Non-invasive alloimmune risk stratification of long-term liver transplant recipients.

BACKGROUND & AIMS: Management of long-term immunosuppression following liver transplantation (LT) remains empirical. Surveillance liver biopsies in combination with transcriptional profiling could overcome this challenge by identifying recipients with active alloimmune-mediated liver damage despite normal liver tests, but this approach lacks applicability. Our aim was to investigate the utility of non-invasive tools for the stratification of stable long-term survivors of LT, according to their immunological risk and need for immunosuppression. METHODS: We conducted a cross-sectional multicentre study of 190 adult LT recipients assessed to determine their eligibility to participate in an immunosuppression withdrawal trial. Patients had stable liver allograft function and had been transplanted for non-autoimmune non-replicative viral liver disease >3 years before inclusion. We performed histological, immunogenetic and serological studies and measured the intrahepatic transcript levels of an 11-gene classifier highly specific for T cell-mediated rejection (TCMR). RESULTS: In this cohort, 35.8% of patients harboured clinically silent fibro-inflammatory liver lesions (13.7% had mild damage and 22.1% had moderate-to-severe damage). The severity of liver allograft damage was positively associated with TCMR-related transcripts, class II donor-specific antibodies (DSAs), ALT, AST, and liver stiffness measurement (LSM), and negatively correlated with serum creatinine and tacrolimus trough levels. Liver biopsies were stratified according to their TCMR transcript levels using a cut-off derived from biopsies with clinically significant TCMR. Two multivariable prediction models, integrating ALT+LSM or ALT+class II DSAs, had a high discriminative capacity for classifying patients with or without alloimmune damage. The latter model performed well in an independent cohort of 156 liver biopsies obtained from paediatric liver recipients with similar inclusion/exclusion criteria. CONCLUSION: ALT, class II DSAs and LSM are valuable tools to non-invasively identify stable LT recipients without significant underlying alloimmunity who could benefit from minimisation of immunosuppression. LAY SUMMARY: A large proportion of liver transplant patients with normal liver tests have inflammatory liver lesions, which in 17% of cases are molecularly indistinguishable from those seen at the time of rejection. ALT, class II donor-specific antibodies and liver stiffness are useful in identifying patients with this form of subclinical rejection. We propose these markers as a useful tool to help clinicians determine if the immunosuppression administered is adequate.

A retrospective analysis of post-transplant lymphoproliferative disorder following liver transplantation.

OBJECTIVE: To evaluate response rates and survival in adults developing post-transplant lymphoproliferative disorder (PTLD) following liver transplantation. METHODS: Patients were identified retrospectively and data collected through local liver and haematology electronic databases and pharmacy records. RESULTS: Forty-five patients were identified. The median age at first transplant and at development of PTLD was 48 and 54 years, respectively, with the median time from transplant to PTLD diagnosis of 56 months. The majority of cases (76%) were monomorphic B-cell lymphomas, and 36% of tumours were EBV positive. Treatment involved reduction in immune-suppression (RIS) in 30 (67%) with RIS the only treatment in 3. Ten (22%) patients were treated with rituximab alone, 13 (29%) with chemotherapy alone and 14 (31%) patients were treated with rituximab and chemotherapy. Twenty-six (58%) patients achieved a complete response (CR). At a median follow-up of 27 months, the median overall survival (OS) was 50 months. Response and OS were not associated with clinical factors or the use of rituximab. CONCLUSION: Outcomes reported in this study are favourable and comparable to those reported previously. The addition of rituximab did not appear to have improved outcomes in this series, although a significant proportion of patients were able to avoid chemotherapy.

Hepatocyte senescence predicts progression in non-alcohol-related fatty liver disease.

