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Objectives Because females are under-represented in coronary trials, this study sought to assess the relative safety and efficacy of Absorb bioresorbable vascular scaffold (BVS) (Abbott Vascular, Santa Clara, California) and the Xience everolimus-eluting stent in females compared with males. Background The Absorb everolimus-eluting BVS provides drug delivery and mechanical support similar to a metallic drug-eluting stent, followed by resorption and restoration of more normal vascular structure with the potential to improve late clinical outcomes. Methods The ABSORB II, ABSORB III, ABSORB Japan, and ABSORB China trials were pooled. Baseline clinical, angiography, procedural variables, and 2-year outcomes were analyzed by sex and device. Results Among 3,384 randomized patients, 932 (27.5%) were female. Females were older, more often had diabetes and hypertension, but had less everolimus-eluting stent, 3-vessel disease, and smoking compared with males (all p≤0.001). The 2-year rates of target lesion failure with BVS versus everolimus-eluting stent in females were 8.9% versus 6.2% (study-level adjusted hazard ratio: 1.47; 95% confidence interval [CI]: 0.88 to 2.46) and 8.9% versus 6.4% in males (HR: 1.40; 95% CI: 1.02 to 1.92; pinteraction = 0.85). There were no significant interactions between sex and device type for any of the components of target lesion failure. Conclusions The relative treatment effects of BVS and everolimus-eluting stent for the 2-year rates of target lesion failure and other cardiovascular outcomes were consistent in females and males.

Because females are under-represented in coronary trials, this study sought to assess the relative safety and efficacy of Absorb bioresorbable vascular scaffold (BVS) (Abbott Vascular, Santa Clara, California) and the Xience everolimus-eluting stent in females compared with males.The Absorb everolimus-eluting BVS provides drug delivery and mechanical support similar to a metallic drug-eluting stent, followed by resorption and restoration of more normal vascular structure with the potential to improve late clinical outcomes.The ABSORB II, ABSORB III, ABSORB Japan, and ABSORB China trials were pooled. Baseline clinical, angiography, procedural variables, and 2-year outcomes were analyzed by sex and device.Among 3,384 randomized patients, 932 (27.5%) were female. Females were older, more often had diabetes and hypertension, but had less everolimus-eluting stent, 3-vessel disease, and smoking compared with males (all p≤0.001). The 2-year rates of target lesion failure with BVS versus everolimus-eluting stent in females were 8.9% versus 6.2% (study-level adjusted hazard ratio: 1.47; 95% confidence interval [CI]: 0.88 to 2.46) and 8.9% versus 6.4% in males (HR: 1.40; 95% CI: 1.02 to 1.92; pThe relative treatment effects of BVS and everolimus-eluting stent for the 2-year rates of target lesion failure and other cardiovascular outcomes were consistent in females and males.

The Absorb bioresorbable vascular scaffold (BVS) (Abbott Vascular, Santa Clara, California) is designed to reduce chronic adverse cardiovascular events that may occur from the permanent presence of a metallic stent and/or durable polymer. The systemic pharmacokinetic (PK) profile of BVS has not been

NK cells are important in the immune response against tumors and virally infected cells. A balance between inhibitory and activating receptors controls the effector functions of NK cells. We examined the fate of circulating NK cells and the expression of the NK cell-activating receptors in pediatric liver transplant recipients. Blood specimens were collected from 38 pediatric liver transplant recipients before transplant, and at one wk, one, three, six, and nine months, and one yr post-transplant. PBMCs were isolated and analyzed for the levels of NK cell activation receptors NKp30, NKp46, and NKG2D in the CD56(dim) CD16(+) and CD56(bright) CD16(+/-) subsets of NK cells. We demonstrated that there is a significant decrease in the percentage of circulating NK cells post-transplant (pretransplant 7.69 ± 1.54 vs. one wk post-transplant 1.73 ± 0.44) in pediatric liver transplant recipients. Interestingly, NKp30 expression is significantly increased, while NKp46 and NKG2D levels remain stable on the NK cells that persist at one wk post-transplant. These data indicate that the numbers and subsets of circulating NK cells are altered in children after liver transplantation.

