A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.

The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.

Long-term ex situ normothermic machine perfusion allows regeneration of human livers with severe bile duct injury.

Bile duct regeneration is hypothesized to prevent biliary strictures, a leading cause of morbidity after liver transplantation. Assessing the capacity for biliary regeneration may identify grafts as suitable for transplantation that are currently declined, but this has been unfeasible until now. This study used long-term ex situ normothermic machine perfusion (LT-NMP) to assess biliary regeneration. Human livers that were declined for transplantation were perfused at 36 °C for up to 13.5 days. Bile duct biopsies, bile, and perfusate were collected throughout perfusion, which were examined for features of injury and regeneration. Biliary regeneration was defined as new Ki-67-positive biliary epithelium following severe injury. Ten livers were perfused for a median duration of 7.5 days. Severe bile duct injury occurred in all grafts, and biliary regeneration occurred in 70% of grafts. Traditional biomarkers of biliary viability such as bile glucose improved during perfusion but this was not associated with biliary regeneration (P > .05). In contrast, the maintenance of interleukin-6 and vascular endothelial growth factor-A levels in bile was associated with biliary regeneration (P = .017 for both cytokines). This is the first study to demonstrate biliary regeneration during LT-NMP and identify a cytokine signature in bile as a novel biomarker for biliary regeneration during LT-NMP.

Incidence, epidemiology, and outcomes of acute allograft rejection following liver transplantation in Australia.

Acute allograft rejection is a well-known complication of liver transplantation (LT). The incidence, epidemiology, and outcomes of acute rejection have not been well described in Australia. We retrospectively studied consecutive adults who underwent deceased donor LT at a single center between 2010 and 2020. Donor and recipient data at the time of LT and recipient outcomes were collected from a prospective LT database. Liver biopsy reports were reviewed, and only a graft's first instance of biopsy-proven acute rejection was analyzed. During the study period, 796 liver transplants were performed in 770 patients. Biopsy-proven rejection occurred in 34.9% of transplants. There were no significant changes in the incidence of rejection over time (linear trend p =0.11). The median time to the first episode of rejection was 71 days after LT: 2.2% hyperacute, 50.4% early (≤90 d), and 47.5% late rejection (>90 d). Independent risk factors for rejection were younger recipient age at transplant (aHR 0.98 per year increase, 95% CI: 0.97-1.00, p =0.01), and ABO-incompatible grafts (aHR 2.55 vs. ABO-compatible, 95% CI: 1.27-5.09, p <0.01) while simultaneous multiorgan transplants were protective (aHR 0.21 vs. LT only, 95% CI: 0.08-0.58, p <0.01). Development of acute rejection (both early and late) was independently associated with significantly reduced graft (aHR 3.13, 95% CI: 2.21-4.42, p <0.001) and patient survival (aHR 3.42, 95% CI: 2.35-4.98, p <0.001). In this 11-year Australian study, acute LT rejection occurred in 35%, with independent risk factors of younger recipient age and ABO-incompatible transplant, while having a simultaneous multiorgan transplant was protective. Acute rejection was independently associated with reduced graft and patient survival after adjustment for other factors.

Impact of Share 35 liver transplantation allocation in Australia and New Zealand.

Patients with high model for end-stage liver disease (MELD) scores waiting for liver transplantation in Australia and New Zealand (ANZ) have had limited access to deceased donor livers and therefore binational sharing of livers, for patients with a MELD score ≥35 was introduced in February 2016. Waiting list mortality, post-transplant outcomes and intention-to-treat survival were compared between patients whose MELD score reached 35 on the waiting list between October 2013 and April 2015 (Pre-Share 35 group, n = 23) and patients who were Share 35 listed between February 2016 and May 2022 (Share 35 group, n = 112). There was significantly reduced waiting list mortality in share 35 listed patients in comparison to the pre-Share 35 group (11.7% vs. 52.2%, OR .120 95% CI .044-.328, P < .001). Post-transplant patient and graft survival were not significantly different between the groups (5-year patient survival 82% vs. 84%, P = .991, 5-year graft survival 82% vs. 76%, P = .543). Intention-to-treat survival was superior in the Share 35 group (HR .302, 95% CI .149-.614, P < .001). Introduction of Share 35 in ANZ resulted in a 78% risk reduction in waiting list mortality, equivalent post-transplant survival and an improvement in intention-to-treat survival.

