Telomerase reverse transcriptase activates transcription of miR500A to inhibit Hedgehog signalling and promote cell invasiveness.

Telomerase reverse transcriptase (TERT) maintains telomere homeostasis, thus ensuring chromosome stability and cell proliferation. In addition, several telomere-independent functions of human TERT have been described. In this study, we report that TERT binds directly to the TCF binding elements located upstream of the oncomiR miR500A, and induces its transcription. This function was independent of the telomerase activity, as shown with experiments using catalytically inactive TERT and inhibitors of TERT and the TERT RNA component. miR500A was in turn found to target three key components of the Hedgehog signalling pathway: Patched 1; Gli family zinc finger 3; and Cullin 3, thereby promoting cell invasion. Our results point to the crucial role of the TERT-miR500A-Hedgehog axis in tumour aggressiveness and highlight the therapeutic potential of targeting noncanonical TERT functions in cancer.

Allogeneic Bone Marrow Mesenchymal Stem Cell Transplantation in Tooth Extractions Sites Ameliorates the Incidence of Osteonecrotic Jaw-Like Lesions in Zoledronic Acid-Treated Rats.

Medication-related osteonecrosis of the jaw (MRONJ) is defined as the exposed necrotic bone involving the maxillofacial structures in bisphosphonate treated patients, and the pathophysiology of this disease remains unclear. The aim of this study was to assess the effects of the allogeneic transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) in a model of Wistar mice with induced MRONJ disease. BM-MSCs from five male Wistar rats were characterized and cultured on ß-tricalcium phosphate (ß-TCP) granules. Thirty female Wistar rats were injected intraperitoneally with zoledronic acid and afterwards upper jaw molars were extracted. The animals were randomized to receive: Group 1: 1 × 106 BM-MSCs/ß-TCP construct in the alveolar socket; and Group 2: Saline solution/ß-TCP construct. A clinical and histological analysis was performed. Nested polymerase chain reaction (PCR) was assessed to verify the presence of transplanted male rat cells in the female recipient jaws. Clinical and histological findings evidenced that none of the animals in Group 1 exhibited uncovered sockets or bone exposure associated to MRONJ, whereas we detected 33% of MRONJ cases in Group 2. In addition, male rat cells were detected in the maxillae site four weeks after transplantation in the BM-MSCs-group. Allogeneic BM-MSCs in extractions sites ameliorates MRONJ incidence in zoledronic acid-treated rats compared to non-MSC treatments.

High temperature CaSiO3-Ca3(PO4)2 ceramic promotes osteogenic differentiation in adult human mesenchymal stem cells.

Silicophosphate calcium ceramics are widely used in orthopedic and oral surgery applications because of their properties for stimulating bone formation and bone bonding. These bioceramics, together with multipotent undifferentiated adult human mesenchymal stem cells, are serious candidates in the field of bone tissue engineering and regenerative medicine. For this reason, the influence of a novel 30 wt%CaSiO3 - 70 wt%Ca3(PO4)2 ceramic over a primary adult human mesenchymal stem cells culture has been investigated in this study, observing a total colonization of the biomaterial by cells at 21 days. The osteoinductive capacity of the materials was also studied: alkaline phosphatase activity, gene quantification of osteoblastic genes and calcium deposits stained by Alizarin Red test, showed evidences of osteogenic differentiation of adult human mesenchymal stem cells seeded with this bioceramic both in growth medium and osteogenic medium. Therefore, the 30 wt%CaSiO3 - 70 wt%Ca3(PO4)2 bioceramic represents a potential scaffold which could be used in the field of biomaterials for bone tissue engineering, allowing cell adhesion, proliferation and promoting osteogenic differentiation of adult human mesenchymal stem cells.

Tolerance Biomarkers in Liver Transplantation: Independent External Validation of the Predictive Strength of SENP6 and FEM1C Gene Expression.

