The role of the coagulation cascade in liver injury

Liver disease affects approximately 600,000 people in England and Wales and is the third biggest cause of premature death. Acute and chronic liver injury occur due to a number of aetiologies and affect the function of the liver in the short and long term through pathological inflammation and fibrosis. Given the variety of aetiologies and growing burden of disease in the population, there is a need to find universal therapies that alter the progression of liver injury and fibrosis beyond the initiating insult. This work focuses on investigating the role of the coagulation cascade, specifically Tissue Factor Pathway Inhibitor (TFPI), in liver injury and it’s use as a potential therapeutic target for altering the progression of acute and chronic liver injury. Using two transgenic strains of mice, expressing TFPI in a cell specific manner I have shown that TFPI decreases the extent and progression of liver injury in models of acute liver injury but does not modify the development of liver fibrosis in a model of chronic liver injury. In the models of acute liver injury there was decreased liver injury associated with decreased fibrin deposition, decreased hepatic stellate cell activation, decreased total liver macrophage content and decreased PAR2 expression. The pattern of changes suggests that TFPI acts early in the injury process, limiting total hepatocyte injury and resulting in a decrease in hepatic stellate cell activation and macrophage recruitment, rather than the other way round. Further work should focus on defining the inflammatory cytokine profile of the liver of these transgenic mice in acute liver injury with the aim of describing the biological mechanism for the action of TFPI in acute liver injury and taking forward the trial of TFPI administration to control mice in a manner that could be carried forward to human studies.Open Acces

Biallelic TMEM260 variants cause truncus arteriosus, with or without renal defects

Only two families have been reported with biallelic TMEM260 variants segregating with structural heart defects and renal anomalies syndrome (SHDRA). With a combination of genome, exome sequencing and RNA studies, we identified eight individuals from five families with biallelic TMEM260 variants. Variants included one multi-exon deletion, four nonsense/frameshifts, two splicing changes and one missense change. Together with the published cases, analysis of clinical data revealed ventricular septal defects (12/12), mostly secondary to truncus arteriosus (10/12), elevated creatinine levels (6/12), horse-shoe kidneys (1/12) and renal cysts (1/12) in patients. Three pregnancies were terminated on detection of severe congenital anomalies. Six patients died between the ages of 6 weeks and 5 years. Using a range of stringencies, carrier frequency for SHDRA was estimated at 0.0007–0.007 across ancestries. In conclusion, this study confirms the genetic basis of SHDRA, expands its known mutational spectrum and clarifies its clinical features. We demonstrate that SHDRA is a severe condition associated with substantial mortality in early childhood and characterised by congenital cardiac malformations with a variable renal phenotype

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure.

OBJECTIVE: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. DESIGN: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer-/-) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. RESULTS: We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer-/- mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. CONCLUSIONS: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury

Biallelic TMEM260 variants cause truncus arteriosus, with or without renal defects

From Wiley via Jisc Publications RouterHistory: received 2021-08-18, rev-recd 2021-09-22, accepted 2021-10-02, pub-electronic 2021-10-11Article version: VoRPublication status: PublishedFunder: Cancer Research UK; Id: http://dx.doi.org/10.13039/501100000289Funder: European Union's Horizon 2020; Grant(s): 779257Funder: Medical Research Council; Id: http://dx.doi.org/10.13039/501100007155Funder: NHS EnglandFunder: NIHR Oxford Biomedical Research Centre ProgrammeFunder: Society for the Relief of Disabled Children, Hong KongFunder: Wellcome Trust; Id: http://dx.doi.org/10.13039/100010269; Grant(s): 203141/Z/16/ZAbstract: Only two families have been reported with biallelic TMEM260 variants segregating with structural heart defects and renal anomalies syndrome (SHDRA). With a combination of genome, exome sequencing and RNA studies, we identified eight individuals from five families with biallelic TMEM260 variants. Variants included one multi‐exon deletion, four nonsense/frameshifts, two splicing changes and one missense change. Together with the published cases, analysis of clinical data revealed ventricular septal defects (12/12), mostly secondary to truncus arteriosus (10/12), elevated creatinine levels (6/12), horse‐shoe kidneys (1/12) and renal cysts (1/12) in patients. Three pregnancies were terminated on detection of severe congenital anomalies. Six patients died between the ages of 6 weeks and 5 years. Using a range of stringencies, carrier frequency for SHDRA was estimated at 0.0007–0.007 across ancestries. In conclusion, this study confirms the genetic basis of SHDRA, expands its known mutational spectrum and clarifies its clinical features. We demonstrate that SHDRA is a severe condition associated with substantial mortality in early childhood and characterised by congenital cardiac malformations with a variable renal phenotype