Validation of a novel point-of-care test for alanine aminotransferase measurement: A pilot cohort study.

BACKGROUND: Alanine aminotransferase (ALT) measurement is essential for evaluation of liver disease. We validated a novel rapid point-of-care (POC) test for ALT1 against laboratory ALT. METHODS: Stored plasma samples from adults with chronic liver disease (Test cohort n = 240; Validation cohort n = 491) were analysed using the BioPoint® antigen immunoassay POC ALT1 lateral flow test, which provides quantitative ALT results (Axxin handheld reader) or semi-quantitative results (visual read, cut off 40 IU/ml). The accuracy of POC ALT1 to detect ALT > 40 IU/L was determined by ROC analysis. In patients with chronic hepatitis B, treatment eligibility (EASL criteria) was determined using POC ALT1 and compared to laboratory ALT. RESULTS: POC ALT1 test had good accuracy for laboratory ALT > 40 IU/L: AUROC 0.93 (95% CI: 0.89-0.96) in the Test cohort and AUROC 0.92 (95% CI: 0.88-0.95) in the Validation cohort. POC ALT1 cut off of 0.8 for ALT > 40 IU/L maximised sensitivity (97%) and specificity (71%) in the Test cohort (42% laboratory ALT > 40 IU/L) and yielded PPV 84% and NPV 91% in the Validation cohort (19% laboratory ALT > 40 IU/L). Semi-quantitative POC ALT1 had good accuracy for laboratory ALT in the Validation cohort (AUROC 0.85, 95% CI: 0.81-0.99; sensitivity 77% and specificity 93%). Combined with HBV DNA and transient elastography, both quantitative and semi-quantitative POC ALT1 tests had good accuracy for excluding hepatitis B treatment needs (sensitivity 96%, specificity 78% and NPV 99%). CONCLUSION: The POC ALT1 test had good accuracy for elevated ALT levels and for determining treatment eligibility among people with chronic hepatitis B.

Epidemiology, characteristics, and outcomes of patients with acute-on-chronic liver failure in Australia.

BACKGROUND AND AIM: Acute-on-chronic liver failure (ACLF) is distinct from acute decompensation (AD) of cirrhosis in its clinical presentation, pathophysiology, and prognosis. There are limited published Australian ACLF data. METHODS: We performed a single-center retrospective cohort study of all adults with cirrhosis admitted with a decompensating event to a liver transplantation (LT) centre between 2015 and 2020. ACLF was defined using the European Association for the Study of the Liver-Chronic Liver Failure (EASL-CLIF) definition while those who did not meet the definition were classified as AD. The primary outcome of interest was 90-day LT-free survival. RESULTS: A total of 615 patients had 1039 admissions for a decompensating event. On their index admission, 34% (209/615) of patients were classified as ACLF. Median admission model for end-stage liver disease (MELD) and MELD-Na scores were higher in ACLF patients compared with AD (21 vs 17 and 25 vs 20 respectively, both P < 0.001). Both the presence and severity of ACLF (grade ≥ 2) significantly predicted worse LT-free survival compared with patients with AD. The EASL-CLIF ACLF score (CLIF-C ACLF), MELD and MELD-Na scores performed similarly in predicting 90-day mortality. Patients with index ACLF had a higher risk of 28-day mortality (28.1% vs 5.1%, P < 0.001) and shorter times to readmission compared with those with AD. CONCLUSION: ACLF complicates over a third of hospital admissions for cirrhosis with decompensating events and is associated with a high short-term mortality. The presence and grade of ACLF predicts 90-day mortality and should be identified as those at greatest risk of poor outcome without intervention such as LT.

Liver-specific deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7.

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

The liver contains distinct interconnected networks of CX3CR1+ macrophages, XCR1+ type 1 and CD301a+ type 2 conventional dendritic cells embedded within portal tracts.

