Mechanisms associated with type 1 diabetes (T1D) development remain incompletely defined. Using a sensitive array-based bioassay where patient plasma is used to induce transcriptional responses in healthy leukocytes, we previously reported disease-specific, partially interleukin (IL)-1−dependent signatures associated with preonset and recent onset (RO) T1D relative to unrelated healthy control subjects (uHC). To better understand inherited susceptibility in T1D families, we conducted cross-sectional and longitudinal analyses of healthy autoantibody-negative (AA−) high HLA−risk siblings (HRS) (DR3 and/or DR4) and AA− low HLA−risk siblings (LRS) (non-DR3/non-DR4). Signatures, scored with a novel ontology-based algorithm, and confirmatory studies differentiated the RO T1D, uHC, HRS, and LRS plasma milieus. Relative to uHC, T1D family members exhibited an elevated inflammatory state, consistent with innate receptor ligation that was independent of HLA, AA, or disease status and included elevated plasma IL-1α, IL-12p40, CCL2, CCL3, and CCL4 levels. Longitudinally, signatures of T1D progressors exhibited increasing inflammatory bias. Conversely, HRS possessing decreasing AA titers revealed emergence of an IL-10/transforming growth factor-β−mediated regulatory state that paralleled temporal increases in peripheral activated CD4+/CD45RA−/FoxP3high regulatory T-cell frequencies. In AA− HRS, the familial innate inflammatory state also was temporally supplanted by immunoregulatory processes, suggesting a mechanism underlying the decline in T1D susceptibility with age.

CD200 (OX-2) is a transmembrane glycoprotein that transmits an immunoregulatory signal through the CD200 receptor (CD200R) to attenuate inflammatory reactions and promote immune tolerance. CD200 expression in the skin has not been described previously. We now report that freshly isolated cells of the murine epidermis contain a subpopulation of major histocompatibility complex (MHC) class II-negative, CD3-negative keratinocytes that are CD200-positive. CD200 expression was accentuated in keratinocytes comprising the outer root sheath of the murine hair follicle (HF). When syngeneic skin grafts were exchanged between gender-matched wild-type (WT) and CD200-deficient C57BL/6 mice, significant perifollicular and intrafollicular inflammation was observed, eventually leading to the destruction of virtually all HF (alopecia) without significant loss of the CD200-negative grafts. Minimal and transient inflammation was observed in WT grafts, which persisted long term with hair. There was a 2-fold increase in graft-infiltrating T cells in CD200-deficient skin at 14 d. Alopecia and skin lesions were induced in CD200-deficient hosts by adoptive transfer of splenocytes from WT mice previously grafted with CD200-negative skin, but not from mice grafted with WT skin. Collectively, these results suggest that the expression of CD200 in follicular epithelium attenuates inflammatory reactions and may play a role in maintaining immune tolerance to HF-associated autoantigens.

ObjectiveChemotherapy doses are limited by toxicity to normal tissues. Intravenous glutamine protects liver cells from oxidant injury by increasing intracellular glutathione (GSH) content. The authors hypothesized that supplemental oral glutamine (GLN) would increase the therapeutic index of methotrexate (MTX) by improving host tolerance through changes in glutathione metabolism. The authors examined the effects of oral glutamine on tumor and host glutathione metabolism and response to methotrexate. MethodsThirty-six 300-g Fischer 344 rats were implanted with fibrosarcomas. On day 21 after implantation, rats were randomized to receive isonitrogenous isocaloric diets containing 1 g/kg/day glutamine or glycine (GLY) by gavage. On day 23 after 2 days of prefeeding, rats were randomized to one of the following four groups receiving an intraperitoneal injection of methotrexate (20 mg/kg) or saline (CON): GLN + MTX, GLY + MTX, GLN-CON, or GLY-CON. On day 24, rats were killed and studied for arterial glutamine concentration, tumor volume, tumor, kidney and gut glutaminase activity, and glutathione content (tumor, gut, heart, liver, muscle, kidney, and lung). ConclusionThese data suggest that oral glutamine supplementation will enhance the selectivity of antitumor drugs by protecting normal tissues from and possibly sensitizing tumor cells to chemotherapy treatment-related injury. jected to alternate 12-hour periods of dark/light cycle and given at least 1 week to acclimate to the animal care facilities. During that time, the rats were allowed ad libitum intake of standard rat chow and water. Animals were randomized during the study period to receive isonitrogenous isocaloric chow diets supplemented with 1 g/kg/day elemental GLN or glycine (GLY) by gavage.Tumor Cell ImplantationAfter 1 week of acclimation to the animal care facility and on day 0 of the study, 36 rats were randomized to flank implantation of a 2 X 2 X 2 mm3 of viable methylcholanthrene-induced fibrosarcoma cells. This tumor model has been used previously by the author7"15-'6'20,2' to study tumor host metabolism interaction. This tumor-cell line is fast-growing and locally aggressive, metastasizes rarely, and never regresses spontaneously. Study ProcedureOn day 21 after tumor cell implantation, rats were randomized to receive pair-fed chow diets with supplemental GLN or GLY by gavage. On day 23, after 2 days of prefeeding, rats were randomized to one ofthe following four groups receiving an intraperitoneal injection of MTX (20 mg/kg) or saline (CON): GLN + MTX, GLY + MTX, GLN-CON, or GLY-CON. Each group contained nine rats. On day 24, all rats were weighed and anesthesia was obtained with ketamine (7.5 mg/100 g body weight) and acepromazine (0.1 mg/100 g body weight). Under sterile conditions, a mid-line incision was made, and the rat was heparinized. Arterial blood was withdrawn from the aorta using a 25-gauge needle attached to a 1-mL syringe. Blood was processed for arterial GLN content. The jejunum and kidney were removed and processed for glutaminas...

