Abstract
We previously reported that active sensitization of rats resulted in the appearance of a unique system for rapid and specific antigen uptake across intestinal epithelial cells. The current studies used rats sensitized to horseradish peroxidase (HRP) to define the essential components of this antigen transport system. Sensitization of rats to HRP stimulated increased HRP uptake into enterocytes (significantly larger area of HRP-containing endosomes) and more rapid transcellular transport compared with rats sensitized to an irrelevant protein or naive control rats. Whole serum but not IgE-depleted serum from sensitized rats was able to transfer the enhanced antigen transport phenomenon. Immunohistochemistry demonstrated that sensitization induced expression of CD23, the low-affinity IgE receptor (FcεRII), on epithelial cells. The number of immunogold-labeled CD23 receptors on the enterocyte microvillous membrane was significantly increased in sensitized rats and was subsequently reduced after antigen challenge when CD23 and HRP were localized within the same endosomes. Finally, pretreatment of tissues with luminally added anti-CD23 antibody significantly inhibited both antigen transport and the hypersensitivity reaction. Our results provide evidence that IgE antibodies bound to low-affinity receptors on epithelial cells are responsible for the specific and rapid nature of this novel antigen transport system.
Authors
Ping-Chang Yang, M. Cecilia Berin, Linda C.H. Yu, Daniel H. Conrad, Mary H. Perdue
Abstract
To investigate the role of IL-6 in alcohol-mediated osteoporosis, we measured a variety of bone remodeling parameters in wild-type (il6+/+) or IL-6 gene knockout (il6–/–) mice that were fed either control or ethanol liquid diets for 4 months. In the il6+/+ mice, ethanol ingestion decreased bone mineral density, as determined by dual-energy densitometry; decreased cancellous bone volume and trabecular width and increased trabecular spacing and osteoclast surface, as determined by histomorphometry of the femur; increased urinary deoxypyridinolines, as determined by ELISA; and increased CFU-GM formation and osteoclastogenesis as determined ex vivo in bone marrow cell cultures. In contrast, ethanol ingestion did not alter any of these parameters in the il6–/– mice. Ethanol increased receptor activator of NF-κB ligand (RANKL) mRNA expression in the bone marrow of il6+/+ but not il6–/– mice. Additionally, ethanol decreased several osteoblastic parameters including osteoblast perimeter and osteoblast culture calcium retention in both il6+/+ and il6–/– mice. These findings demonstrate that ethanol induces bone loss through IL-6. Furthermore, they suggest that IL-6 achieves this effect by inducing RANKL and promoting CFU-GM formation and osteoclastogenesis.
Authors
Jinlu Dai, Dinlii Lin, Jian Zhang, Paula Habib, Peter Smith, Jill Murtha, Zheng Fu, Zhi Yao, Yinghua Qi, Evan T. Keller
Abstract
ATP-sensitive potassium channels play a major role in linking metabolic signals to the exocytosis of insulin in the pancreatic β cell. These channels consist of two types of protein subunit: the sulfonylurea receptor SUR1 and the inward rectifying potassium channel Kir6.2. Mutations in the genes encoding these proteins are the most common cause of congenital hyperinsulinism (CHI). Since 1973, we have followed up 38 pediatric CHI patients in Finland. We reported previously that a loss-of-function mutation in SUR1 (V187D) is responsible for CHI of the most severe cases. We have now identified a missense mutation, E1506K, within the second nucleotide binding fold of SUR1, found heterozygous in seven related patients with CHI and in their mothers. All patients have a mild form of CHI that usually can be managed by long-term diazoxide treatment. This clinical finding is in agreement with the results of heterologous coexpression studies of recombinant Kir6.2 and SUR1 carrying the E1506K mutation. Mutant KATP channels were insensitive to metabolic inhibition, but a partial response to diazoxide was retained. Five of the six mothers, two of whom suffered from hypoglycemia in infancy, have developed gestational or permanent diabetes. Linkage and haplotype analysis supported a dominant pattern of inheritance in a large pedigree. In conclusion, we describe the first dominantly inherited SUR1 mutation that causes CHI in early life and predisposes to later insulin deficiency.
