Early γGT and bilirubin levels as biomarkers for regeneration and outcomes in damaged bile ducts after liver transplantation.

BACKGROUND: Early patient and allograft survival after liver transplantation (LT) depend primarily on parenchymal function, but long-term allograft success relies often on biliary-tree function. We examined parameters related to cholangiocyte damage that predict poor long-term LT outcomes after donation after brain death (DBD). METHODS: Sixty bile ducts (BD) were assessed by a BD damage-score and divided into groups with "major" BD-damage (n = 33) and "no relevant" damage (n = 27) during static cold storage. Patients with "major" BD damage were further investigated by measuring biliary excretion parameters in the first 14 days post-LT (followed-up for 60-months). RESULTS: Patients who received LT showing "major" BD damage had significantly worse long-term patient survival, versus grafts with "no relevant" damage (p = .03). When "major" BD damage developed, low bilirubin levels (p = .012) and high gamma-glutamyl transferase (GGT)/bilirubin ratio (p = .0003) were evident in the early post-LT phase (7-14 days) in patients who survived (> 60 months), compared to those who did not. "High risk" patients with bile duct damage and low GGT/bilirubin ratio had significantly shorter overall survival (p < .0001). CONCLUSIONS: Once "major" BD damage occurs, a high GGT/bilirubin ratio in the early post-operative phase is likely indicator of liver and cholangiocyte regeneration, and thus a harbinger of good overall outcomes. "Major" BD damage without markers of regeneration identifies LT patients that could benefit from future repair therapies.

A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

Steatotic Livers Are More Susceptible to Ischemia Reperfusion Damage after Transplantation and Show Increased γδ T Cell Infiltration.

Liver transplantation (LTx) is often the only possible therapy for many end-stage liver diseases, but successful long-term transplant outcomes are limited by multiple factors, including ischemia reperfusion injury (IRI). This situation is aggravated by a shortage of transplantable organs, thus encouraging the use of inferior quality organs. Here, we have investigated early hepatic IRI in a retrospective, exploratory, monocentric case-control study considering organ marginality. We analyzed standard LTx biopsies from 46 patients taken at the end of cold organ preparation and two hours after reperfusion, and we showed that early IRI was present after two hours in 63% of cases. Looking at our data in general, in accordance with Eurotransplant criteria, a marginal transplant was allocated at our institution in about 54% of cases. We found that patients with a marginal-organ LTx showing evidence of IRI had a significantly worse one-year survival rate (51% vs. 75%). As we saw in our study cohort, the marginality of these livers was almost entirely due to steatosis. In contrast, survival rates in patients receiving a non-marginal transplant were not influenced by the presence or absence of IRI. Poorer outcomes in marginal organs prompted us to examine pre- and post-reperfusion biopsies, and it was revealed that transplants with IRI demonstrated significantly greater T cell infiltration. Molecular analyses showed that higher mRNA expression levels of CXCL-1, CD3 and TCRγ locus genes were found in IRI livers. We therefore conclude that the marginality of an organ, namely steatosis, exacerbates early IRI by enhancing effector immune cell infiltration. Preemptive strategies targeting immune pathways could increase the safety of using marginal organs for LTx.

Cross-presentation of dead-cell-associated antigens by DNGR-1+ dendritic cells contributes to chronic allograft rejection in mice.

The purpose of this study was to elucidate whether DC NK lectin group receptor-1 (DNGR-1)-dependent cross-presentation of dead-cell-associated antigens occurs after transplantation and contributes to CD8+ T cell responses, chronic allograft rejection (CAR), and fibrosis. BALB/c or C57BL/6 hearts were heterotopically transplanted into WT, Clec9a-/- , or Batf3-/- recipient C57BL/6 mice. Allografts were analyzed for cell infiltration, CD8+ T cell activation, fibrogenesis, and CAR using immunohistochemistry, Western blot, qRT2 -PCR, and flow cytometry. Allografts displayed infiltration by recipient DNGR-1+ DCs, signs of CAR, and fibrosis. Allografts in Clec9a-/- recipients showed reduced CAR (p < 0.0001), fibrosis (P = 0.0137), CD8+ cell infiltration (P < 0.0001), and effector cytokine levels compared to WT recipients. Batf3-deficiency greatly reduced DNGR-1+ DC-infiltration, CAR (P < 0.0001), and fibrosis (P = 0.0382). CD8 cells infiltrating allografts of cytochrome C treated recipients, showed reduced production of CD8 effector cytokines (P < 0.05). Further, alloreactive CD8+ T cell response in indirect pathway IFN-γ ELISPOT was reduced in Clec9a-/- recipient mice (P = 0.0283). Blockade of DNGR-1 by antibody, similar to genetic elimination of the receptor, reduced CAR (P = 0.0003), fibrosis (P = 0.0273), infiltration of CD8+ cells (p = 0.0006), and effector cytokine levels. DNGR-1-dependent alloantigen cross-presentation by DNGR-1+ DCs induces alloreactive CD8+ cells that induce CAR and fibrosis. Antibody against DNGR-1 can block this process and prevent CAR and fibrosis.

