The ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse "trans vivo" delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen-linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-β or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.

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This functional model of hepatocyte transplantation is validated for the study of host immune responses to hepatocellular grafts and to assess efficacy of strategies designed to alter these in vivo immune responses. The immunologic rejection of allogeneic hepatocytes appears to be T cell-mediated.

Abstract-In mammalian organs under normoxic conditions, O 2 concentration ranges from 12% to Ͻ0.5%, with O 2 Ϸ14%in arterial blood and Ͻ10% in the myocardium. During mild hypoxia, myocardial O 2 drops to Ϸ1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO 2 results in perceived hyperoxia. These effects were independent of NADPH oxidase function. CFs exposed to high O 2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-␤1, (2) lowered telomerase activity, and (3) Key Words: redox Ⅲ free radicals Ⅲ heart Ⅲ cell culture C ellular O 2 concentrations are maintained within a narrow range (normoxia) because of the risk of oxidative damage from excess O 2 (hyperoxia) and of metabolic demise from insufficient O 2 (hypoxia). 1 PO 2 ranges from 90 to Ͻ3 mm Hg in mammalian organs under normoxic conditions, with arterial PO 2 of Ϸ100 mm Hg or Ϸ14% O 2 . 2 Thus, "normoxia" for cells is a variable that is dependent on the specific localization of the cell in organs and functional status of the specific tissue. O 2 sensing is required to adjust to physiological or pathophysiological variations in PO 2 . Current work in this field is almost exclusively focused on the study of hypoxia. Reoxygenation, on the other hand, has been mostly investigated in the context of oxidative injury. Over 25 years ago, it was observed that PO 2 beyond the comfort of the "perceived normoxic range" is a significant stressor, leading to growth arrest. 3 The molecular bases of such observations remain to be characterized in light of current knowledge of signal transduction.During chronic hypoxia in the heart, cells adjust their normoxic set point such that the return to normoxic PO 2 after chronic hypoxia is perceived as relative hyperoxia. 4,5 We hypothesized that such challenge triggers changes in signal transduction processes. Although acute insult caused during reperfusion may be lethal to cells localized at the focus of insult, elevation of O 2 tension in the surrounding ischemic tissue triggers phenotypic changes in the surviving cells that may be associated with tissue remodeling.Ischemia in the heart results in a hypoxic area containing a central focus of near-zero O 2 pressure bordered by tissue with diminished but nonzero O 2 pressures. These border zones extend for several millimeters from the hypoxic core, with the O 2 pressures progressively increasing from the focus to the normoxic region. 6 Moderate hypoxia is associated with a 30% to 60% decrease (Ϸ1% to 3% O 2 ) in PO 2 . 7 Cardiac fibroblasts (CFs) are mainly responsible for the synthesis of major extracellular matrix (ECM) in the myocardium, including fibrillar collagen types I and III and fibronectin. More than 90% of the interstitial cells of the myocardium are fibroblasts, 8 which actively e...

The mouse vascular smooth muscle alpha-actin (SMA) gene enhancer is activated in fibroblasts by transforming growth factor beta1 (TGFbeta1), a potent mediator of myofibroblast differentiation and wound healing. The SMA enhancer contains tandem sites for the Sp1 transcriptional activator protein and Puralpha and beta repressor proteins. We have examined dynamic interplay between these divergent proteins to identify checkpoints for possible control of myofibroblast differentiation during chronic inflammatory disease. A novel element in the SMA enhancer named SPUR was responsible for both basal and TGFbeta1-dependent transcriptional activation in fibroblasts and capable of binding Sp1 and Pur proteins. A novel Sp1:Pur:SPUR complex was dissociated when SMA enhancer activity was increased by TGFbeta1 or Smad protein overexpression. Physical association of Pur proteins with Smad2/3 was observed as was binding of Smads to an upstream enhancer region that undergoes DNA duplex unwinding in TGFbeta1-activated myofibroblasts. Purbeta repression of the SMA enhancer could not be relieved by TGFbeta1, whereas repression mediated by Puralpha was partially rescued by TGFbeta1 or overexpression of Smad proteins. Interplay between Pur repressor isoforms and Sp1 and Smad coactivators may regulate SMA enhancer output in TGFbeta1-activated myofibroblasts during episodes of wound repair and tissue remodeling.