Aurora A kinase inhibition destabilizes PAX3-FOXO1 and MYCN and synergizes with Navitoclax to induce Rhabdomyosarcoma cell death

The clinically aggressive alveolar rhabdomyosarcoma subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacological combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as a top compound in a screen of 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS

Defective monocyte oxidative burst predicts infection in alcoholic hepatitis and is associated with reduced expression of NADPH oxidase

ObjectiveIn order to explain the increased susceptibility to serious infection in alcoholic hepatitis, we evaluated monocyte phagocytosis, aberrations of associated signalling pathways and their reversibility, and whether phagocytic defects could predict subsequent infection.DesignMonocytes were identified from blood samples of 42 patients with severe alcoholic hepatitis using monoclonal antibody to CD14. Phagocytosis and monocyte oxidative burst (MOB) were measured ex vivo using flow cytometry, luminometry and bacterial killing assays. Defects were related to the subsequent development of infection. Intracellular signalling pathways were investigated using western blotting and PCR. Interferon-γ (IFN-γ) was evaluated for its therapeutic potential in reversing phagocytic defects. Paired longitudinal samples were used to evaluate the effect of in vivo prednisolone therapy.ResultsMOB, production of superoxide and bacterial killing in response toEscherichia coliwere markedly impaired in patients with alcoholic hepatitis. Pretreatment MOB predicted development of infection within two weeks with sensitivity and specificity that were superior to available clinical markers. Accordingly, defective MOB was associated with death at 28 and 90 days. Expression of the gp91phoxsubunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was reduced in patients with alcoholic hepatitis demonstrating defective MOB. Monocytes were refractory to IFN-γ stimulation and showed high levels of a negative regulator of cytokine signalling, suppressor of cytokine signalling-1. MOB was unaffected by 7 days in vivo prednisolone therapy.ConclusionsMonocyte oxidative burst and bacterial killing is impaired in alcoholic hepatitis while bacterial uptake by phagocytosis is preserved. Defective MOB is associated with reduced expression of NADPH oxidase in these patients and predicts the development of infection and death.</jats:sec

Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells

BACKGROUND: rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (&lt; 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours.RESULTS: we demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS.CONCLUSIONS: the results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients.</p

Achieving an early pregnancy following allogeneic uterine transplantation in a rabbit model

[EN] Objective: Uterine transplantation (UTx) has been proposed as a treatment option for women diagnosed with absolute uterine factor infertility (AUFI). The goal of UTx remains achieving pregnancy and live birth of a healthy neonate following allogeneic UTx. Our aim was to assess whether fertility was possible following allogeneic uterine transplantation (UTx), when the recipient had demonstrated long-term survival and had been administered immunosuppression. Study design: Nine allogeneic UTx in New Zealand White rabbits were performed using a pre-determined protocol. Tacrolimus was the immunosuppressant selected. Embryos were transferred into both cornua of the sole living recipient via a mini-midline laparotomy. The pregnancy was monitored with regular reproductive profiles and serial trans-abdominal ultrasound to measure conceptus growth (gestation sac and crown rump length (CRL)). Results: In the sole surviving doe a gestation sac was visualised on ultrasound from Day 9 (D9) after embryo transfer. Gestation sac diameter and CRL increased from D9 to D16 but by D18 the gestation sac had reduced in size. The fetus was no longer visible, suggesting fetal resorption had occurred. Subsequent scans on D22 and D25 did not demonstrate a gestation sac. Scheduled necropsy on D27 and histopathology confirmed evidence of a gravid uterus and presence of a gestational sac. A single episode of acute rejection occurred on D13. Conclusion: Pregnancy was achieved after rabbit allogeneic UTx but serial ultrasound suggested that fetal demise occurred prior to scheduled necropsy. The study represents only the third example of conception and pregnancy following an animal allogeneic UTx.The authors would like to thank all the staff and veterinarians at the Royal Veterinary College (London, UK) who have been with us from the beginning and have worked tirelessly at ensuring we had the best possible conditions to carry out our transplant work. ALD receives funding at UCLH/UCL via the Department of Health's NIHR Biomedical Research Centres funding scheme.Saso, S.; Petts, G.; David, AL.; Thum, M.; Chatterjee, J.; Vicente Antón, JS.; Marco Jiménez, F.... (2015). Achieving an early pregnancy following allogeneic uterine transplantation in a rabbit model. European Journal of Obstetrics and Gynecology and Reproductive Biology. (185):164-169. doi:10.1016/j.ejogrb.2014.12.017S16416918