BACKGROUND & AIMS: Models of non-alcohol-related fatty liver disease (NAFLD) reveal features of accelerated ageing, such as impaired regeneration, and an increased risk of hepatocellular carcinoma. The relation between accelerated ageing, disease progression and clinical outcome has not been previously investigated and is the subject of the current study. METHODS: Liver sections from 70 patients with NAFLD (105 biopsies) and 60 controls were studied for telomere length, nuclear area, DNA damage and cell cycle phase markers, using quantitative fluorescent in situ hybridization and immunohistochemistry. RESULTS: Hepatocyte telomeres were shorter in NAFLD than controls (p <0.0001). Hepatocytes in NAFLD demonstrated lack of cell cycle progression beyond G1/S phase and high-level expression of p21, the universal cell cycle inhibitor (p=0.001). γ-H(2)AX expression increased with steatosis (p=0.01), indicating DNA damage, and was associated with shorter hepatocyte telomeres (p <0.0001). Hepatocyte p21 expression correlated with fibrosis stage and diabetes mellitus, independently (p <0.001 and p=0.002, respectively). Further analysis revealed that an adverse liver-related outcome was strongly associated with higher hepatocyte p21 expression and greater hepatocyte nuclear area (p=0.02 and p=0.006), but not with telomere length. In paired biopsies, changes in hepatocyte p21 expression and nuclear area mirrored changes in fibrosis stage (p=0.01 and p=0.006, respectively). CONCLUSIONS: These findings are consistent with hepatocyte senescence and permanent cell cycle arrest in NAFLD. Hepatocyte senescence correlated closely with fibrosis stage, diabetes mellitus, and clinical outcome. Hepatocyte p21 expression could be used as a prognostic marker and for stratification in clinical studies.

Sustained telomere length in hepatocytes and cholangiocytes with increasing age in normal liver.

UNLABELLED: Telomeres, a validated biomarker of aging, comprise multiple nucleotide repeats capping chromosomes that shorten with each cell cycle until a critical length is achieved, precipitating cell senescence. Only two previous studies focused on the effect of aging in "normal" liver tissue, but these studies were compromised by small sample size, limited age range, tissue derived from individuals with an increased risk of senescence, and the use of liver homogenates. We developed a robust large-volume, four-color quantitative fluorescent in situ hybridization technique to measure telomere length in large numbers of hepatocytes, Kupffer cells, hepatic stellate cells, CD4-positive and CD8-positive lymphocytes, and cholangiocytes. Following validation against the gold standard (Southern blotting), the technique was applied to normal archived paraffin-embedded liver tissue obtained following reperfusion of implanted donor liver. We studied 73 highly selected donors aged 5-79 years with a short medical illness preceding death and no history of liver disease, reperfusion injury, or steatosis and normal graft function 1-year posttransplantation. Cholangiocytes had significantly longer telomeres compared with all other intrahepatic lineages over a wide age range (P < 0.05). Age-related telomere attrition was restricted to sinusoidal cells (i.e., Kupffer cells [P = 0.0054] and stellate cells [P = 0.0001]). Cholangiocytes and hepatocytes showed no age-related telomere shortening. CONCLUSION: In normal liver and over a broad age range, cholangiocytes have longer telomeres than all other intrahepatic lineages. Age-related telomere length decline is restricted to Kupffer cells and stellate cells.

Chronic Hepatitis B Virus Infection: The Relation between Hepatitis B Antigen Expression, Telomere Length, Senescence, Inflammation and Fibrosis.

BACKGROUND: Chronic Hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that HBV might accelerate hepatocyte ageing and investigated the effect of HBV on hepatocyte cell cycle state and biological age. We also investigated the relation between inflammation, fibrosis and cell cycle phase. METHODS: Liver samples from patients with chronic HBV (n = 91), normal liver (n = 55) and regenerating liver (n = 15) were studied. Immunohistochemistry for cell cycle phase markers and HBV antigens was used to determine host cell cycle phase. Hepatocyte-specific telomere length was evaluated by quantitative fluorescent in-situ hybridization (Q-FISH) in conjunction with hepatocyte nuclear area and HBV antigen expression. The effects of induced cell cycle arrest and induced cellular senescence on HBV production were assessed in vitro. RESULTS: 13.7% hepatocytes in chronic HBV had entered cell cycle, but expression of markers for S, G2 and M phase was low compared with regenerating liver. Hepatocyte p21 expression was increased (10.9%) in chronic HBV and correlated with liver fibrosis. Mean telomere length was reduced in chronic HBV compared to normal. However, within HBV-affected livers, hepatocytes expressing HBV antigens had longer telomeres. Telomere length declined and hepatocyte nuclear size increased as HBV core antigen (HBcAg) expression shifted from the nucleus to cytoplasm. Nuclear co-expression of HBcAg and p21 was not observed. Cell cycle arrest induced in vitro was associated with increased HBV production, in contrast to in vitro induction of cellular senescence, which had no effect. CONCLUSION: Chronic HBV infection was associated with hepatocyte G1 cell cycle arrest and accelerated hepatocyte ageing, implying that HBV induced cellular senescence. However, HBV replication was confined to biologically younger hepatocytes. Changes in the cellular location of HBcAg may be related to the onset of cellular senescence.