Differential effects of heat-inactivated, secretome-deficient MSC and metabolically active MSC in sepsis and allogenic heart transplantation.

Mesenchymal stem cells (MSCs) are used in various clinical and preclinical models for immunomodulation. However, it remains unclear how the immunomodulatory effect of MSC is communicated. MSC-induced immunomodulation is known to be mediated through both MSC-secreted cytokines and direct cell-cell interactions. Recently, it has been demonstrated that metabolically inactive, heat-inactivated MSCs (HI-MSCs) have similar anti-inflammatory capacities in LPS-induced sepsis compared with viable MSC. To further investigate the immunomodulatory effects of MSC, we introduced MSC and HI-MSC in two animal models with different immunological causes. In the first model, allogeneic hearts were transplanted from C57BL/6 mice to BALB/c recipients. MSC in combination with mycophenolate mofetil (MMF) significantly improved graft survival compared with MMF alone, whereas the application of HI-MSC had no effect on graft survival. We revealed that control MSC dose-dependently inhibited CD3+ and CD8+ T-cell proliferation in vitro, whereas HI-MSC had no effect. In the second model, sepsis was induced in mice via cecal ligation and puncture. HI-MSC treatment significantly improved the overall survival, whereas control MSCs had no effect. in vitro studies demonstrated that HI-MSCs are more effectively phagocytosed by monocytes than control MSCs and induced cell death in particular of activated CD16+ monocytes, which may explain the immune protective effect of HI-MSC in the sepsis model. The results of our study demonstrate that MSC-mediated immunomodulation in sepsis is dependent on a passive recognition of MSC by monocytes, whereas fully functional MSCs are required for inhibition of T-cell-mediated allograft rejection.

Alpha-syntrophin deficiency protects against non-alcoholic steatohepatitis associated increase of macrophages, CD8+ T-cells and galectin-3 in the liver.

Non-alcoholic steatohepatitis (NASH) is characterized by immune cell infiltration. Loss of the scaffold protein alpha-syntrophin (SNTA) protected mice from hepatic inflammation in the methionine-choline-deficient (MCD) diet model. Here, we determined increased numbers of macrophages and CD8+ T-cells in MCD diet induced NASH liver of wild type mice. In the mutant animals these NASH associated changes in immune cell composition were less pronounced. Further, there were more γδ T-cells in the NASH liver of the null mice. Galectin-3 protein in the hepatic non-parenchymal cell fraction was strongly induced in MCD diet fed wild type but not mutant mice. Antioxidant enzymes declined in NASH liver with no differences between the genotypes. To identify the target cells responsive to SNTA loss in-vitro experiments were performed. In the human hepatic stellate cell line LX-2, SNTA did not regulate pro-fibrotic or antioxidant proteins like alpha-smooth muscle actin or catalase. Soluble galectin-3 was, however, reduced upon SNTA knock-down and increased upon SNTA overexpression. SNTA deficiency neither affected cell proliferation nor cell death of LX-2 cells. In the macrophage cell line RAW264.7 low SNTA indeed caused higher galectin-3 production whereas release of TNF and cell viability were normal. Moreover, SNTA had no effect on hepatocyte chemerin and CCL2 expression. Overall, SNTA loss improved NASH without causing major effects in macrophage, hepatocyte and hepatic stellate cell lines. SNTA null mice fed the MCD diet had less body weight loss and this seems to contribute to improved liver health of the mutant mice.

Augmenter of Liver Regeneration Reduces Ischemia Reperfusion Injury by Less Chemokine Expression, Gr-1 Infiltration and Oxidative Stress.