Authors
Hanna Huopio, Frank Reimann, Rebecca Ashfield, Jorma Komulainen, Hanna-Liisa Lenko, Jaques Rahier, Ilkka Vauhkonen, Juha Kere, Markku Laakso, Frances Ashcroft, Timo Otonkoski
Abstract
Chromogranin A (CgA) is the major soluble protein in the core of catecholamine-storage vesicles and is also distributed widely in secretory vesicles throughout the neuroendocrine system. CgA contains the sequences for peptides that modulate catecholamine release, but the proteases responsible for the release of these bioactive peptides from CgA have not been established. We show here that the major fibrinolytic enzyme, plasmin, can cleave CgA to form a series of large fragments as well as small trichloroacetic acid-soluble peptides. Peptides generated by plasmin-mediated cleavage of CgA significantly inhibited nicotinic cholinergic stimulation of catecholamine release from PC12 cells and primary bovine adrenal chromaffin cells. We also show that the zymogen, plasminogen, as well as tissue plasminogen activator bind saturably and with high capacity to catecholaminergic (PC12) cells. Occupancy of cell surface binding sites promoted the cleavage of CgA by plasmin. Positive and negative modulation of the local cellular fibrinolytic system resulted in substantial alterations in catecholamine release. These results suggest that catecholaminergic cells express binding sites that localize fibrinolytic molecules on their surfaces to promote plasminogen activation and proteolytic processing of CgA in the environment into which CgA is secreted to generate peptides which may regulate neuroendocrine secretion. Interactions between CgA and plasmin(ogen) define a previously unrecognized autocrine/paracrine system that may have a dramatic impact upon catecholamine secretion.
Authors
Robert J. Parmer, Manjula Mahata, Yun Gong, Sushil K. Mahata, Qijiao Jiang, Daniel T. O’Connor, Xiao-Ping Xi, Lindsey A. Miles
Abstract
The phosphatase Cdc25A plays an important role in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases, and it has been shown to transform diploid murine fibroblasts in cooperation with activated Ras. Here we show that Cdc25A is overexpressed in primary breast tumors and that such overexpression is correlated with higher levels of cyclin-dependent kinase 2 (Cdk2) enzymatic activity in vivo. Furthermore, in the breast cancer cell line MCF-7, Cdc25A activity is necessary for both the activation of Cdk2 and the subsequent induction of S-phase entry. Finally, in a series of small (< 1 cm) breast carcinomas, overexpression of Cdc25A was found in 47% of patients and was associated with poor survival. These data suggest that overexpression of Cdc25A contributes to the biological behavior of primary breast tumors and that both Cdc25A and Cdk2 are suitable therapeutic targets in early-stage breast cancer.
Authors
M. Giulia Cangi, Barry Cukor, Peggy Soung, Sabina Signoretti, Gilberto Moreira Jr., Moksha Ranashinge, Blake Cady, Michele Pagano, Massimo Loda
Abstract
The DF3/MUC1 gene is aberrantly overexpressed in human breast and other carcinomas. Previous studies have demonstrated that the DF3/MUC1 promoter/enhancer confers selective expression of diverse transgenes in MUC1-positive breast cancer cells. In this study, we show that an adenoviral vector (Ad.DF3-E1) in which the DF3/MUC1 promoter drives expression of E1A selectively replicates in MUC1-positive breast cancer cells. We also show that Ad.DF3-E1 infection of human breast tumor xenografts in nude mice is associated with inhibition of tumor growth. In contrast to a replication-incompetent adenoviral vector that infects along the injection track, Ad.DF3-E1 infection was detectable throughout the tumor xenografts. To generate an Ad.DF3-E1 vector with the capacity for incorporating therapeutic products, we inserted the cytomegalovirus (CMV) promoter upstream of the TNF cDNA. Infection with Ad.DF3-E1/CMV-TNF was associated with selective replication and production of TNF in cells that express MUC1. Moreover, treatment of MUC1-positive, but not MUC1-negative, xenografts with a single injection of Ad.DF3-E1/CMV-TNF was effective in inducing stable tumor regression. These findings demonstrate that the DF3/MUC1 promoter confers competence for selective replication of Ad.DF3-E1 in MUC1-positive breast tumor cells, and that the antitumor activity of this vector is potentiated by integration of the TNF cDNA.