BACKGROUND: Numerous studies have emphasized the genetic and phenotypic profiles of tolerant transplant patients. Moreover, different groups have defined several biomarkers, trying to distinguish patients who are going to be tolerant from those who are going to reject. However, most of these biomarkers have not been validated by other groups or even established for clinical practice. METHODS: We reanalyzed and stratified the predictive capacity of 20 previously described biomarkers for liver transplantation tolerance in a cohort of 17 liver transplant patients subjected to an independent, nonrandomized, prospective study of immunosuppression drug withdrawal. RESULTS: Only 4 of the 20 studied biomarkers (expression of SENP6, FEM1C, miR31, and miR95) showed a strong predictive capacity in the present study. miR31 and FEM1C presented an area under the ROC curve of 96.7%, followed by SENP1 with 93.3%. Finally, miR95 had an area under the ROC curve value <86.7%. CONCLUSIONS: Even though this independent analysis seems to confirm the predictive strength of SENP6 and FEM1C in liver transplantation tolerance, there are also risks in establishing biomarkers for clinical phenotypes without an understanding of how they are biologically relevant. Future collaborations between groups should be promoted so that the most promising biomarkers can be validated and implemented in daily clinical practice.

Extracellular adenosine reversibly inhibits the activation of human regulatory T cells and negatively influences the achievement of the operational tolerance in liver transplantation.

The artificial induction of tolerance in transplantation is gaining strength. In mice, a differential role of extracellular adenosine (eADO) for regulatory and effector T cells (Tregs and Teffs, respectively) has been proposed: inhibiting Teffs and inducing Tregs. The aim of this study was to analyze the action of extracellular nucleotides in human T cells and, moreover, to examine the influence of CD39 and CD73 ectonucleotidases and subsequent adenosine signaling through adenosine 2 receptor (A2 R) in the induction of clinical tolerance after liver transplant. The action of extracellular nucleotides in human T cells was analyzed by in vitro experiments with isolated T cells. Additionally, 17 liver transplant patients were enrolled in an immunosuppression withdrawal trial, and the differences in the CD39-CD73-A2 R axis were compared between tolerant and nontolerant patients. In contrast to the mice, the activation of human Tregs was inhibited similarly to Teffs in the presence of eADO. Moreover, the expression of the enzyme responsible for the degradation of ADO, adenosine deaminase, was higher in tolerant patients with respect to the nontolerant group along the immunosuppression withdrawal. Our data support the idea that eADO signaling and its degradation may play a role in the complex system of regulation of liver transplant tolerance.

Role of Kupffer cells in the progression of CRC liver metastases after the first stage of ALPPS.

Associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) has been suggested as a potential therapy for extensive bilobar liver tumors, although in some circumstances this technique may induce tumor progression, a fact still not well studied. Our aim was to study tumor hepatic progression induced by the first step of ALPPS in a WAG/Rij rat syngenic model of metastatic colorectal carcinoma by subcapsular CC531 cell line inoculation. ALPPS induced: tumor progression on deportalized lobe and metastases; expression of hepatic vasculogenic factors (HIF1-α and VEGF); and a dramatic increase of Kupffer cells (KCs) and tumor-associated macrophages (TAMs). Interestingly, KCs expressed COX-2 (M1 polarization), while TAMs expressed mainly arginase-1 (M2 polarization). ALPPS also induced a decrease of tumor-infiltrating lymphocytes and an increase of intrahepatic T lymphocytes. Thus, ALPPS technique seems to induce a hypoxic environment, which enhances hepatic HIF1-α and VEGF expression and may promote KCs and TAMs polarization. Consequently, the regenerative stimulus seems to be driven by a pro-inflammatory and hypoxic environment, in which M1 intrahepatic macrophages expressing COX-2 and T-Lymphocytes play a key role, facts which may be related with the tumor progression observed.

Extracellular ATP Activates the NLRP3 Inflammasome and Is an Early Danger Signal of Skin Allograft Rejection.

