In Situ-Targeting of Dendritic Cells with Donor-Derived Apoptotic Cells Restrains Indirect Allorecognition and Ameliorates Allograft Vasculopathy

Chronic allograft vasculopathy (CAV) is an atheromatous-like lesion that affects vessels of transplanted organs. It is a component of chronic rejection that conventional immuno-suppression fails to prevent, and is a major cause of graft loss. Indirect allo-recognition through T cells and allo-Abs are critical during CAV pathogenesis. We tested whether the indirect allo-response and its impact on CAV is down-regulated by in situ-delivery of donor Ags to recipient's dendritic cells (DCs) in lymphoid organs in a pro-tolerogenic fashion, through administration of donor splenocytes undergoing early apoptosis. Following systemic injection, donor apoptotic cells were internalized by splenic CD11chi CD8α+ and CD8− DCs, but not by CD11cint plasmacytoid DCs. Those DCs that phagocytosed apoptotic cells in vivo remained quiescent, resisted ex vivo-maturation, and presented allo-Ag for up to 3 days. Administration of donor apoptotic splenocytes, unlike cells alive, (i) promoted deletion, FoxP3 expression and IL-10 secretion, and decreased IFN-γ-release in indirect pathway CD4 T cells; and (ii) reduced cross-priming of anti-donor CD8 T cells in vivo. Targeting recipient's DCs with donor apoptotic cells reduced significantly CAV in a fully-mismatched aortic allograft model. The effect was donor specific, dependent on the physical characteristics of the apoptotic cells, and was associated to down-regulation of the indirect type-1 T cell allo-response and secretion of allo-Abs, when compared to recipients treated with donor cells alive or necrotic. Down-regulation of indirect allo-recognition through in situ-delivery of donor-Ag to recipient's quiescent DCs constitutes a promising strategy to prevent/ameliorate indirect allorecognition and CAV

Pancreatic β-Cell Death in Response to Pro-Inflammatory Cytokines Is Distinct from Genuine Apoptosis

A reduction in functional β-cell mass leads to both major forms of diabetes; pro-inflammatory cytokines, such as interleukin-1beta (IL-1β) and gamma-interferon (γ-IFN), activate signaling pathways that direct pancreatic β-cell death and dysfunction. However, the molecular mechanism of β-cell death in this context is not well understood. In this report, we tested the hypothesis that individual cellular death pathways display characteristic phenotypes that allow them to be distinguished by the precise biochemical and metabolic responses that occur during stimulus-specific initiation. Using 832/13 and INS-1E rat insulinoma cells and isolated rat islets, we provide evidence that apoptosis is unlikely to be the primary pathway underlying β-cell death in response to IL-1β+γ-IFN. This conclusion was reached via the experimental results of several different interdisciplinary strategies, which included: 1) tandem mass spectrometry to delineate the metabolic differences between IL-1β+γ-IFN exposure versus apoptotic induction by camptothecin and 2) pharmacological and molecular interference with either NF-κB activity or apoptosome formation. These approaches provided clear distinctions in cell death pathways initiated by pro-inflammatory cytokines and bona fide inducers of apoptosis. Collectively, the results reported herein demonstrate that pancreatic β-cells undergo apoptosis in response to camptothecin or staurosporine, but not pro-inflammatory cytokines

Genome-wide identification and transcriptional analysis of folate metabolism-related genes in maize kernels

BACKGROUND: Maize is a major staple food crop globally and contains various concentrations of vitamins. Folates are essential water-soluble B-vitamins that play an important role as one-carbon (C1) donors and acceptors in organisms. To gain an understanding of folate metabolism in maize, we performed an intensive in silico analysis to screen for genes involved in folate metabolism using publicly available databases, followed by examination of the transcript expression patterns and profiling of the folate derivatives in the kernels of two maize inbred lines. RESULTS: A total of 36 candidate genes corresponding to 16 folate metabolism-related enzymes were identified. The maize genome contains all the enzymes required for folate and C1 metabolism, characterized by highly conserved functional domains across all the other species investigated. Phylogenetic analysis revealed that these enzymes in maize are conserved throughout evolution and have a high level of similarity with those in sorghum and millet. The LC-MS analyses of two maize inbred lines demonstrated that 5-methyltetrahydrofolate was the major form of folate derivative in young seeds, while 5-formyltetrahydrofolate in mature seeds. Most of the genes involved in folate and C1 metabolism exhibited similar transcriptional expression patterns between these two maize lines, with the highest transcript abundance detected on day after pollination (DAP) 6 and the decreased transcript abundance on DAP 12 and 18. Compared with the seeds on DAP 30, 5-methyltetrahydrofolate was decreased and 5-formyltetrahydrofolate was increased sharply in the mature dry seeds. CONCLUSIONS: The enzymes involved in folate and C1 metabolism are conserved between maize and other plant species. Folate and C1 metabolism is active in young developing maize seeds at transcriptional levels

Sustained NF-\u3baB activation produces a short-term cell proliferation block in conjunction with repressing effectors of cell cycle progression controlled by E2F or FoxM1

NF-kappaB transcription factors induce a host of genes involved in pro-inflammatory/stress-like responses; but the collateral effects and consequences of sustained NF-kappaB activation on other cellular gene expression programming remain less well understood. Here enforced expression of a constitutively active IKKbeta T-loop mutant (IKKbetaca) drove murine fibroblasts into transient growth arrest that subsided within 2-3 weeks of continuous culture. Proliferation arrest was associated with a G1/S phase block in immortalized and primary early passage MEFs. Molecular analysis in immortalized MEFs revealed that inhibition of cell proliferation in the initial 1-2 weeks after their IKKbetaca retroviral infection was linked to the transient, concerted repression of essential cell cycle effectors that are known targets of either E2F or FoxM1. Co-expression of a phosphorylation resistant IkappaBalpha super repressor and IKKbetaca abrogated growth arrest and cell cycle effector repression, thereby linking IKKbetaca's effects to canonical NF-kappaB activation. Transient growth arrest of IKKbetaca cells was associated with enhanced p21 (cyclin-dependent kinase inhibitor 1A) protein expression, due in part to transcriptional activation by NF-kappaB and also likely due to strong repression of Skp2 and Csk1, both of which are FoxM1 direct targets mediating proteasomal dependent p21 turnover. Ablation of p21 in immortalized MEFs reduced their IKKbetaca mediated growth suppression. Moreover, trichostatin A inhibition of HDACs alleviated the repression of E2F and FoxM1 targets induced by IKKbetaca, suggesting chromatin mediated gene silencing in IKKbetaca's short term repressive effects on E2F and FoxM1 target gene expression