Hepatic ischemia reperfusion injury (IRI) is a major complication in liver resection and transplantation. Here, we analyzed the impact of recombinant human augmenter of liver regeneration (rALR), an anti-oxidative and anti-apoptotic protein, on the deleterious process induced by ischemia reperfusion (IR). Application of rALR reduced tissue damage (necrosis), levels of lipid peroxidation (oxidative stress) and expression of anti-oxidative genes in a mouse IRI model. Damage associated molecule pattern (DAMP) and inflammatory cytokines such as HMGB1 and TNFα, were not affected by rALR. Furthermore, we evaluated infiltration of inflammatory cells into liver tissue after IRI and found no change in CD3 or γδTCR positive cells, or expression of IL17/IFNγ by γδTCR cells. The quantity of Gr-1 positive cells (neutrophils), and therefore, myeloperoxidase activity, was lower in rALR-treated mice. Moreover, we found under hypoxic conditions attenuated ROS levels after ALR treatment in RAW264.7 cells and in primary mouse hepatocytes. Application of rALR also led to reduced expression of chemo-attractants like CXCL1, CXCL2 and CCl2 in hepatocytes. In addition, ALR expression was increased in IR mouse livers after 3 h and in biopsies from human liver transplants with minimal signs of tissue damage. Therefore, ALR attenuates IRI through reduced neutrophil tissue infiltration mediated by lower expression of key hepatic chemokines and reduction of ROS generation.

Postoperative cellular stress in the kidney is associated with an early systemic γδ T-cell immune cell response.

BACKGROUND: Basic science data suggest that acute kidney injury (AKI) induced by ischemia-reperfusion injury (IRI) is an inflammatory process involving the adaptive immune response. Little is known about the T-cell contribution in the very early phase, so we investigated if tubular cellular stress expressed by elevated cell cycle biomarkers is associated with early changes in circulating T-cell subsets, applying a bedside-to-bench approach. METHODS: Our observational pilot study included 20 consecutive patients undergoing endovascular aortic repair for aortic aneurysms affecting the renal arteries, thereby requiring brief kidney hypoperfusion and reperfusion. Clinical-grade flow cytometry-based immune monitoring of peripheral immune cell populations was conducted perioperatively and linked to tubular cell stress biomarkers ([TIMP-2]•[IGFBP7]) immediately after surgery. To confirm clinical results and prove T-cell infiltration in the kidney, we simulated tubular cellular injury in an established mouse model of mild renal IRI. RESULTS: A significant correlation between tubular cell injury and a peripheral decline of γδ T cells, but no other T-cell subpopulation, was discovered within the first 24 hours (r = 0.53; p = 0.022). Turning to a mouse model of kidney warm IRI, a similar decrease in circulating γδ T cells was found and concomitantly was associated with a 6.65-fold increase in γδ T cells (p = 0.002) in the kidney tissue without alterations in other T-cell subsets, consistent with our human data. In search of a mechanistic driver of IRI, we found that the damage-associated molecule high-mobility group box 1 protein HMGB1 was significantly elevated in the peripheral blood of clinical study subjects after tubular cell injury (p = 0.019). Correspondingly, HMGB1 RNA content was significantly elevated in the murine kidney. CONCLUSIONS: Our investigation supports a hypothesis that γδ T cells are important in the very early phase of human AKI and should be considered when designing clinical trials aimed at preventing kidney damage. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01915446 . Registered on 5 Aug 2013.

Extracellular Citrate Affects Critical Elements of Cancer Cell Metabolism and Supports Cancer Development In Vivo.

Glycolysis and fatty acid synthesis are highly active in cancer cells through cytosolic citrate metabolism, with intracellular citrate primarily derived from either glucose or glutamine via the tricarboxylic acid cycle. We show here that extracellular citrate is supplied to cancer cells through a plasma membrane-specific variant of the mitochondrial citrate transporter (pmCiC). Metabolomic analysis revealed that citrate uptake broadly affected cancer cell metabolism through citrate-dependent metabolic pathways. Treatment with gluconate specifically blocked pmCiC and decreased tumor growth in murine xenografts of human pancreatic cancer. This treatment altered metabolism within tumors, including fatty acid metabolism. High expression of pmCiC was associated with invasion and advanced tumor stage across many human cancers. These findings support the exploration of extracellular citrate transport as a novel potential target for cancer therapy.Significance: Uptake of extracellular citrate through pmCiC can be blocked with gluconate to reduce tumor growth and to alter metabolic characteristics of tumor tissue. Cancer Res; 78(10); 2513-23. ©2018 AACR.