Portal tracts are key intrahepatic structures where leukocytes accumulate during immune responses. They contain the blood inflow, which includes portal blood from the gut, and lymphatic and biliary outflow of the liver, and as such represent a key interface for potential pathogen entry to the liver. Myeloid cells residing in the interstitium of the portal tract might play an important role in the surveillance or prevention of pathogen dissemination; however, the exact composition and localization of this population has not been explored fully. Our in-depth characterization of portal tract myeloid cells revealed that in addition to T lymphocytes, portal tracts contain a heterogeneous population of MHCIIhigh myeloid cells with potential antigen presenting cell (APC) function. These include a previously unreported subset of CSF1R-dependent CX3CR1+ macrophages that phenotypically and morphologically resemble liver capsular macrophages, as well as the two main dendritic cell subsets (cDC1 and cDC2). These cells are not randomly distributed, but each subset forms interconnected networks intertwined with specific components of the portal tract. The CX3CR1+ cells were preferentially detected along the outer border of the portal tracts, and also in the portal interstitium adjacent to the portal vein, bile duct, lymphatic vessels and hepatic artery. cDC1s abounded along the lymphatic vessels, while cDC2s mostly surrounded the biliary tree. The specific distributions of these discrete subsets predict that they may serve distinct functions in this compartment. Overall, our findings suggest that portal tracts and their embedded cellular networks of myeloid cells form a distinctive lymphoid compartment in the liver that has the potential to orchestrate immune responses in this organ.

Is it safe to expand the indications for split liver transplantation in adults? A single-center analysis of 155 in-situ splits.

INTRODUCTION: Split liver transplantation (SLT) enables two recipients to be transplanted using a single donor liver; typically, an adult and a child. Despite equivalent long-term outcomes to whole grafts in selected adults, the use of these grafts in high-risk adult recipients with high model for end-stage liver disease (MELD) scores (≥30), a poor pre-transplant clinical status (ICU or hospital-bound), acute liver failure or retransplantation remains controversial. METHODS: We retrospectively analyzed all deceased donor adult liver transplants performed between July 2002 and November 2019 at a single high-volume center and performed a propensity score-matched analysis. A subgroup analysis was performed to assess utility of these grafts for high-risk recipients. RESULTS: A total of 1090 adult liver transplants were performed, including 155 SLT (14%). Graft survival at 1-, 3- and 5-years were comparable between recipients of split and whole liver grafts (82%, 79% and 74% vs. 86%, 81% and 77%, respectively, log rank P = .537), as was patient survival at 1-, 3- and 5-years. Recipients of split grafts were more likely to have biliary complications and hepatic artery thrombosis, but equivalent long-term survival. Recipients with high MELD scores or a poor pre-transplant clinical status had similar patient and graft survival and complication profiles irrespective of whether they received split or whole grafts. CONCLUSIONS: SLT is an important method for addressing donor shortages and provides comparable long-term outcomes in adult recipients despite an increase in short-term complications. SLT use in high-risk recipients should be considered to allow for sickest-first allocation policies.

Prevalence and outcomes of Clostridioides difficile infection in liver transplant recipients.

BACKGROUND AND AIM: Data are limited on whether Clostridioides difficile infection (CDI) in the first year after liver transplantation (LT) is associated with increased mortality. In an Australian setting without hypervirulent strain of C. difficile we investigated the prevalence, risk factors, and patient survival associated with CDI in LT. METHODS: Consecutive patients who underwent deceased-donor LT from 2007 to 2017 were studied retrospectively. Prevalence and long-term outcomes of LT recipients with and without CDI were examined in the entire LT cohort. A case-control study was performed to investigate risk factors associated with CDI. RESULTS: Six hundred and forty-nine patients underwent LT, of which 32 (4.9%) were diagnosed with CDI within the first 12 months post-LT. There was no difference in patient survival in the overall LT cohort on Kaplan-Meier analysis when stratified by CDI status (log-rank test, p = .08). Furthermore, age was the only predictor of mortality on Cox regression (hazard ratio (HR) 1.06, 95% confidence interval (CI) 1.00-1.13, p = .03). On multivariable logistic regression, rifaximin pre-LT reduced risk (odds ratio (OR) 0.22, 95% CI 0.65-0.74, p = .01) whereas antibiotics pre-LT (OR 7.02, 95% CI 1.26-39.01, p = .03) and length of hospital stay after LT (OR 1.03, 95% CI 1.01-1.06, p = .02) were associated with increased risk of CDI. CONCLUSIONS: Within the local setting of our study, CDI within 12 months post-LT is of low severity, associated with pre-LT antibiotic exposure and longer hospital stay but no survival impact after LT. Rifaximin use pre-LT reduced the risk of CDI post-LT.

