Ted M. Dawson, Valina L. Dawson
Christelle Guégan, Serge Przedborski
Harry Ischiropoulos, Joseph S. Beckman
S. Bruce King
Kenneth R. Chien
George S. Eisenbarth, Brian L. Kotzin
Lawrence Steinman, Paul Conlon, Rich Maki, Alan Foster
Hematopoietic progenitor cells (HPCs) normally reside in the bone marrow (BM) but can be mobilized into the peripheral blood (PB) after treatment with GCSF or chemotherapy. In previous studies, we showed that granulocyte precursors accumulate in the BM during mobilization induced by either GCSF or cyclophosphamide (CY), leading to the accumulation of active neutrophil proteases in this tissue. We now report that mobilization of HPCs by GCSF coincides in vivo with the cleavage of the N-terminus of the chemokine receptor CXCR4 on HPCs resident in the BM and mobilized into the PB. This cleavage of CXCR4 on mobilized HPCs results in the loss of chemotaxis in response to the CXCR4 ligand, the chemokine stromal cell–derived factor-1 (SDF-1/CXCL12). Furthermore, the concentration of SDF-1 decreased in vivo in the BM of mobilized mice, and this decrease coincided with the accumulation of serine proteases able to directly cleave and inactivate SDF-1. Since both SDF-1 and its receptor, CXCR4, are essential for the homing and retention of HPCs in the BM, the proteolytic degradation of SDF-1, together with that of CXCR4, could represent a critical step leading to the mobilization of HPCs into the PB in response to GCSF or CY.
Jean-Pierre Lévesque, Jean Hendy, Yasushi Takamatsu, Paul J. Simmons, Linda J. Bendall
This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-α–mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-α, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-α–induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-α–mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-α or LPS to galactosamine-pretreated ASMase+/+ mice caused liver damage, ASMase–/– mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-α–mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-α, protected mGSH-depleted ASMase+/+ hepatocytes from TNF-α despite enhancement of TNF-α–stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase+/+ but not in ASMase–/– hepatocytes. However, treatment of ASMase–/– hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-α–induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.
Carmen García-Ruiz ... Carlos Enrich, José C. Fernández-Checa
Restrictive cardiomyopathy (RCM) is an uncommon heart muscle disorder characterized by impaired filling of the ventricles with reduced volume in the presence of normal or near normal wall thickness and systolic function. The disease may be associated with systemic disease but is most often idiopathic. We recognized a large family in which individuals were affected by either idiopathic RCM or hypertrophic cardiomyopathy (HCM). Linkage analysis to selected sarcomeric contractile protein genes identified cardiac troponin I (TNNI3) as the likely disease gene. Subsequent mutation analysis revealed a novel missense mutation, which cosegregated with the disease in the family (lod score: 4.8). To determine if idiopathic RCM is part of the clinical expression of TNNI3 mutations, genetic investigations of the gene were performed in an additional nine unrelated RCM patients with restrictive filling patterns, bi-atrial dilatation, normal systolic function, and normal wall thickness. TNNI3 mutations were identified in six of these nine RCM patients. Two of the mutations identified in young individuals were de novo mutations. All mutations appeared in conserved and functionally important domains of the gene.
Jens Mogensen ... Perry Elliott, William J. McKenna
Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic β cells by T lymphocytes. Prediction of cell-mediated autoimmune diseases by direct detection of autoreactive T cells in peripheral blood has proved elusive, in part because of their low frequency and reduced avidity for peptide MHC ligands.
Jacqueline D. Trudeau ... Pere Santamaria, Rusung Tan
The adipose-derived hormone resistin is postulated to link obesity to insulin resistance and diabetes. Here, the infusion of either resistin or the resistin-like molecule–β (RELMβ) rapidly induced severe hepatic but not peripheral insulin resistance. In the presence of physiologic hyperinsulinemia, the infusion of purified recombinant resistin, increasing circulating resistin levels by approximately twofold to 15-fold, inhibited glucose metabolism such that lower rates of glucose infusion were required to maintain the plasma glucose concentration at basal levels. The effects of resistin and RELMβ on in vivo insulin action were completely accounted for by a marked increase in the rate of glucose production. These results support the notion that a novel family of fat- and gut-derived circulating proteins modulates hepatic insulin action.
Michael W. Rajala, Silvana Obici, Philipp E. Scherer, Luciano Rossetti
Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of γ-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced γ-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.
Vladan P. Cokic ... Mark T. Gladwin, Alan N. Schechter
In the work presented here, we explored the influence of leptin on the kinetics of experimental autoimmune encephalomyelitis (EAE) onset, in the EAE-associated inflammatory anorexia, and in the development of pathogenic T cell responses. We found that the expression of serum leptin increased before the clinical onset of EAE in disease-susceptible C57BL/6J (H-2b) and SJL/J (H-2s) strains of mice, which are models of chronic-progressive and relapsing-remitting EAE, respectively. This increase in serum leptin correlated with disease susceptibility, reduction in food intake, and decrease in body weight. Indeed, acute starvation, which is able to prevent the increase in serum leptin, delayed disease onset and attenuated clinical symptoms by inducing a T helper 2 cytokine switch. Furthermore, immunohistochemical analysis revealed a parallel in situ production of leptin in inflammatory infiltrates and in neurons only during the acute/active phase of both chronic-progressive and relapsing-remitting EAE. We also found that leptin secretion by activated T cells sustained their proliferation in an autocrine loop, since antileptin receptor antibodies were able to inhibit the proliferative response of autoreactive T cells in vitro. Given that leptin appears to regulate EAE susceptibility, inflammatory anorexia, and pathogenic T-cell immune function, we postulate that it may offer a potential target in the treatment of multiple sclerosis.