NK cells are important in the immune response against tumors and virally infected cells. A balance between inhibitory and activating receptors controls the effector functions of NK cells. We examined the fate of circulating NK cells and the expression of the NK cell-activating receptors in pediatric liver transplant recipients. Blood specimens were collected from 38 pediatric liver transplant recipients before transplant, and at one wk, one, three, six, and nine months, and one yr post-transplant. PBMCs were isolated and analyzed for the levels of NK cell activation receptors NKp30, NKp46, and NKG2D in the CD56(dim) CD16(+) and CD56(bright) CD16(+/-) subsets of NK cells. We demonstrated that there is a significant decrease in the percentage of circulating NK cells post-transplant (pretransplant 7.69 ± 1.54 vs. one wk post-transplant 1.73 ± 0.44) in pediatric liver transplant recipients. Interestingly, NKp30 expression is significantly increased, while NKp46 and NKG2D levels remain stable on the NK cells that persist at one wk post-transplant. These data indicate that the numbers and subsets of circulating NK cells are altered in children after liver transplantation.

NKG2D is a receptor expressed by NK cells and subsets of T lymphocytes. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We cloned and expressed two rat NKG2D ligands, both members of the RAE1 family, RAE1L and RRLT, and demonstrate that these ligands can induce IFN-gamma secretion and cytotoxicity by rat NK cells. To examine changes in expression of NKG2D and the NKG2D ligands RAE1L and RRLT after transplantation, we used a Dark Agouti (DA)-->Lewis rat model of liver transplantation. NKG2D expression was significantly increased in allogeneic liver grafts by day 7 post-transplant. Ligands of NKG2D, absent in normal liver, were readily detected in both syngeneic and allogeneic liver grafts by day 1 post-transplant. By day 7 post-transplant, hepatocyte RAE1L and RRLT expression was significantly and specifically increased in liver allografts. In contrast to acute rejection that develops in the DA-->Lewis model, transplantation of Lewis livers into DA recipients (Lewis-->DA) results in spontaneous tolerance. Interestingly, expression of RAE1L and RRLT is low in Lewis-->DA liver allografts, but significantly increased in DA-->Lewis liver allografts undergoing rejection. In conclusion, our results suggest that expression of NKG2D ligands may be important in allograft rejection.

International audience; Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions. In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C. Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta. The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta. We discuss the implications of our findings within the current framework of our understanding of DNA replication.

International audience; We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase delta. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2+/rhp9+. This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30 degrees C, but were defective in this checkpoint function when treated with MMS at 37 degrees C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37 degrees C. Furthermore, the crb2+ gene was required, together with the cds1+ gene, for the S/M checkpoint at 30 degrees C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the poldeltats3 background at both 30 degrees C and 37 degrees C. The rapid death phenotype was independent of the checkpoint functions.

International audience; We have isolated and characterized DNA polymerase delta (pol delta) from two thermosensitive Schizosaccharomyces pombe strains, poldeltats1 and poldeltats3, mutated in two different evolutionarily conserved domains of the catalytic subunit. At the restrictive temperature of 37 degreesC poldeltats1 and poldeltats3 mutant strains arrest growth in the S phase of the cell cycle. We show that at low levels of primer ends, in vitro stimulation by proliferating cell nuclear antigen (PCNA) of mutant enzymes is lower than stimulation of wild-type pol delta. Affinity for primer (3'-OH) ends and processivity of mutant enzymes do not appear different from wild-type pol delta. In contrast, Vmax values are lower than the wild-type value. The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulting in reduced velocity of DNA synthesis. In addition, ts1 pol delta is not stimulated by low PCNA concentration at 37 degreesC, although low concentrations stimulate activity at 25 degreesC, suggesting that this thermolability at low levels of primer ends could be its critical defect in vivo. Thus, both ts1 and ts3 pol delta mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.

International audience; We have generated proliferating cell nuclear antigen (PCNA) mutants by low fidelity PCR and screened for lethal mutations by testing for lack of complementation of a Schizosaccharomyces pombe strain disrupted for the pcn1 + gene. We thus identified eight lethal mutants out of the 50 cDNAs tested. Six were truncated in their C-terminal region due to the introduction of a stop codon within their coding sequences. Two were full-length with a single point mutation at amino acid 68 or 69. The two latter mutants were overexpressed in insect cells via a recombinant baculovirus and were purified. They were unable to stimulate DNA polymerase delta DNA replication activity on a poly(dA).oligo(dT) template. Cross-linking experiments showed that this was due to their inability to form trimers. Since these two mutations are adjacent and not located in a domain of the protein putatively involved in inter-monomer interactions, our results show that the beta-sheet betaF1 to which they belong must play an essential role in maintaining the 3-dimensional structure of S.pombe PCNA.

