy (CT) with gemcitabine and cisplatin. Phase III evidence for a second-line (2L) CT is lacking. We aimed to investigate the feasibility of a 2L CT, to estimate the outcome and to identify prognostic markers. METHODS: Patients of our institution with advanced BTC between 2000 and 2015 receiving CT were included. Data were analysed in univariate and multivariate analysis. RESULTS: Three-hundred and fifteen and 144 patients (45.7%) received first-line (1L) and 2L CT respectively. The OS of patients receiving 2L CT was 16.67 and 9.9 months from the beginning of 1L and 2L CT respectively. The overall response rate and the disease control rate after 3 months were 9.7% and 33.6% respectively. Adverse events of grade 3 or more were observed in 26.1%. One patient died of gemcitabine-related haemolytic uraemic syndrome. Age of more than 70 years was not associated with a poor outcome. In multivariate analysis, CEA levels of >3 µg/L (P = 0.004, hazard ratio [HR] 1.89, 95% CI 1.22, 2.91), cholinesterase (CHE) levels of <5 kU/L (P = 0.001, HR 2.11, 95% CI 1.34, 3.31) and leukocytosis (P = 0.001, HR 2.90, 95% CI 1.51, 5.56) were associated with poor survival. CONCLUSIONS: Despite a relevant toxicity, our data suggest that 2L CT may be feasible in fit BTC patients. CEA elevation, leukocytosis and low CHE levels are unfavourable prognostic markers. Results from prospective randomized trials are urgently awaited.

An inducible model for genetic manipulation and fate-tracing of PDGFRß-expressing fibrogenic cells in the liver.

Myofibroblasts are the source of extracellular matrix protein during liver fibrogenesis. Fibroblasts, hepatic stellate cells (HSCs) and vascular smooth muscle cells are mesenchymal subpopulations in the liver that are characterized by the expression of PDGFRß and contribute to the pool of these myofibroblasts. Conditional knockout models are important to better understand the function of specific liver cell populations including mesenchymal cells. While there is a limited number of constitutive mouse models for liver mesenchymal cell specific transgene expression, there is no established model for inducible gene targeting in HSCs or PDGFRß-expressing mesenchymal cell populations in the liver. To address this, we investigated whether the tamoxifen inducible PDGFRß-P2A-CreERT2 mouse can be used as a reliable tool to specifically express transgens in liver mesenchymal cells. Our data demonstrate, that PDGFRß-P2A-CreERT2 specifically and efficiently marks over 90% of retinoid positive HSCs in healthy and fibrotic liver in mice upon tamoxifen injection, and that those cells give rise to Col1a1-expressing myofibroblasts in different models of liver fibrosis. Together with a negligible background recombination of only about 0.33%, this confirms that the PDGFRß-P2A-CreERT2 mouse is nearly as efficient as established constitutive LratCre and PDGFRß-Cre mouse models for recombination in HSCs, and that it is a powerful model for mesenchymal liver cell studies that require an inducible Cre approach.

Pulmonary Arterial Hypertension and Consecutive Right Heart Failure Lead to Liver Fibrosis.

Hepatic congestion occurs in patients with right heart failure and can ultimately lead to liver fibrosis or cardiac cirrhosis. Elevated pulmonary arterial pressure is found in patients with hepatic congestion. However, whether pulmonary arterial hypertension (PAH) can be a cause of liver fibrosis is unknown. The aim of this study was to investigate whether rats in the SuHx model with severe PAH develop liver fibrosis and to explore the mechanisms of congestive hepatic fibrosis both in rats and humans. To achieve this, PAH was induced in six to eight-week old male Sprague Dawley rats by a single subcutaneous injection of the VEGFR 2 inhibitor SU5416 and subsequent hypoxia for 3 weeks, followed by a 6-week period in room air. SuHx-exposed rats developed severe PAH, right ventricular hypertrophy (RVH), and consecutive right ventricular failure. Cardiac magnetic resonance imaging (MRI) and histological analysis revealed that PAH rats developed both hepatic congestion and liver fibrosis. Gene set enrichment analysis (GSEA) of whole liver RNA sequencing data identified a hepatic stellate cell specific gene signature in PAH rats. Consistently, tissue microarray from liver of patients with histological evidence of hepatic congestion and underlying heart disease revealed similar fibrogenic gene expression patterns and signaling pathways. In conclusion, severe PAH with concomitant right heart failure leads to hepatic congestion and liver fibrosis in the SU5416/hypoxia rat PAH model. Patients with PAH should therefore be screened for unrecognized liver fibrosis.