Platelet activating factor receptor acts to limit colitis-induced liver inflammation.

Liver inflammation is a common extraintestinal manifestation in inflammatory bowel disease (IBD), yet, the mechanisms driving gut-liver axis inflammation remain poorly understood. IBD leads to a breakdown in the integrity of the intestinal barrier causing an increase in portal and systemic gut-derived antigens, which challenge the liver. Here, we examined the role of platelet activating factor receptor (PAFR) in colitis-associated liver damage using dextran sulfate sodium (DSS) and anti-CD40-induced colitis models. Both DSS and anti-CD40 models exhibited liver inflammation associated with colitis. Colitis reduced global PAFR protein expression in mouse livers causing an exclusive re-localization of PAFR to the portal triad. The global decrease in liver PAFR was associated with increased sirtuin 1 while relocalized PAFR expression was limited to Kupffer cells (KCs) and co-localized with toll-like receptor 4. DSS activated the NLRP3-inflammasome and increased interleukin (IL)-1ß in the liver. Antagonism of PAFR amplified the inflammasome response by increasing NLRP3, caspase-1, and IL-1ß protein levels in the liver. LPS also increased NLRP3 response in human hepatocytes, however, overexpression of PAFR restored the levels of NLPR3 and caspase-1 proteins. Interestingly, KCs depletion also increased IL-1ß protein in mouse liver after DSS challenge. These data suggest a protective role for PAFR-expressing KCs during colitis and that regulation of PAFR is important for gut-liver axis homeostasis.

An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha.

Fibroblast activation protein alpha (FAP) is a cell-surface expressed type II glycoprotein that has a unique proteolytic activity. FAP has active soluble forms that retain the extracellular portion but lack the transmembrane domain and cytoplasmic tail. FAP expression is normally very low in adult tissue but is highly expressed by activated fibroblasts in sites of tissue remodelling. Thus, FAP is a potential biomarker and pharmacological target in liver fibrosis, atherosclerosis, cardiac fibrosis, arthritis and cancer. Understanding the biological significance of FAP by investigating protein structure, interactions and activities requires reliable methods for the production and purification of abundant pure and stable protein. We describe an improved production and purification protocol for His6-tagged recombinant soluble human FAP. A modified baculovirus expression construct was generated using the pFastBac1 vector and the gp67 secretion signal to produce abundant active soluble recombinant human FAP (residues 27-760) in insect cells. The FAP purification protocol employed ammonium sulphate precipitation, ion exchange chromatography, immobilised metal affinity chromatography and ultrafiltration. High purity was achieved, as judged by gel electrophoresis and specific activity. The purified 82 kDa FAP protein was specifically inhibited by a FAP selective inhibitor, ARI-3099, and was inhibited by zinc with an IC50 of 25 µM. Our approach could be adopted for producing the soluble portions of other type II transmembrane glycoproteins to study their structure and function.

Drug-induced liver injury in Australia, 2009-2020: the increasing proportion of non-paracetamol cases linked with herbal and dietary supplements.