RORγt(+) IL-22-producing NKp46(+) cells protect from hepatic ischemia reperfusion injury in mice.

BACKGROUND & AIMS: NKp46(+) cells are major effector cells in the pathogenesis of hepatic ischemia reperfusion injury (IRI). Nevertheless, the precise role of unconventional subsets like the IL-22-producing NKp46(+) cells (NK22) remains unknown. The purpose of this study was to examine the role of NK22 cells in IRI in transplantation, particularly with respect to regulation by the transcription factor ROR-gamma-t (RORγt). METHODS: To explore the role of NK22 cells in IRI in the absence of adaptive immunity, B6.RORγt-(gfp/wt)-reporter and B6.RORγt-(gfp/gfp)-knockout (KO) mice on a Rag KO background underwent 90min partial warm ischemia, followed by 24h of reperfusion. RESULTS: Rag KO mice that possess fully functional NKp46(+) cells, and Rag-common-γ-chain-double-KO (Rag-γc-DKO) mice that lack T, B and NKp46(+) cells, were used as controls. We found that Rag-γc-DKO mice lacking NK22 cells show more severe levels of hepatocellular damage (GPT, histological injury) when compared to both Rag-RORγt-reporter and Rag KO mice that possess NK22 cells. Importantly, Rag-RORγt-reporter and Rag KO mice undergoing IRI expressed high protein levels of both IL-22 and GFP (RORγt), suggesting a protective role for RORγt(+) NK22 cells in IRI. Therefore, we tested the hypothesis that RORγt critically protects from IRI through the induction of hepatic NK22 cells by studying Rag-Rorγt-DKO mice under IRI conditions. We found that the lack of RORγt(+) NK22 cells in Rag-Rorγt-DKO mice significantly enhanced IR-induced hepatocellular injury, a phenotype that could be reversed upon adoptive transfer of Rag-Rorγt-reporter NK22 cells into DKO mice. CONCLUSIONS: RORγt(+) NK22 cells play an important protective role in IRI in mice.

CD27low natural killer cells prolong allograft survival in mice by controlling alloreactive CD8+ T cells in a T-bet-dependent manner.

BACKGROUND: Natural killer (NK) cells play a dichotomous role in alloimmune responses because they are known to promote both allograft survival and rejection. The aim of this study was to investigate the role of functionally distinct NK cell subsets in alloimmunity with the hypothesis that this dichotomy is explained by the functional heterogeneity of distinct NK cell subsets. METHODS: Because T-bet controls thematuration of NK cells from CD27high to terminally differentiated CD27low NK cells, we used Rag−/−T-bet−/− mice that lackmature CD27low NK cells to study the distinct roles of CD27low versus CD27high NK cells in a model of Tcell­mediated skin transplant rejection under costimulatory blockade conditions. RESULTS: We found that T cell­reconstituted Rag1−/− recipients (possessing CD27low NK cells) show significantly prolonged allograft survival on costimulatory blockade when compared to Rag1−/−T-bet−/− mice (lacking CD27low NK cells), indicating that CD27low but not CD27high NK cells enhance allograft survival. Critically, Rag1−/−T-bet−/− recipients showed strikingly increased alloreactive memory CD8+ Tcell responses, as indicated by increased CD8+ Tcell proliferation and interferon-γ production. Therefore, we speculated that CD27low NK cells directly regulate alloreactive CD8+ Tcell responses under costimulatory blockade conditions. To test this, we adoptively transferred CD27low NK cells into Rag1−/−T-bet−/− skin transplant recipients and found that the CD27low NK cells restore better allograft survival by inhibiting the proliferation of alloreactive interferon-γ+CD8+ T cells. CONCLUSIONS: In summary, mature CD27low NK cells promote allograft survival under costimulatory blockade conditions by regulating alloreactive memory CD8+ T-cell responses.

Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.

Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4(-/-) mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4(-/-) mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.

The life and fate of mesenchymal stem cells.