Immune cells are equipped with a number of receptors that recognize sterile injury and pathogens. We find that host immune cells release ATP as an inflammatory signal in response to allogeneic transplantation. ATP then acts via a feedback mechanism on the P2X7 channel to activate the NLRP3 inflammasome and subsequently process and release interleukin (IL)-18. This process is a necessary stage in the deleterious Th1 response against allotransplantation via interferon-γ production. Lack of IL-18 resulted in a decrease in graft-infiltrating CD8 cells but an increase in regulatory T cells. In human liver transplant patients undergoing progressive immunosuppressive drug withdrawal, we found that patients experiencing acute rejection had higher levels of the P2X7 receptor in circulating inflammatory monocytes compared to tolerant patients. These data suggest that the pharmacological inhibition of the P2X7 receptor or the NLRP3 inflammasome will aid in inducing transplant tolerance without complete immunoparalysis.

PARP-1/PARP-2 double deficiency in mouse T cells results in faulty immune responses and T lymphomas.

The maintenance of T-cell homeostasis must be tightly regulated. Here, we have identified a coordinated role of Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 in maintaining T-lymphocyte number and function. Mice bearing a T-cell specific deficiency of PARP-2 in a PARP-1-deficient background showed defective thymocyte maturation and diminished numbers of peripheral CD4+ and CD8+ T-cells. Meanwhile, peripheral T-cell number was not affected in single PARP-1 or PARP-2-deficient mice. T-cell lymphopenia was associated with dampened in vivo immune responses to synthetic T-dependent antigens and virus, increased DNA damage and T-cell death. Moreover, double-deficiency in PARP-1/PARP-2 in T-cells led to highly aggressive T-cell lymphomas with long latency. Our findings establish a coordinated role of PARP-1 and PARP-2 in T-cell homeostasis that might impact on the development of PARP-centred therapies.

Differential profile of activated regulatory T cell subsets and microRNAs in tolerant liver transplant recipients.

Regulatory T cells (Tregs) play a potential role in operational tolerance in liver transplantation (LT) patients, and microRNAs (miRNAs) are known to be involved in immunological responses and tolerance. Thus, we analyzed the implication of different peripheral blood Treg subsets and miRNAs on LT tolerance in 24 tolerant (Tol) and 23 non-tolerant (non-Tol) LT recipients by cellular, genetic, and epigenetic approximation. Non-Tol patients had a lower demethylation rate of the forkhead box P3 (FOXP3) regulatory T cell-specific demethylated region (TSDR) than Tol patients that correlated with the frequency of circulating Tregs. Tol patients presented a different signature of Treg subset markers compared with non-Tol patients with increased expression of HELIOS and FOXP3 and a higher proportion of latency-associated peptide (LAP)+ Tregs and CD45RA- human leukocyte antigen D related (HLA-DR)+ activated effector-memory Tregs. The expression of miR95, miR24, miR31, miR146a, and miR155 was higher in Tol than in non-Tol patients and was positively correlated with activated Treg markers. In conclusion, these data suggest that activated effector-memory Tregs and a TSDR-demethylation state of Tregs may play a role in the complex system of regulation of LT tolerance. In addition, we describe a set of miRNAs differentially expressed in human LT Tol patients providing suggestive evidence that miRNAs are implied in the preservation of self-tolerance as mediated by Tregs. Liver Transplantation 23 933-945 2017 AASLD.

Intraarticular and intravenous administration of 99MTc-HMPAO-labeled human mesenchymal stem cells (99MTC-AH-MSCS): In vivo imaging and biodistribution.

