Abstract
Although L-arginine is the only substrate for nitric oxide (NO) production, no studies have yet been reported on the effect of an L-arginine deficiency on vascular function in humans. Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of dibasic amino acid transport caused by mutations in the SLC7A7 gene, resulting in an L-arginine deficiency. Vascular endothelial function was examined in an LPI patient who was shown to be a compound heterozygote for two mutations in the gene (5.3-kbp Alu-mediated deletion, IVS3+1G→Α). The lumen diameter of the brachial artery was measured in this patient and in healthy controls at rest, during reactive hyperemia (endothelium-dependent vasodilation [EDV]), and after sublingual nitroglycerin administration (endothelium-independent vasodilation [EIV]) using ultrasonography. Both EDV and NOx concentrations were markedly reduced in the patient compared with those for the controls. They became normal after an L-arginine infusion. EIV was not significantly different between the patient and controls. Positron emission tomography of the heart and a treadmill test revealed ischemic changes in the patient, which were improved by the L-arginine infusion. Thus, in the LPI patient, L-arginine deficiency caused vascular endothelial dysfunction via a decrease in NO production.
Authors
Yoshihiro Kamada, Hiroyuki Nagaretani, Shinji Tamura, Tohru Ohama, Takao Maruyama, Hisatoyo Hiraoka, Shizuya Yamashita, Akira Yamada, Shinichi Kiso, Yoshiaki Inui, Nobuyuki Ito, Yoshiro Kayanoki, Sumio Kawata, Yuji Matsuzawa
Abstract
The immortalization of human cells is a critical step in multistep carcinogenesis. Oral-esophageal carcinomas, a model system to investigate molecular mechanisms underlying squamous carcinogenesis, frequently involve cyclin D1 overexpression and inactivation of the p53 tumor suppressor. Therefore, our goal was to establish the functional role of cyclin D1 overexpression and p53 inactivation in the immortalization of primary human oral squamous epithelial cells (keratinocytes) as an important step toward malignant transformation. Cyclin D1 overexpression alone was found to induce extension of the replicative life span of normal oral keratinocytes, whereas the combination of cyclin D1 overexpression and p53 inactivation led to their immortalization. This study also demonstrates that immortalization of oral keratinocytes can be independent of telomerase activation, involving an alternative pathway of telomere maintenance (ALT).
Authors
Oliver G. Opitz, Yasir Suliman, William C. Hahn, Hideki Harada, Hubert E. Blum, Anil K. Rustgi
Abstract
The mechanism underlying the regulation of basal metabolic rate by thyroid hormone remains unclear. Although it has been suggested that thyroid hormone might uncouple substrate oxidation from ATP synthesis, there are no data from studies on humans to support this hypothesis. To examine this possibility, we used a novel combined 13C/31P nuclear magnetic resonance (NMR) approach to assess mitochondrial energy coupling in skeletal muscle of seven healthy adults before and after three days of triiodothyronine (T3) treatment. Rates of ATP synthesis and tricarboxylic acid (TCA) cycle fluxes were measured by 31P and 13C NMR spectroscopy, respectively, and mitochondrial energy coupling was assessed as the ratio. Muscle TCA cycle flux increased by approximately 70% following T3 treatment. In contrast, the rate of ATP synthesis remained unchanged. Given the disproportionate increase in TCA cycle flux compared with ATP synthesis, these data suggest that T3 promotes increased thermogenesis in part by promoting mitochondrial energy uncoupling in skeletal muscle.
Authors
Vincent Lebon, Sylvie Dufour, Kitt Falk Petersen, Jianming Ren, Beat M. Jucker, Lori A. Slezak, Gary W. Cline, Douglas L. Rothman, Gerald I. Shulman
Abstract
Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4+ T cells secreting Th1 cytokines, while recovery from disease is associated with expression of Th2 cytokines. Investigations into the role of individual cytokines in disease induction have yielded contradictory results. Here we used animals with targeted deletion of the STAT4 or STAT6 genes to determine the role of these signaling molecules in EAE. The STAT4 pathway controls the differentiation of cells into a Th1 phenotype, while the STAT6 pathway controls the differentiation of cells into a Th2 phenotype. We found that mice deficient in STAT4 are resistant to the induction of EAE, with minimal inflammatory infiltrates in the central nervous system. In contrast, STAT6-deficient mice, which have a predominantly Th1 phenotype, experience a more severe clinical course of EAE as compared with wild-type or STAT4 knockout mice. In addition, adoptive transfer studies confirm the regulatory functions of a Th2 environment in vivo. These novel data indicate that STAT4 and STAT6 genes play a critical role in regulating the autoimmune response in EAE.