Mesenchymal stem cells (MSC) are present throughout the body and are thought to play a role in tissue regeneration and control of inflammation. MSC can be easily expanded in vitro and their potential as a therapeutic option for degenerative and inflammatory disease is therefore intensively investigated. Whilst it was initially thought that MSC would replace dysfunctional cells and migrate to sites of injury to interact with inflammatory cells, experimental evidence indicates that the majority of administered MSC get trapped in capillary networks and have a short life span. In this review, we discuss current knowledge on the migratory properties of endogenous and exogenous MSC and confer on how culture-induced modifications of MSC may affect these properties. Finally, we will discuss how, despite their limited survival, administered MSC can bring about their therapeutic effects.

KLRG1+ NK cells protect T-bet-deficient mice from pulmonary metastatic colorectal carcinoma.

We studied the developmental and functional mechanisms behind NK cell-mediated antitumor responses against metastatic colorectal carcinoma (CRC) in mice. In particular, we focused on investigating the significance of T-box transcription factors and the immunotherapeutic relevance of IL-15 in the development and function of tumor-reactive NK cells. Pulmonary CRC metastases were experimentally seeded via an adoptive i.v. transfer of luciferase-expressing CT26 CRC cells that form viewable masses via an in vivo imaging device; genetically deficient mice were used to dissect the antitumor effects of developmentally different NK cell subsets. IL-15 precomplexed to IL-15 receptor-α was used in immunotherapy experiments. We found that mice deficient for the T-box transcription factor T-bet lack terminally differentiated antitumor CD27(low)KLRG1(+) NK cells, leading to a terminal course of rapid-onset pulmonary CRC metastases. The importance of this NK cell subset for effective antitumor immunity was shown by adoptively transferring purified CD27(low)KLRG1(+) NK cells into T-bet-deficient mice and, thereby, restoring immunity against lung metastasis formation. Importantly, immunity to metastasis formation could also be restored in T-bet-deficient recipients by treating mice with IL-15 precomplexed to IL-15 receptor-α, which induced the development of eomesodermin(+)KLRG1(+) NK cells from existing NK cell populations. Thus, contingent upon their T-bet-dependent development and activation status, NK cells can control metastatic CRC in mice, which is highly relevant for the development of immunotherapeutic approaches in the clinic.

Double deficiency for RORγt and T-bet drives Th2-mediated allograft rejection in mice.

Although Th1, Th2, and Th17 cells are thought to be major effector cells in adaptive alloimmune responses, their respective contribution to allograft rejection remains unclear. To precisely address this, we used mice genetically modified for the Th1 and Th17 hallmark transcription factors T-bet and RORγt, respectively, which allowed us to study the alloreactive role of each subset in an experimental transplant setting. We found that in a fully mismatched heterotopic mouse heart transplantation model, T cells deficient for T-bet (prone to Th17 differentiation) versus RORγt (prone to Th1 differentiation) rejected allografts at a more accelerated rate, indicating a predominance of Th17- over Th1-driven alloimmunity. Importantly, T cells doubly deficient for both T-bet and RORγt differentiated into alloreactive GATA-3-expressing Th2 cells, which promptly induced allograft rejection characterized by a Th2-type intragraft expression profile and eosinophilic infiltration. Mechanistically, Th2-mediated allograft rejection was contingent on IL-4, as its neutralization significantly prolonged allograft survival by reducing intragraft expression of Th2 effector molecules and eosinophilic allograft infiltration. Moreover, under IL-4 neutralizing conditions, alloreactive double-deficient T cells upregulated Eomesodermin (Eomes) and IFN-γ, but not GATA-3. Thus, in the absence of T-bet and RORγt, Eomes may salvage Th1-mediated alloimmunity that underlies IL-4 neutralization-resistant allograft rejection. We summarize that, whereas Th17 cells predictably promote allograft rejection, IL-4-producing GATA-3(+) Th2 cells, which are generally thought to protect allogeneic transplants, may actually be potent facilitators of organ transplant rejection in the absence of T-bet and RORγt. Moreover, Eomes may rescue Th1-mediated allograft rejection in the absence of IL-4, T-bet, and RORγt.

Heart grafts tolerized through third-party multipotent adult progenitor cells can be retransplanted to secondary hosts with no immunosuppression.