Pediatric organ transplant recipients are at highest risk of developing morbidity and mortality from EBV infection. There is broad consensus that T cell immunity is important in controlling viral replication and expansion of infected B cells. In this study, we simultaneously analyze virus-specific T cells’ phenotypes, functions, and epitope specificity using 37-parameter mass cytometry in the setting of pediatric organ transplant recipients, followed over the first two years post-transplant. We compared T cell responses to peptide pools derived from lytic and latent EBV antigens in both healthy EBV+ donors and pediatric transplant patients. The results suggest that healthy EBV+ donors can mount CD8+ T cell responses to both lytic and latent EBV antigens, with production of multiple cytokines. In contrast, pediatric patients have much weaker T cell responses, especially to lytic antigens. Based on current data, we hypothesize a polyfunctional T cell response to lytic antigens may preferentially protect against EBV viremia. We also use the SPADE algorithm for unsupervised clustering to compare the distribution of different T cell subsets in cytokine-producing cells. In addition, we analyze these T cells’ functional capacity for cytokine production by visualization in a heatmap. By employing highly multi-parameter mass cytometry and longitudinal sampling, we have created a more detailed picture of the T cell responses to EBV in the setting of pediatric transplant recipients.

NKG2D is expressed by Natural Killer (NK cells) and subsets of T cells. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We have demonstrated increased expression of NKG2D ligands on rejecting grafts and enhanced infiltration of NKG2D-expressing cells in allografts during rejection. To further examine the role of NKG2D in graft survival we utilized Klrk1 mice that are deficient for NKG2D as recipients of cardiac or small intestine allografts. Groups (n=4-6) of C57BL/6 (H-2b) or NKG2D deficient Klrk1 mice (H-2b) received heterotopic donor hearts or vascularized, orthotopic small intestine grafts from BALB/c (H-2d) or BALB/c x C57BL/6 F1 mice (H-2d/b). Some groups of mice also received 200μg (heart) or 500μg (intestine) anti-CD40L mAb (MR1) at transplant. Cardiac allograft survival was similar in wild-type and Klrk1 mice although intestinal allograft survival was prolonged in Klrk1 mice. Treatment with MR1 prolonged cardiac allograft survival that was further enhanced in the absence of NKG2D. Treatment with MR1 did not prolong survival of intestine allografts in wild-type recipients. Quite dramatically, however, treatment with MR1, significantly (p=0.007) prolonged intestinal allograft survival in Klrk1 mice. Prolongation of intestinal graft survival in the absence of NKG2D and CD40-CD40L interactions suggest that the addition of therapeutics to block NKG2D has great potential for improving costimulation blockade based therapies

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by translational repression or mRNA degradation. We defined a profile of miRNAs expression during graft rejection and suggested that the miRNA miR-182, which is a member of the mir-183-miR-96-miR-182 cluster, is significantly increased in the allograft, graft infiltrating cells, spleen and peripheral blood lymphocytes during rejection. To further define the function of miR-182 in alloimmune responses, splenocytes isolated from C57BL/6 or BALB/c mice were used as responders or stimulators in a one-way MLR. Proliferation was measured by [3H]-thymidine uptake on days 0 to 7 and cells were collected at each time point and RNA isolated to detect kinetic expression of miR-182 and its potential targets. Expression of miR-182 increased specifically in allo MLR between days 2-3 and reached peak expression on day 5. Computational target prediction suggested the genes Foxo1, Rac1, Bcl2 and Grb2 as putative targets of miR-182. The four genes all decreased from days 4 to 6 specifically in the allo MLR group supporting that increased expression of miR-182 may inhibit expression of these genes. CONCLUSION. Our results suggest an important role of miR-182 in alloimmune responses. Further studies may result in novel therapeutics to control the proliferation of alloreactive cells.