Authors
Tanuja Chitnis, Nader Najafian, Christina Benou, Alan D. Salama, Michael J. Grusby, Mohamed H. Sayegh, Samia J. Khoury
Abstract
We studied the distribution of 5-hydroxytryptamine– (5-HT-) containing cells in the guinea pig pancreas and examined the effects of 5-HT on fluid secretion by interlobular pancreatic ducts. The 5-HT–immunoreactive cells with morphological characteristics of enterochromaffin (EC) cells were scattered throughout the duct system and were enriched in islets of Langerhans. The fluid secretory rate in the isolated interlobular ducts was measured by videomicroscopy. Basolateral applications of 5-HT strongly but reversibly reduced HCO3-dependent, as well as secretin- and acetylcholine- (ACh-) stimulated, fluid secretion, whereas 5-HT applied into the lumen had no such effects. Secretin-stimulated fluid secretion could be inhibited by a 5-HT3 receptor agonist, but not by agonists of the 5-HT1, 5-HT2, or 5-HT4 receptors. Under the stimulation with secretin, 5-HT decreased the intracellular pH (pHi) and reduced the rate of pHi recovery after acid loading with NH4+, suggesting that 5-HT inhibits the intracellular accumulation of HCO3–. The elevation of intraductal pressure in vivo reduced secretin-stimulated fluid secretion, an effect that could be attenuated by a 5-HT3 receptor antagonist. Thus, 5-HT, acting through basolateral 5-HT3 receptors, strongly inhibits spontaneous, secretin-, and ACh-stimulated fluid secretion by guinea pig pancreatic ducts. 5-HT released from pancreatic ductal EC cells on elevation of the intraductal pressure may regulate fluid secretion of neighboring duct cells in a paracrine fashion.
Authors
Atsushi Suzuki, Satoru Naruse, Motoji Kitagawa, Hiroshi Ishiguro, Toshiyuki Yoshikawa, Shigeru B.H. Ko, Akiko Yamamoto, Hiroyuki Hamada, Tetsuo Hayakawa
Abstract
Failure of CD4+ T cells to proliferate in response to antigenic stimulation is a characteristic of HIV infection. Analysis of the proliferation defect has been hampered by an inability to identify CD4+ cells with T cell receptor specificity for antigen. To focus only on cells that had been stimulated through the T cell receptor, CD4+ T cells were stimulated with an anti-Vβ3 Ab that activates approximately 3–5% of peripheral blood T cells. This approach revealed proliferation defects in cells from HIV-infected patients that were not appreciated using anti-CD3 Ab stimulation and provided the capacity to examine responses on a single cell basis. After anti-Vβ3 Ab stimulation, CD4+Vβ3+ cells from HIV-infected patients demonstrated defects in expression of cell cycle–associated proteins, D-type cyclins, and cyclin A. However, the expression of early activation markers, CD69 and CD25, was not significantly impaired in cells from most patients. Thus, CD4+ T cell proliferation failure in HIV disease is characterized by dysregulated activation that precludes cell cycle progression. This proliferation defect was most apparent in patients with diminished CD4+ T cell numbers and higher plasma HIV RNA levels. CD4+ T cell proliferation failure may be a key determinant of immune impairment in HIV disease.
Authors
Scott F. Sieg, Clifford V. Harding, Michael M. Lederman
Abstract
Immune mediation of aplastic anemia (AA) has been inferred from clinical responsiveness to immunosuppressive therapies and a large body of circumstantial laboratory evidence. However, neither the immune response nor the nature of the antigens recognized has been well characterized. We established a large number of CD4 and CD8 T cell clones from a patient with AA and analyzed their T cell receptor (TCR) usage. Most CD4 clones displayed BV5, whereas most CD8 clones displayed BV13. We found sequence identity for complementarity determining region 3 (CDR3) among a majority of CD4 clones; the same sequence was present in marrow lymphocytes from four other patients with AA but was not detected in controls. The dominant CD4 clone showed a Th1 secretion pattern, lysed autologous CD34 cells, and inhibited their hematopoietic colony formation. In three of four patients, successful immunosuppressive treatment led to marked decrease in clones bearing the dominant CDR3 BV5 sequence. These results suggest surprisingly limited heterogeneity of the T cell repertoire in an individual patient and similarity at the molecular level of the likely pathological lymphocyte response among multiple patients with AA, consistent with recognition of limited numbers of antigens shared by individuals with the same HLA type in this disease.
