During human uterine spiral artery (SpA) remodeling, vascular smooth muscle cells (VSMCs) are lost and replaced by fibrinoid, incorporating extravillous trophoblast (EVT) cells. The aim of the current study was to determine the relative contributions of apoptosis and migration to VSMC loss during SpA remodeling. Immunohistochemistry (Apoptag, active caspase 3, lamin) of placental bed biopsies (8-20 wk gestation) demonstrated apoptotic cells in all samples; double immunolabeling identified these as trophoblasts, leukocytes, and endothelial cells. In total, 294 SpAs were studied, and only one apoptotic VSMC was identified. H-caldesmon-immunopositive VSMCs were observed surrounding and separate from SpA walls in partially remodeled vessels; the highest level of VSMC migration was observed in vessels with associated EVT cells (number of migrated cells 6.4±1.2; distance migrated 3.5±0.3 pixels) compared with those without (number of migrated cells 3.6±0.5, P<0.001; distance migrated 2.8±0.1 pixels, P<0.0001). VEGF-A, VEGF-C, TGF-β1, and Ang-2 all stimulated human aorta VSMC invasion in vitro, although EVT cell culture supernatants did not. In summary, apoptosis is unlikely to play a major role in loss of VSMCs from SpAs during remodeling in normal pregnancy, but VSMCs appear to migrate away from the wall of the SpA, an effect enhanced by the presence of EVT cells.-Bulmer, J. N., Innes, B. A., Levey, J., Robson, S. C., Lash, G. E. The role of vascular smooth muscle cell apoptosis and migration during uterine spiral artery remodeling in normal human pregnancy.

Although Th17 cells are known to promote tissue inflammation and autoimmunity, their role during cancer progression remains elusive. Here, we showed that in vitro Th17 cells generated with the cytokines IL-6 and TGF-β expressed CD39 and CD73 ectonucleotidases, leading to adenosine release and the subsequent suppression of CD4(+) and CD8(+) T cell effector functions. The IL-6-mediated activation of the transcription factor Stat3 and the TGF-β-driven downregulation of Gfi-1 transcription factor were both essential for the expression of ectonucleotidases during Th17 cell differentiation. Stat3 supported whereas Gfi-1 repressed CD39 and CD73 expression by binding to their promoters. Accordingly, Th17 cells differentiated with IL-1β, IL-6, and IL-23 but without TGF-β did not express ectonucleotidases and were not immunosuppressive. Finally, adoptive transfer of Th17 cells induced by TGF-β and IL-6 promoted tumor growth in a CD39-dependent manner. Thus, ectonucleotidase expression supports the immunosuppressive fate of Th17 cells in cancer.

Objectives: Intracoronary administration of glycosaminoglycan analogs, including the complement inhibitor dextran sulfate, attenuates myocardial ischemia/reperfusion injury (I/R injury). However, dextran sulfate has a distinct anticoagulatory effect, possibly limiting its use in specific situations in vivo. We therefore developed multimeric tyrosine sulfate (sTyr-PAA), a novel, minimally anticoagulatory, fully synthetic non-carbohydrate-containing polyacrylamide conjugate, for in vivo testing in an acute closed-chest porcine model of acute myocardial infarction. Methods: Following balloon occlusion of the left anterior descending artery just after the first diagonal branch (60-minute ischemia), sTyr-PAA (approx. 10 mg/kg bodyweight, fraction with strongest complement-inhibitory and minimal anticoagulatory properties, n = 11) or phosphate-buffered saline (controls, n = 9) was administered intracoronarily into ischemic myocardium prior to 120 min of reperfusion. Results: sTyr-PAA significantly reduced infarct size (from 61.0 ± 12.0% of the ischemic area at risk to 39.4 ± 17.0%), plasma creatine kinase, local complement deposition and tissue factor upregulation, without affecting systemic coagulation. Protection was associated with significantly reduced myocardial neutrophil extravasation and translated into a significant improvement of ejection fraction and left ventricular enddiastolic pressure. Conclusions: sTyr-PAA protected significantly against myocardial I/R injury without substantially affecting systemic coagulation. Local intravascular sTyr-PAA administration may prove advantageous in situations where bleeding complications are likely or are to be avoided at all costs.

BACKGROUND & AIMS:: Patients with autoimmune hepatitis (AIH) have reduced numbers and function of CD4(+)CD25(high)FOXP3(+) T regulatory (Treg) cells. Treg cells can be generated from CD25-(ngTreg) cells, which suppress the immune response less efficiently than Treg cells. We investigated whether their differentiation into T-helper (Th)17 cells, an effector subset that has the same CD4(+) progenitors as Treg cells, accounts for the reduced suppressive functions of ngTreg cells. We investigated whether blocking interleukin (IL)-17 increased the immunosuppressive activity of Treg cells. METHODS:: ngTreg cells were generated from 36 patients with AIH and 23 healthy subjects (controls). During Treg cell differentiation, expression of IL-17 was inhibited by physical removal of IL-17-secreting cells, exposure to recombinant transforming growth factor-β or neutralizing antibodies against IL-6 and IL-1β to promote differentiation of ngTreg vs Th17 cells), small inhibitory RNAs specific for the Th17 transcription factor RORC, or a combination of all these approaches. RESULTS:: ngTreg cells from patients with AIH contained greater proportions of IL-17(+) and RORC(+) cells than Treg cells from controls. All approaches to inhibit IL-17 increased expression of FOXP3 by ngTreg cells and their suppressive functions. Inhibition of IL-17 led to development of ngTreg cells that were phenotypically stable and did not acquire pro-inflammatory properties after exposure to IL-6 and IL-1β. CONCLUSION:: Blocking Th17 allows ngTreg cells to differentiate into functionally stable immune inhibitory cells; this approach might be developed for therapy of patients with AIH.

Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice, and is triggered by stimulation of immune cells. The purinergic receptor P2X7, is upstream of the NLRP3 inflammasome in immune cells, and is activated by ATP and NAD that serve as DAMP (damage associated molecular patterns). APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7 -/-), and in wild type mice. P2X7 -/- mice had significantly decreased APAP induced liver necrosis. In addition, APAP poisoned mice were treated with the specific P2X7 antagonist A438079, or etheno-NAD, a competitive antagonist of NAD. Pre or post-treatment with A438079 significantly decreased APAP induced necrosis and hemorrhage in APAP liver injury in wild type but not P2X7 -/- mice. Pre-treatment with etheno-NAD also significantly decreased APAP induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild type mice. CD39 -/- mice had increased APAP induced hemorrhage and mortality whereas apyrase also decreased APAP induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7 dependent inflammasome activation. P2X7 was required for ATP stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP induced hepatotoxicity.

Invasion of uterine tissues by extravillous trophoblast cells (EVT) is essential for successful human pregnancy. EVT invasion is tightly regulated by a number of factors including growth factors and cytokines but the mechanisms that underlie their regulatory effect remain poorly understood. Interleukin (IL)-6 has been suggested to play a role in controlling EVT invasion. We hypothesised that IL-6 produced by cells in uterine decidua would regulate EVT invasiveness via IL-6Rα and gp130 receptors expressed by trophoblast cells. The effect of IL-6 on EVT signalling and cytokine production was also studied. Supernatants from disaggregated 'total' decidual cells, CD8+ T cells, CD10+ decidual stromal cells, CD14 macrophages, CD56+ uterine natural killer cells, cytotrophoblast and extravillous trophoblast cells contained large quantities of IL-6 protein at both 8-10 and 12-14 weeks gestational age. IL-6Rα and gp130 was immunolocalised to EVT in placental bed biopsies from 8-20 weeks gestation and IL-6Rα expression was confirmed by Western Blotting. IL-6 had no effect on the invasive potential of EVT from chorionic villi or the immortalised EVT cell line HTR-8/SVneo in a Matrigel® invasion assay. IL-6 stimulated phosphorylation of several cell signalling proteins in EVT (8-14 weeks' gestation), although significance was lost after correction for multiple comparisons. Incubation with IL-6 decreased secretion of RANTES by EVT cells. In conclusion, although IL-6 did not affect trophoblast cell invasion, it stimulated EVT cellular cascades and inhibited secretion of RANTES involved in a number of cellular processes.

Scalea J, Hanecamp I, Robson SC, Yamada K. T-cell-mediated immunological barriers to xenotransplantation. Xenotransplantation 2012; 19: 23-30. © 2012 John Wiley & Sons A/S. Abstract:  Xenotransplantion remains the most viable option for significant expansion of the donor organ pool in clinical transplantation. With the advent of nuclear transfer technologies, the production of transgenic swine has become a possibility. These animals have allowed transplant investigators to overcome humoral mechanisms of hyperacute xenograft rejection in experimental pig-to-non-human primate models. However, other immunologic barriers preclude long-term acceptance of xenografts. This review article focuses on a major feature of xenogeneic rejection: xenogeneic T cell responses. Evidence obtained from both small and large animal models, particularly those using either islet cells or kidneys, have demonstrated that T cell responses play a major role in xenogeneic rejection, and that immunosuppression alone is likely incapable of completely suppressing these responses. Additionally, both the direct and indirect pathway of antigen presentation appear to be involved in these anti donor processes. Enhanced understanding of (i) CD47 and its role in transduced xeno-bone marrow (ii) CD39 and its role in coagulation dysregulation and (iii) thymic transplantation have provided us with encouraging results. Presently, experiments evaluating the possibility of xenogeneic tolerance are underway.

CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60min followed by 3hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2±4.3% vs. Control: 44.7±5.2%, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.

OBJECTIVE:: This study was designed to evaluate the clinical outcome of patients undergoing portal vein embolization (PVE) and autologous CD133 bone marrow-derived stem cell (CD133 BMSC) application before extended right hepatectomy. BACKGROUND:: We have previously shown that portal venous infusion of CD133 BMSCs substantially increases hepatic proliferation, when compared with PVE alone. METHODS:: Among 40 consecutive patients with a median follow-up of 28 months (7.4-57.2) scheduled for extended right hepatectomy, we compared a preconditioned group with PVE and CD133 BMSC cotreatment (PVE+SC group, n = 11) and a group pretreated only with PVE (PVE group, n = 11). Functional and overall outcomes after extended right hepatectomy were evaluated. Patients without presurgical treatment served as controls (n = 18). RESULTS:: In preconditioned patients, mean hepatic growth of segments II/III 14 days after PVE in the PVE+SC group was significantly higher (138.66 mL ± 66.29) when compared with that of PVE group patients (62.95 mL ± 40.03; P = 0.004). There were no significant differences among all 3 groups regarding general and oncological characteristics and functional parameters on postoperative day (POD) 7. Lack of hepatic preconditioning, extrahepatic extension of resection, and postoperative complications were of negative prognostic value, using univariate analysis (P < 0.05). In multivariate analysis, freedom from postoperative major complications (P = 0.012), coagulation status on POD 7 (international normalized ratio < 1.4; P = 0.027), and presurgical expansion of the future liver remnant volume (P = 0.048) were positively associated with overall survival. Post hoc analysis revealed a better survival for the PVE+SC group (P = 0.028) compared with the PVE group (P = 0.094) and compared with controls. CONCLUSION:: Promising data from this survival analysis suggest that PVE, together with CD133 BMSC pretreatment, could positively impact overall outcomes after extended right hepatectomy.