Dulaglutide Alone and in Combination with Empagliflozin Attenuate Inflammatory Pathways and Microbiome Dysbiosis in a Non-Diabetic Mouse Model of NASH.

Dysregulation of glucose homeostasis plays a major role in the pathogenesis of non-alcoholic steatohepatitis (NASH) as it activates proinflammatory and profibrotic processes. Beneficial effects of antiglycemic treatments such as GLP-1 agonist or SGLT-2 inhibitor on NASH in patients with diabetes have already been investigated. However, their effect on NASH in a non-diabetic setting remains unclear. With this aim, we investigated the effect of long-acting GLP1-agonist dulaglutide and SGLT-2 inhibitor empagliflozin and their combination in a non-diabetic mouse model of NASH. C57BL/6 mice received a high-fat-high-fructose (HFHC) diet with a surplus of cholesterol for 16 weeks. After 12 weeks of diet, mice were treated with either dulaglutide, empagliflozin or their combination. Dulaglutide alone and in combination with empagliflozin led to significant weight loss, improved glucose homeostasis and diminished anti-inflammatory and anti-fibrotic pathways. Combination of dulaglutide and empagliflozin further decreased MoMFLy6CHigh and CD4+Foxp3+ T cells. No beneficial effects for treatment with empagliflozin alone could be shown. While no effect of dulaglutide or its combination with empaglifozin on hepatic steatosis was evident, these data demonstrate distinct anti-inflammatory effects of dulaglutide and their combination with empagliflozin in a non-diabetic background, which could have important implications for further treatment of NASH.

The PNPLA3 rs738409 GG genotype is associated with poorer prognosis in 239 patients with autoimmune hepatitis.

BACKGROUND: Fibrosis progression in autoimmune hepatitis can be attenuated by immunosuppressive treatment; however, some patients progress despite therapy. Single nucleotide polymorphisms (SNPs) such as PNPLA3-rs738409, TM6SF2-rs58542926 and MBOAT7-rs641738 are associated with non-alcoholic fatty liver disease and fibrosis progression, whereas a splice variant in HSD17B13-rs72613567:TA has been shown to be protective. AIM: To analyse the impact of different SNPs on the long-term outcome of patients with autoimmune hepatitis. METHODS: We included 239 patients into this study who had been treated between 1983 and 2018 for autoimmune hepatitis. Genomic DNA was isolated from whole blood and SNPs were determined by PCR analysis. Liver biopsies were available for 215/239 patients (90%). Clinical and laboratory patient data were assessed by chart review. RESULTS: Mean age at baseline was 42.1 years with 74.1% being female. The median follow-up was 9.4 years (IQR 3.5-15.0), 11.7% of the patients (n = 28) died or required liver transplantation. In the Kaplan-Meier analysis of the combined endpoint time to liver transplantation or death, we observed that patients with the PNPLA3-rs738409 GG variant met more frequently the primary endpoint (P = 0.005). In Cox regression analysis PNPLA3-rs738409 GG as well as liver cirrhosis were identified as strong predictors for time to liver transplantation or death (HR 4.5 [CI 1.48-13.72], P = 0.008 and HR 9.24 [CI 2.11-40.44], P = 0.003, respectively). Neither steatosis, diabetes mellitus nor obesity were associated with outcome. CONCLUSIONS: PNPLA3-rs738409 variant GG is a predictor for time to liver transplantation or death and may help to identify autoimmune hepatitis patients at risk for disease progression.

Transient increase of activated regulatory T cells early after kidney transplantation.

Regulatory T cells (Tregs) are crucial in controlling allospecific immune responses. However, studies in human kidney recipients regarding the contribution of polyspecific Tregs have provided differing results and studies on alloreactive Tregs are missing completely. In this retrospective study, we specifically analyzed activated CD4+CD25highFOXP3+GARP+ Tregs in 17 patients of a living donor kidney transplantation cohort longitudinally over 24 months by flow cytometry (FOXP3: forkhead box protein 3, GARP: glycoprotein A repetitions predominant). We could demonstrate that Tregs of patients with end-stage renal disease (ESRD) are already pre-activated when compared to healthy controls. Furthermore, even though total CD4+CD25highFOXP3+ Treg numbers decreased in the first three months after transplantation, frequency of activated Tregs increased significantly representing up to 40% of all peripheral Tregs. In a cohort of living donor kidney transplantation recipients with stable graft function, frequencies of activated Tregs did not correlate with the occurrence of acute cellular rejection or chronic graft dysfunction. Our results will be important for clinical trials using adoptive Treg therapy after kidney transplantation. Adoptively transferred Tregs could be important to compensate the Treg loss at month 3, while they have to compete within the Treg niche with a large number of activated Tregs.