Veronica Sanna ... Serafino Zappacosta, Giuseppe Matarese
Current therapeutic strategies for genetic skin disorders rely on the complex process of grafting genetically engineered tissue to recipient wound beds. Because fibroblasts synthesize and secrete extracellular matrix, we explored their utility in recessive dystrophic epidermolysis bullosa (RDEB), a blistering disease due to defective extracellular type VII collagen. Intradermal injection of RDEB fibroblasts overexpressing type VII collagen into intact RDEB skin stably restored correctly localized type VII collagen expression in vivo and normalized hallmark RDEB disease features, including subepidermal blistering and anchoring fibril defects.
Susana Ortiz-Urda ... M. Peter Marinkovich, Paul A. Khavari
Rapid oscillations of visceral lipolysis have been reported. To examine the putative role of the CNS in oscillatory lipolysis, we tested the effects of β3-blockade on pulsatile release of FFAs. Arterial blood samples were drawn at 1-minute intervals for 120 minutes from fasted, conscious dogs (n = 7) during the infusion of saline or bupranolol (1.5 μg/kg/min), a high-affinity β3-blocker. FFA and glycerol time series were analyzed and deconvolution analysis was applied to estimate the rate of FFA release. During saline infusion FFAs and glycerol oscillated in phase at about eight pulses/hour. Deconvolution analysis showed bursts of lipolysis (nine pulses/hour) with time-dependent variation in burst frequency. Bupranolol completely removed rapid FFA and glycerol oscillations. Despite removal of lipolytic bursts, plasma FFAs (0.31 mM) and glycerol (0.06 mM) were not totally suppressed and deconvolution analysis revealed persistent non-oscillatory lipolysis (0.064 mM/min). These results show that lipolysis in the fasting state consists of an oscillatory component, which appears to be entirely dependent upon sympathetic innervation of the adipose tissue, and a non-oscillatory, constitutive component, which persists despite β3-blockade. The extinction of lipid fuel bursts by β3-blockade implies a role for the CNS in the maintenance of cyclic provision of lipid fuels.
Katrin Hücking, Marianthe Hamilton-Wessler, Martin Ellmerer, Richard N. Bergman
IGF-II is a mitogenic peptide that has been implicated in hepatocellular oncogenesis. Since the silencing of gene expression is frequently associated with cytosine methylation at cytosine-guanine (CpG) dinucleotides, we designed a methylated oligonucleotide (MON1) complementary to a region encompassing IGF2 promoter P4 in an attempt to induce DNA methylation at that locus and diminish IGF2 mRNA levels. MON1 specifically inhibited IGF2 mRNA accumulation in vitro, whereas an oligonucleotide (ON1) with the same sequence but with nonmethylated cytosines had no effect on IGF2 mRNA abundance. MON1 treatment led to the specific induction of de novo DNA methylation in the region of IGF2 promoter hP4. Cells from a human hepatocellular carcinoma (HCC) cell line, Hep 3B, were implanted into the livers of nude mice, resulting in the growth of large tumors. Animals treated with MON1 had markedly prolonged survival as compared with those animals treated with saline or a truncated methylated oligonucleotide that did not alter IGF2 mRNA levels in vitro. This study demonstrates that a methylated sense oligonucleotide can be used to induce epigenetic changes in the IGF2 gene and that inhibition of IGF2 mRNA accumulation may lead to enhanced survival in a model of HCC.
Xiaoming Yao ... Tao Li, Andrew R. Hoffman
Prolactin is a peptide hormone produced by the anterior pituitary gland that is critical in lactation. Prolactin can also be produced by lymphocytes, and both B and T cells express prolactin receptors. These findings have suggested that prolactin has immunomodulatory functions. Studies in spontaneously autoimmune hosts have demonstrated a role for prolactin in augmenting autoreactivity. We chose to analyze prolactin effects on anti-DNA B cells in nonspontaneously autoimmune female BALB/c mice transgenic for the heavy chain of an anti-DNA antibody. Treatment with prolactin for 4 weeks induced a lupus-like phenotype with an increased number of transgene-expressing B cells, elevated serum anti-DNA antibody titers, and glomerular immunoglobulin deposits. Prolactin caused a decrease in the population of transitional B cells and an increase in mature follicular and marginal zone B cells. The DNA-reactive B cells had a follicular cell phenotype. Anti-DNA hybridomas demonstrated that prolactin alters selection of the naive B cell repertoire. The expansion and activation of anti-DNA B cells in prolactin-treated R4A-γ2b BALB/c mice was dependent on the presence of CD4+ T cells. Finally, treatment with prolactin was unable to break tolerance in R4A-γ2b transgenic C57Bl/6 mice, suggesting that responsiveness of the immune system to prolactin is genetically determined.
Elena Peeva, Daniel Michael, James Cleary, Jeffrey Rice, Xian Chen, Betty Diamond
Copyright © 2014 American Society for Clinical Investigation