OBJECTIVE: To compare the characteristics and outcomes of drug-induced liver injury (DILI) caused by paracetamol and non-paracetamol medications, particularly herbal and dietary supplements. DESIGN: Retrospective electronic medical record data analysis. SETTING, PARTICIPANTS: Adults admitted with DILI to the Gastroenterology and Liver Centre at the Royal Prince Alfred Hospital, Sydney (a quaternary referral liver transplantation centre), 2009-2020. MAIN OUTCOME MEASURES: 90-day transplant-free survival; drugs implicated as causal agents in DILI. RESULTS: A total of 115 patients with paracetamol-related DILI and 69 with non-paracetamol DILI were admitted to our centre. The most frequently implicated non-paracetamol medications were antibiotics (19, 28%), herbal and dietary supplements (15, 22%), anti-tuberculosis medications (six, 9%), and anti-cancer medications (five, 7%). The number of non-paracetamol DILI admissions was similar across the study period, but the proportion linked with herbal and dietary supplements increased from 2 of 13 (15%) during 2009-11 to 9 of 19 (47%) during 2018-20 (linear trend: P = 0.011). Despite higher median baseline model for end-stage liver disease (MELD) scores, 90-day transplant-free survival for patients with paracetamol-related DILI was higher than for patients with non-paracetamol DILI (86%; 95% CI, 79-93% v 71%; 95% CI, 60-82%) and herbal and dietary supplement-related cases (59%; 95% CI, 34-85%). MELD score was an independent predictor of poorer 90-day transplant-free survival in both paracetamol-related (per point increase: adjusted hazard ratio [aHR], 1.19; 95% CI, 1.09-3.74) and non-paracetamol DILI (aHR, 1.24; 95% CI, 1.14-1.36). CONCLUSION: In our single centre study, the proportion of cases of people hospitalised with DILI linked with herbal and dietary supplements has increased since 2009. Ninety-day transplant-free survival for patients with non-paracetamol DILI, especially those with supplement-related DILI, is poorer than for those with paracetamol-related DILI.

Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics.

Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

Differential Impact of Extended Criteria Donors After Brain Death or Circulatory Death in Adult Liver Transplantation.

Using grafts from extended criteria donors (ECDs) and donation after circulatory death (DCD) donors is a strategy to address organ shortage in liver transplantation (LT). We studied the characteristics and outcomes of ECD and DCD grafts. We retrospectively studied consecutive adults who underwent deceased donor LT between 2006 and 2019. ECD was defined using modified Eurotransplant criteria. Our primary outcomes were graft and patient survival. A total of 798 grafts were used for LT, of which 93.1% were donation after brain death (DBD; 59.9% were also ECD) and 6.9% were DCD grafts (49.1% were also ECD). Among DBD graft recipients, donors having >33% liver steatosis or 3 ECD criteria resulted in poorer graft survival. Otherwise ECD graft recipients had similar graft and patient survival compared with non-ECD graft recipients. DCD graft recipients also had similar patient survival compared with DBD recipients. However, DCD grafts from an ECD appeared to have worse outcomes. DCD graft recipients experienced higher rates of early allograft dysfunction (50.9% versus 24.7%; P < 0.001) and ischemic biliopathy (16.4% versus 1.5%; P < 0.001) compared with DBD graft recipients. Use of DBD grafts from ECDs did not impact outcomes unless there was significant donor steatosis or 3 Eurotransplant criteria were met. DCD graft recipients have similar patient survival compared with DBD graft recipients as long as the donor was not an ECD. We recommend that DBD donors with 3 or more ECD features or >33% steatosis and DCD donors with any ECD features be used with caution in adult LT.

Efficacy and safety profile of calcineurin inhibitor salvage therapy in autoimmune hepatitis.

BACKGROUND: As data is limited on the outcomes of calcineurin inhibitors (CNI) in autoimmune hepatitis (AIH), we evaluated the efficacy and safety of CNI in AIH patients who failed prior treatment(s). METHODS: A retrospective study was performed of AIH patients who received cyclosporine A (CsA) and/or tacrolimus (TAC) after prior treatment(s) failure. Records were reviewed for baseline demographic and clinical characteristics, and treatment outcomes. The primary outcome was biochemical remission.Results: Thirty-three AIH patients received CNI across seven liver centers:17 received CsA, 21 TAC and 5 TAC after CsA failure/intolerance. 82% received CNI for an insufficient response to treatment(s). Overall, 48% of CNI treated patients achieved biochemical remission including 41% in prior non-responders and 83% in treatment intolerant patients. Remission rates with CNI as second-line and third-line therapy were 63% and 29% respectively. There were no baseline predictors of response to CNI on multivariate analysis. Eighteen (55%) patients developed significant side effects and 8 (24%) discontinued due to intolerance. Three patients required liver transplantation for decompensated cirrhosis and 6 patients died including one from malignancy possibly related to CNI. CONCLUSION: CNI salvage therapy is well tolerated and moderately effective achieving remission in around 50% of AIH who failed standard therapy.