Authors
Weihua Zeng, Jaroslaw P. Maciejewski, Guibin Chen, Neal S. Young
A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis
Abstract
In normal epidermis, β1 integrin expression is confined to the basal layer, whereas in hyperproliferative epidermis, integrins are also expressed in the suprabasal layers. Transgenic mice in which integrins are expressed suprabasally via the involucrin promoter have a sporadic psoriatic phenotype; however, the mechanism by which integrins contribute to the pathogenesis of psoriasis is unknown. We observed activation of mitogen-activated protein kinase (MAPK) in basal and suprabasal keratinocytes of human and transgenic mouse psoriatic lesions and healing mouse skin wounds, correlating in each case with suprabasal integrin expression. Phenotypically normal human and transgenic mouse epidermis did not contain activated MAPK. Transgene-positive keratinocytes produced more IL-1α than controls did, and keratinocyte MAPK could be activated by ligation of suprabasal integrins or treatment with IL-1α. Constitutive activation of MAPK increased the growth rate of human keratinocytes and delayed the onset of terminal differentiation, recreating many of the histological features of psoriatic epidermis. We propose that activation of MAPK by integrins, either directly or through increased IL-1α production, is responsible for epidermal hyperproliferation in psoriasis and wound healing, and that the sporadic phenotype of the transgenic mice may reflect the complex mechanisms by which IL-1 release and responsiveness are controlled in skin.
Authors
Ingo Haase, Robin M. Hobbs, M. Rosario Romero, Simon Broad, Fiona M. Watt
Abstract
The thrombomodulin (TM) gene was ablated in mice in a cell type–restricted manner from vascular endothelium by Cre-recombinase–mediated excision controlled by the endothelial cell lineage–specific Tie2 promoter. Forty percent of mutant (TMLox-) mice display a distinct lethal embryonic phenotype not observed in completely TM-deficient embryos. The remaining 60% of TMLox mice survive beyond birth, but invariably succumb to a severe hypercoagulable state and massive thrombosis after 3 weeks, terminating in a lethal consumptive coagulopathy. The progression of thrombosis was age- and sex-dependent. Disruption of the TM/protein C pathway was not associated with a latent proinflammatory state. Disease onset and progression could be prevented by warfarin anticoagulation. These results show that in mice, loss of endothelial cell TM function causes spontaneous and fatal thrombosis in the arterial and venous circulation, resulting from unfettered activation of the coagulation system. The combination of complete disease penetrance, uniform disease onset at young age, large vessel thrombosis of the extremities and multiple organ systems, and consumptive coagulopathy as the disease end-point provides a unique mouse model of human thrombotic disease.
Authors
Berend Isermann, Sara B. Hendrickson, Mark Zogg, Mark Wing, Marjorie Cummiskey, Yaz Y. Kisanuki, Masashi Yanagisawa, Hartmut Weiler
Abstract
Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival after lung transplantation. Acute rejection, its main risk factor, is characterized by perivascular/bronchiolar leukocyte infiltration. BOS is characterized by persistent peribronchiolar leukocyte recruitment leading to airway fibrosis and obliteration. The specific mechanism(s) by which these leukocytes are recruited are unknown. Because MCP-1, acting through its receptor CCR2, is a potent mononuclear cell chemoattractant, we hypothesized that expression of this chemokine during an allogeneic-response promotes persistent recruitment of leukocytes and, ultimately, rejection. We found that elevated levels of biologically active MCP-1 in human bronchial lavage fluid (BALF) were associated with the continuum from acute to chronic allograft rejection. Translational studies in a murine model of BOS demonstrated increased MCP-1 expression paralleling mononuclear cell recruitment and CCR2 expression. Loss of MCP-1/CCR2 signaling, as seen in CCR2–/– mice or in WT mice treated with neutralizing antibodies to MCP-1, significantly reduced recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS. Lymphocyte infiltration was not reduced under these conditions. We suggest that MCP-1/CCR2 signaling plays an important role in recruitment of mononuclear phagocytes, a pivotal event in the pathogenesis of BOS.
Authors
John A. Belperio, Michael P. Keane, Marie D. Burdick, Joseph P. Lynch III, Ying Ying Xue, Aaron Berlin, David J. Ross, Steven L. Kunkel, Israel F. Charo, Robert M. Strieter
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