Multipotent adult progenitor cells (MAPCs) are an adherent stem cell population that belongs to the mesenchymal-type progenitor cell family. Although MAPCs are emerging as candidate agents for immunomodulation after solid organ transplantation, their value requires further validation in a clinically relevant cell therapy model using an organ donor- and organ recipient-independent, third-party cell product. We report that stable allograft survival can be achieved following third-party MAPC infusion in a rat model of fully allogeneic, heterotopic heart transplantation. Furthermore, long-term accepted heart grafts recovered from MAPC-treated animals can be successfully retransplanted to naïve animals without additional immunosuppression. This prolongation of MAPC-mediated allograft acceptance depends upon a myeloid cell population since depletion of macrophages by clodronate abrogates the tolerogenic MAPC effect. We also show that MAPC-mediated allograft acceptance differs mechanistically from drug-induced tolerance regarding marker gene expression, T regulatory cell induction, retransplantability, and macrophage dependence. MAPC-based immunomodulation represents a promising pathway for clinical immunotherapy that has led us to initiate a phase I clinical trial for testing safety and feasibility of third-party MAPC therapy after liver transplantation.

Adiponectin receptor 1 C-terminus interacts with PDZ-domain proteins such as syntrophins.

Adiponectin receptor 1 (AdipoR1) is one of the two signaling receptors of adiponectin with multiple beneficial effects in metabolic diseases. AdipoR1 C-terminal peptide is concordant with the consensus sequence of class I PSD-95, disc large, ZO-1 (PDZ) proteins, and screening of a liver yeast two hybrid library identified binding to ß2-syntrophin (SNTB2). Hybridization of a PDZ-domain array with AdipoR1 C-terminal peptide shows association with PDZ-domains of further proteins including ß1- and α-syntrophin (SNTA). Interaction of PDZ proteins and C-terminal peptides requires a free carboxy terminus next to the PDZ-binding region and is blocked by carboxy terminal added tags. N-terminal tagged AdipoR1 is more highly expressed than C-terminal tagged receptor suggesting that the free carboxy terminus may form a complex with PDZ proteins to regulate cellular AdipoR1 levels. The C- and N-terminal tagged AdipoR1 proteins are mainly localized in the cytoplasma. N-terminal but not C-terminal tagged AdipoR1 colocalizes with syntrophins in adiponectin incubated Huh7 cells. Adiponectin induced hepatic phosphorylation of AMPK and p38 MAPK which are targets of AdipoR1 is, however, not blocked in SNTA and SNTB2 deficient mice. Further, AdipoR1 protein is similarly abundant in the liver of knock-out and wild type mice when kept on a standard chow or a high fat diet. In summary these data suggest that AdipoR1 protein levels are regulated by so far uncharacterized class I PDZ proteins which are distinct from SNTA and SNTB2.

Unconventional RORγt+ T cells drive hepatic ischemia reperfusion injury.

An emerging body of evidence suggests a pivotal role of CD3(+) T cells in mediating early ischemia reperfusion injury (IRI). However, the precise phenotype of T cells involved and the mechanisms underlying such T cell-mediated immune responses in IRI, as well as their clinical relevance, are poorly understood. In this study, we investigated early immunological events in a model of partial warm hepatic IRI in genetically targeted mice to study the precise pathomechanistic role of RORγt(+) T cells. We found that unconventional CD27(-)γδTCR(+) and CD4(-)CD8(-) double-negative T cells are the major RORγt-expressing effector cells in hepatic IRI that play a mechanistic role by being the main source of IRI-mediating IL-17A. We further show that unconventional IRI-mediating T cells are contingent on RORγt, as highlighted by the fact that a genetic deficiency for RORγt, or its therapeutic antagonization via digoxin, is protective against hepatic IRI. Therefore, identification of CD27(-)γδTCR(+) and CD4(-)CD8(-) double-negative T cells as the major source of IL-17A via RORγt in hepatic IRI opens new therapeutic options to improve liver transplantation outcomes.

Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Fourth Meeting: lessons learned from first clinical trials.

The Fourth Expert Meeting of the Mesenchymal Stem Cells in Solid Organ Transplantation (MiSOT) Consortium took place in Barcelona on October 19 and 20, 2012. This meeting focused on the translation of preclinical data into early clinical settings. This position paper highlights the main topics explored on the safety and efficacy of mesenchymal stem cells as a therapeutic agent in solid organ transplantation and emphasizes the issues (proper timing, concomitant immunossupression, source and immunogenicity of mesenchymal stem cells, and oncogenicity) that have been addressed and will be followed up by the MiSOT Consortium in future studies.