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2.

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

Anti-PD-1-induced high-grade hepatitis associated with corticosteroid-resistant T cells: a case report.

Effective treatment or prevention of immune side effects associated with checkpoint inhibitor therapy of cancer is an important goal in this new era of immunotherapy. Hepatitis due to immunotherapy with antibodies against PD-1 is uncommon and generally of low severity. We present an unusually severe case arising in a melanoma patient after more than 6 months uncomplicated treatment with anti-PD-1 in an adjuvant setting. The hepatitis rapidly developed resistance to high-dose steroids, requiring anti-thymocyte globulin (ATG) to achieve control. Mass cytometry allowed comprehensive phenotyping of circulating lymphocytes and revealed that CD4+ T cells were profoundly depleted by ATG, while CD8+ T cells, B cells, NK cells and monocytes were relatively spared. Multiple abnormalities in CD4+ T cell phenotype were stably present in the patient before disease onset. These included a population of CCR4-CCR6- effector/memory CD4+ T cells expressing intermediate levels of the Th1-related chemokine receptor CXCR3 and abnormally high multi-drug resistance type 1 transporter (MDR1) activity as assessed by a rhodamine 123 excretion assay. Expression of MDR1 has been implicated in steroid resistance and may have contributed to the severity and lack of a sustained steroid response in this patient. The number of CD4+ rhodamine 123-excreting cells was reduced > 3.5-fold after steroid and ATG treatment. This case illustrates the need to consider this form of steroid resistance in patients failing treatment with corticosteroids. It also highlights the need for both better identification of patients at risk and the development of treatments that involve more specific immune suppression.

Molecular profiling of postreperfusion milieu determines acute kidney injury after liver transplantation: A prospective study.

Acute kidney injury (AKI) after liver transplantation (LT) is a common event, but its pathogenesis remains unclear. The aim of this prospective study is to investigate the potential relationship between postreperfusion gene expression, serum mediators, and the onset of AKI after LT. Sixty-five consecutive patients undergoing LT were included in the study. Reverse transcription polymerase chain reaction (PCR) was performed on liver biopsies. Gene expression of 23 genes involved in ischemia/reperfusion injury (IRI) was evaluated. The serum concentrations of endothelin (ET)-1 and inflammatory cytokines were analyzed. AKI after LT developed in 21 (32%) recipients (AKI group). Reverse transcription PCR of reperfusion biopsy in the AKI group showed higher expression of several genes involved in IRI compared with the non-AKI group. Fold changes in the gene expression of ET-1, interleukin (IL) 18, and tumor necrosis factor α (TNF-α) were associated with creatinine peak value. AKI patients also had significantly higher ET-1, IL18, and TNF-α postoperative serum levels. Multivariate analysis showed that ET-1 (odds ratio [OR], 16.7; 95% confidence interval [CI], 3.34-83.42; P = 0.001) and IL18 (OR, 5.27; 95% CI, 0.99-27.82, P = 0.048) serum levels on postoperative day 1 were independently predictive of AKI. Receiver operating characteristic analysis demonstrated that the combination of biomarkers ET-1+IL18 was highly predictive of AKI (area under the receiver operating characteristic curve, 0.91; 95% CI, 0.83-0.99). Early allograft dysfunction and chronic kidney disease stage ≥ 2 occurred more frequently in AKI patients. These results suggest that the graft itself, rather than intraoperative hemodynamic instability, plays a main role in AKI after LT. These data may have mechanistic and diagnostic implications for AKI after LT. Liver Transplantation 24 922-931 2018 AASLD.