Spontaneous Incomplete transverse subtrochanteric femoral fracture with cortical thickening possibly secondary to risedronate use: a case report.

INTRODUCTION: Osteoporosis is an asymptomatic disease characterized by bone weakening and predisposition to fragility (insufficiency) fractures and can have devastating effects on individual life and great financial impact on the economy. Bisphosphonates are used worldwide for the primary and secondary prevention of osteoporotic fractures. However, increasing evidence raises concern that bisphosphonates can be associated with atypical fractures. CASE PRESENTATION: A 65-year-old Caucasian woman on long-term steroid treatment for polymyalgia rheumatica was admitted with severe and constant pain in the right hip, radiating to the right knee. She had a history of steroid-induced osteoporosis, for which she was started on risedronate four years earlier. She had no history of trauma. Her blood results were unremarkable. Her X-rays confirmed that she had an incomplete right subtrochanteric femoral fracture. A bone scan confirmed the diagnosis and also ruled out any other associated fractures. Our patient successfully underwent internal nail fixation of the fracture. She was reviewed by a rheumatology team, which stopped the risedronate. She was started on treatment with denosumab injection. CONCLUSIONS: Previous case series have reported that long-term bisphosphonate use is associated with atypical fractures of the femur, and certain criteria have been established to help identify such rare fractures. Delayed union or non-union is expected in such fractures following definitive orthopedic treatment because of the long half life of bisphosphonates. In this case report, we try to raise questions related to this important subject, like the duration and safety of bisphosphonate use and the alternative medications used in osteoporosis in this rare condition. We consider this case report not only interesting but also important and unusual because it is about a patient who developed a potentially rare and serious side effect of long-term bisphosphonate use, estimated to affect 2.3 in every 10,000 patients, and who presented with a pelvic X-ray that showed the characteristic features of atypical fractures secondary to risedronate use. In addition, most of the documented cases have been associated with many years of bisphosphonate use whereas our patient had been on risedronate for only four years.

Population prevalence and symptom associations of autonomic dysfunction in primary biliary cirrhosis.

UNLABELLED: Patients with primary biliary cirrhosis (PBC) frequently experience significant fatigue thought to result from as-yet-unidentified central nervous system (CNS)-mediated processes. Pilot studies have suggested that autonomic dysfunction is a frequent occurrence in PBC and may contribute to the pathogenesis of this fatigue. The degree to which autonomic dysfunction affects the PBC population as a whole, and its interrelationship with other symptoms experienced by PBC patients remains unstudied. In this study, we used a geographically defined, fully representative PBC patient cohort to study the prevalence of symptoms of autonomic dysfunction and its relationship with other symptoms of PBC. Symptoms of cardiovascular autonomic dysfunction (as assessed using the Orthostatic Grading Scale [OGS]) were significantly more frequently reported and significantly more severe in PBC patients than in both matched normal controls (40% versus 6% with moderate or worse orthostasis (P < .0001), mean OGS score 3.2 +/- 3.4 versus 1.3 +/- 1.9, P < .005) and in patients with primary sclerosing cholangitis and in severity were independently associated with severity of fatigue and cognitive symptoms (both r2 = 0.3, P < .0001). Thirteen of 20 patients with an OGS value > 4 (moderate severity and worse) had significant abnormality in autonomic regulation of blood pressure, which was identified on dynamic testing. CONCLUSION: Symptoms suggestive of autonomic dysfunction frequently occur in PBC patients and reflect dysregulation of actual blood pressure. Autonomic dysfunction is independently associated with both fatigue and, importantly, symptoms of cognitive dysfunction, suggesting the potential for significant organic sequelae.