INTRODUCTION: Therapeutic application of intravenous administered (IV) human bone marrow-derived mesenchymal stem cells (ahMSCs) appears to have as main drawback the massive retention of cells in the lung parenchyma, questioning the suitability of this via of administration. Intraarticular administration (IAR) could be considered as an alternative route for therapy in degenerative and traumatic joint lesions. Our work is outlined as a comparative study of biodistribution of 99mTc-ahMSCs after IV and IAR administration, via scintigraphic study in an animal model. METHODS: Isolated primary culture of adult human mesenchymal stem cells was labeled with 99mTc-HMPAO for scintigraphic study of in vivo distribution after intravenous and intra-articular (knee) administration in rabbits. RESULTS: IV administration of radiolabeled ahMSCs showed the bulk of radioactivity in the lung parenchyma while IAR images showed activity mainly in the injected cavity and complete absence of uptake in pulmonary bed. CONCLUSIONS: Our study shows that IAR administration overcomes the limitations of IV injection, in particular, those related to cells destruction in the lung parenchyma. After IAR administration, cells remain within the joint cavity, as expected given its size and adhesion properties. ADVANCES IN KNOWLEDGE: Intra-articular administration of adult human mesenchymal stem cells could be a suitable route for therapeutic effect in joint lesions. IMPLICATIONS FOR PATIENT CARE: Local administration of adult human mesenchymal stem cells could improve their therapeutic effects, minimizing side effects in patients.

Tolerance in liver transplantation: Biomarkers and clinical relevance.

Transplantation is the optimal treatment for end-stage organ failure, and modern immunosuppression has allowed important progress in short-term outcomes. However, immunosuppression poorly influences chronic rejection and elicits chronic toxicity in current clinical practice. Thus, a major goal in transplantation is to understand and induce tolerance. It is well established that human regulatory T cells expressing the transcription factor FoxP3 play important roles in the maintenance of immunological self-tolerance and immune homeostasis. The major regulatory T cell subsets and mechanisms of expansion that are critical for induction and long-term maintenance of graft tolerance and survival are being actively investigated. Likewise, other immune cells, such as dendritic cells, monocyte/macrophages or natural killer cells, have been described as part of the process known as "operational tolerance". However, translation of these results towards clinical practice needs solid tools to identify accurately and reliably patients who are going to be tolerant. In this way, a plethora of genetic and cellular biomarkers is raising and being validated worldwide in large multi-center clinical trials. Few of the studies performed so far have provided a detailed analysis of the impact of immunosuppression withdrawal on pre-existing complications derived from the long-term administration of immunosuppressive drugs and the side effects associated with them. The future of liver transplantation is aimed to develop new therapies which increase the actual low tolerant vs non-tolerant recipients ratio.

Immunosuppressive potency of mechanistic target of rapamycin inhibitors in solid-organ transplantation.

Mammalian target of rapamycin, also known as mechanistic target of rapamycin (mTOR) is a protein kinase that belongs to the PI3K/AKT/mTOR signaling pathway, which is involved in several fundamental cellular functions such as cell growth, proliferation, and survival. This protein and its associated pathway have been implicated in cancer development and the regulation of immune responses, including the rejection response generated following allograft transplantation. Inhibitors of mTOR (mTORi) such as rapamycin and its derivative everolimus are potent immunosuppressive drugs that both maintain similar rates of efficacy and could optimize the renal function and diminish the side effects compared with calcineurin inhibitors. These drugs are used in solid-organ transplantationtoinduceimmunosuppression while also promoting the expansion of CD4+CD25+FOXP3+ regulatory T-cells that could favor a scenery of immunological tolerance. In this review, we describe the mechanisms by which inhibitors of mTOR induce suppression by regulation of these pathways at different levels of the immune response. In addition, we particularly emphasize about the main methods that are used to assess the potency of immunosuppressive drugs, highlighting the studies carried out about immunosuppressive potency of inhibitors of mTOR.

Associated Liver Partition and Portal Vein Ligation (ALPPS) vs Selective Portal Vein Ligation (PVL) for Staged Hepatectomy in a Rat Model. Similar Regenerative Response?

Associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a two-stage hepatectomy technique which can be associated with a hypertrophic stimulus on the future liver remnant (FLR) stronger than other techniques--such as portal vein ligation (PVL). However, the reason of such hypertrophy is still unclear, but it is suggested that liver transection combined with portal vein ligation (ALPPS) during the first stage of this technique may play a key role. The aim of this study is to compare the hypertrophic stimulus on the FLR and the clinical changes associated with both ALPPS and PVL in a rat surgical model. For this purpose, three groups of SD rats were used, namely ALPPS (n = 30), PVL (n = 30) and sham-treated (n = 30). The second stage of ALPPS (hepatectomy of the atrophic lobes), was performed at day 8. Blood and FLR samples were collected at 1, 24, 48 hours, 8 days and 12 weeks after the surgeries. ALPPS provoked a greater degree of hypertrophy of the FLR than the PVL at 48 hours and 8 days (p<0.05). The molecular pattern was also different, with the highest expression of IL-1ß at 24h, IL-6 at 8 days, and HGF and TNF-α at 48 hours and 8 days (p<0.05). ALPPS also brought about a mild proliferative stimulus at 12 weeks, with a higher expression of HGF and TGF-ß (p<0.05) than PVL. Clinically, ALPPS caused a significant liver damage during the first 48 hours, with a recovery of liver function at day 8. In conclusion, ALPPS seems to induce higher functional hypertrophy on the FLR than PVL at day 8. Such regenerative response seems to be leaded by a complex interaction between pro-mitogenic (IL-6, HGF, TNF-α) and antiproliferative (IL1-ß and TGF-ß) cytokines.

Predictive value of MicroRNAs in the progression of barrett esophagus to adenocarcinoma in a long-term follow-up study.

OBJECTIVE: The aim of this study is to identify a set of microRNAs (miRNAs) as prognostic molecular biomarkers for the progression of Barrett esophagus (BE) to esophageal adenocarcinoma (EAC) to rationalize the surveillance programs in patients with BE. BACKGROUND: Histological dysplasia is currently used as the main biomarker to identify the BE patients at high risk for developing EAC. Although miRNA expression profiles in BE and EAC have been reported, it has not been established which set of miRNAs could constitute a robust diagnostic test to predict the progression of BE to EAC. METHODS: miRNAs associated with progression of BE to EAC were identified using miRNA sequencing analysis. Further validation by quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in 2 groups of BE patients who either developed or did not develop adenocarcinoma after at least 5 years of follow-up. RESULTS: Twenty-three miRNAs were identified by miRNA sequencing analysis in the carcinogenesis process associated with BE. qRT-PCR analysis using independent tissue samples confirmed differential expression for 19 of them (miR-let-7c, 7, 146a, 149, 153, 192, 192*, 194, 194*, 196a, 196b, 200a, 203, 205, 215, 424, 625, 625*, and 944). However, only miR-192, 194, 196a, and 196b showed a significantly higher expression in BE samples from patients with progression to EAC compared with those who did not progress to EAC. CONCLUSIONS: These findings suggest that the expression pattern of a modest number of miRNAs in metaplasia biopsies could identify the BE patients at high risk for developing EAC. Therefore, it has potential use for the control and treatment of this malignancy.

[Development of immune tolerance in liver transplantation]. / Desarrollo de inmunotolerancia en el trasplante hepático.

The liver is a privileged organ and has a lower incidence of rejection than other organs. However, immunosuppressive regimens are still required to control the alloreactive T-lymphocyte response after transplantation. These treatments may lead to severe complications, such as infectious diseases, cancers, cardiovascular diseases and chronic renal insufficiency. In clinical transplantation there is increasing evidence that some liver transplant recipients who cease taking immunosuppressive (IS) drugs maintain allograft function, suggesting that tolerance is already present. This strategy is feasible in 25-33% of liver transplant recipients. A series of experimental and clinical observations indicates that liver allografts can even provide "tolerogenic" properties for other organ grafts. In the clinical setting, clinical operational tolerance (COT) is defined as the absence of acute and chronic rejection and graft survival with normal function and histology in an IS-free, fully immunocompetent host, usually as an end result of a successful attempt at IS withdrawal. The exact mechanisms involved in achieving transplant tolerance remain unknown, although animal models suggest a possible role for regulatory T cells (Treg). Recent data have demonstrated an increase in the frequency of CD4+ CD25(high) T cells and FoxP3 transcripts during IS withdrawal in operationally tolerant liver transplant recipients. The data obtained from transcriptional profiling of the peripheral blood of IS-free liver transplant recipients suggest that there is a molecular signature of tolerance that could be employed to identify tolerant liver transplant recipients and that innate immune cells are likely to play a major role in the maintenance of COT after liver transplantation.