Clinical studies have shown gallium nitrate to have significant antitumor activity against non-Hodgkin's lymphoma and bladder cancer, thus indicating that gallium-based drugs have potential for further development as antineoplastic agents. In this study, we compared the cytotoxicity of gallium maltolate, a novel gallium compound, with gallium nitrate in lymphoma cell lines, including p53 variant and unique gallium nitrate-resistant cells. We found that gallium maltolate inhibited cell proliferation and induced apoptosis through the mitochondrial pathway at lower concentrations and more rapidly than gallium nitrate. Gallium maltolate produced an increase in intracellular reactive oxygen species (ROS) within 2 h of incubation with cells; this effect could be blocked by mitoquinone, a mitochondria-targeted antioxidant. The role of the transferrin receptor (TfR) in gallium maltolate's action was examined using monoclonal antibody (MoAb) 42/6 to block TfR function. However, although MoAb 42/6 reduced gallium maltolate-induced caspase-3 activity, it had only a minor effect on cell growth inhibition. Importantly, gallium maltolate induced apoptosis in cells resistant to gallium nitrate, and, unlike gallium nitrate, its cytotoxicity was not affected by cellular p53 status. Cellular gallium uptake was greater with gallium maltolate than with gallium nitrate. We conclude that gallium maltolate inhibits cell proliferation and induces apoptosis more efficiently than gallium nitrate. Gallium maltolate is incorporated into lymphoma cells to a greater extent than gallium nitrate via both TfR-independent and -dependent pathways; it has significant activity against gallium nitrate-resistant cells and acts independently of p53. Further studies to evaluate its antineoplastic activity in vivo are warranted.

During apoptotic cell death, biochemical processes modify self-proteins and create potential autoantigens. To maintain self-tolerance in the face of natural cell turnover, the immune system must prevent or control responses to apoptosisassociated autoantigens or risk autoimmunity. The molecular mechanisms governing this process remain largely unknown. Here, we show that expression of the immunoregulatory protein CD200 increases as murine dendritic cells (DCs) undergo apoptosis. We define CD200 as a p53-target gene and identify both p53-and caspase-dependent pathways that control CD200 expression during apoptosis. CD200 expression on apoptotic DCs diminishes proinflammatory cytokine production in response to self-antigens in vitro and is required for UVB-mediated tolerance to haptenated self-proteins in vivo. Up-regulation of CD200 may represent a novel mechanism, whereby immune reactivity to apoptosis-associated self-antigens is suppressed under steady state conditions. IntroductionAs a result of natural cell turnover, cells in peripheral tissues continually undergo apoptosis. These apoptotic cells are associated with a distinct lack of inflammation, leading to the initial perception that the process of apoptosis is immunologically silent and passive. 1 However, it is now clear that apoptotic cells do not just quietly disappear. Instead, they actively inhibit immune responses by providing immunoregulatory signals directly to cells of the immune system. For example, when added to human lymphocyte cultures stimulated with lipopolysaccharide, apoptotic cells inhibit production of proinflammatory cytokines such as tumor necrosis factor ␣ (TNF-␣), interleukin 1 (IL-1), and IL-12 and promote the production of the anti-inflammatory cytokine IL-10. 2 T-cell activation is inhibited when apoptotic cells are added to splenocytes in the presence of Con A. 3 Macrophages that have ingested apoptotic cells increase production of anti-inflammatory cytokines and inhibit production of proinflammatory cytokines. 4 Furthermore, immature dendritic cells (DCs) that have taken up apoptotic cells have a compromised ability to mature into immunostimulatory antigen-presenting cells (APCs). 5 Given their immunosuppressive capacity, it has been suggested that apoptotic cells generated during natural cell turnover play a central role in the establishment and maintenance of peripheral self-tolerance. 6 Immature DCs and monocytes traffic through tissues, phagocytose apoptotic cells, and migrate to lymphoid tissue 7 ; DCs that have ingested apoptotic cells process and present apoptotic cell-derived peptides on major histocompatibility complex (MHC) class I and class II molecules 8 ; and DCs that have taken up apoptotic cells fail to initiate productive T-cell responses, possibly through the induction of regulatory T cells. 9,10 Patients with autoimmune diseases have abnormal immune responses to self-peptides generated during apoptosis, suggesting that mechanisms exist in healthy individuals that suppress immunoreactivity to apoptosis-associat...