Immunosuppression withdrawal improves long-term metabolic parameters, cardiovascular risk factors and renal function in liver transplant patients.

After liver transplantation, long-term immunosuppression (IS) administration is commonly complicated by renal dysfunction and cardiovascular complications. Twenty liver transplant patients on cyclosporine (CyA)-based IS were followed up prospectively after IS withdrawal. They consisted of 10 electively weaned patients and 10 either forcibly or incidentally weaned patients. Liver biochemical tests, blood pressure, serum creatinine, serum urea, serum uric acid, triglycerides, cholesterol and glucose were monitored after the start of weaning. Eight of the 20 patients (40%) were IS therapy free for a mean period of 61 +/- 39 months (range: 10-132 months). Of the remaining 12 patients, mild or moderate acute rejection occurred in six patients (30%), and mixed inflammatory portal tract infiltrate was seen in another six patients (30%). At the end of the study, mean (SD) serum creatinine had fallen by 0.28 (0.10) mg/dL (p < 0.001) in operationally tolerant (T) patients whereas the serum creatinine level increased in IS-dependent patients [+0.35 (0.35) mg/dL] (p = 0.005). In T patients, serum cholesterol, serum uric acid, fasting glucose and diastolic arterial pressure values significantly decreased. IS withdrawal can be achieved in selected liver transplant patients, and can improve not only kidney function, but also other CyA-associated side effects, such as hypercholesterolemia, hyperuricemia, hypertension and diabetes.

FoxP3 in peripheral blood is associated with operational tolerance in liver transplant patients during immunosuppression withdrawal.

BACKGROUND: Human liver allografts do sometimes survive in a recipient after withdrawal of immunosuppression (IS), commonly referred to as "operational tolerance." Preliminary clinical data have suggested an increase in the frequency of regulatory T cells (Treg) CD4+CD25 high and FoxP3 expression in operationally tolerant liver transplant recipients (Gr-T). In the context of human liver transplantation, the dynamics of Treg have not been studied. We designed a prospective study to ascertain the profile of the Treg population and FoxP3 expression during IS withdrawal. METHODS: To identify such parameters, we analyzed peripheral blood mononuclear cell populations and FoxP3 mRNA expression in 12 liver allograft recipients under cyclosporine A-based IS, who showed stable function of the allograft for more than 2 years. RESULTS: An increase was observed in the frequency of CD4+CD25 high cells when the IS was withdrawn in Gr-T patients (n=5). These patients exhibited a 3.5-fold increase for relative mRNA FoxP3 expression before the complete IS withdrawal and this continued when IS therapy was stopped. In patients who suffered rejection (n=7) there was no increase in the CD4+CD25 high cells or FoxP3 expression. CONCLUSIONS: With the present study, the first evidence is provided that the increase of CD4+CD25 high T cells and FoxP3 transcripts is associated with operational tolerance in liver transplanted patients during IS withdrawal.

Transport of N-acetyl-D-mannosamine and N-acetyl-D-glucosamine in Escherichia coli K1: effect on capsular polysialic acid production.

N-Acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc) are the essential precursors of N-acetylneuraminic acid (NeuAc), the specific monomer of polysialic acid (PA), a bacterial pathogenic determinant. Escherichia coli K1 uses both amino sugars as carbon sources and uptake takes place through the mannose phosphotransferase system transporter, a phosphoenolpyruvate-dependent phosphotransferase system that shows a broad range of specificity. Glucose, mannose, fructose, and glucosamine strongly inhibited the transport of these amino-acetylated sugars and GlcNAc and ManNAc strongly affected ManNAc and GlcNAc uptake, respectively. The ManNAc and the GlcNAc phosphorylation that occurs during uptake affected NeuAc synthesis in vitro. These findings account for the low in vivo PA production observed when E. coli K1 uses ManNAc or GlcNAc as a carbon source for growth.