Authors
Toshikazu Kurihara, Douglas E. Brough, Imre Kovesdi, Donald W. Kufe
Abstract
Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue–induced pancreatitis, the trypsin activity in the pancreas of ctsb–/– animals was more than 80% lower than in ctsb+/+ animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb–/– animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis.
Authors
Walter Halangk, Markus M. Lerch, Barbara Brandt-Nedelev, Wera Roth, Manuel Ruthenbuerger, Thomas Reinheckel, Wolfram Domschke, Hans Lippert, Christoph Peters, Jan Deussing
Abstract
Hyperoxia is an important cause of acute lung injury. To determine whether IL-13 is protective in hyperoxia, we compared the survival in 100% O2 of transgenic mice that overexpress IL-13 in the lung and of nontransgenic littermate controls. IL-13 enhanced survival in 100% O2. One hundred percent of nontransgenic mice died in 4–5 days, whereas 100% of IL-13–overexpressing mice lived for more than 7 days, and many lived 10–14 days. IL-13 also stimulated VEGF accumulation in mice breathing room air, and it interacted with 100% 2 to increase VEGF accumulation further. The 164–amino acid isoform was the major VEGF moiety in bronchoalveolar lavage from transgenic mice in room air, whereas the 120– and 188–amino acid isoforms accumulated in these mice during hyperoxia. In addition, antibody neutralization of VEGF decreased the survival of IL-13–overexpressing mice in 100% 2. These studies demonstrate that IL-13 has protective effects in hyperoxic acute lung injury. They also demonstrate that IL-13, alone and in combination with 100% 2, stimulates pulmonary VEGF accumulation, that this stimulation is isoform-specific, and that the protective effects of IL-13 are mediated, in part, by VEGF.
Authors
Jonathan Corne, Geoffrey Chupp, Chun Guen Lee, Robert J. Homer, Zhou Zhu, Qingsheng Chen, Bing Ma, Yuefen Du, Francoise Roux, John McArdle, Aaron B. Waxman, Jack A. Elias
Abstract
The CCR2-mediated recruitment of monocytes into the vessel wall plays an important role in all stages of atherosclerosis. In recent studies, we have shown that lipoproteins can modulate CCR2 expression and have identified native LDL as a positive regulator. In contrast, oxidized LDL (OxLDL), which is mainly formed in the aortic intima, reduces CCR2 expression, promotes monocyte retention, and may cause pathological accumulation of monocytes in the vessel wall. We now provide evidence that OxLDL reduces monocyte CCR2 expression by activating intracellular signaling pathways that may involve peroxisome proliferator–activated receptor γ (PPARγ). Receptor-mediated uptake of the lipoprotein particle was required and allows for delivery of the exogenous ligand to the nuclear receptor. The suppression of CCR2 expression by OxLDL was mediated by lipid components of OxLDL, such as the oxidized linoleic acid metabolites 9-HODE and 13-HODE, known activators of PPARγ. Modified apoB had no such effect. Consistent with a participation of the PPARγ signaling pathway, BRL49653 reduced CCR2 expression in freshly isolated human monocytes ex vivo and in circulating mouse monocytes in vivo. These results implicate PPARγ in the inhibition of CCR2 gene expression by oxidized lipids, which may help retain monocytes at sites of inflammation, such as the atherosclerotic lesion.
Authors
Ki Hoon Han, Mi Kyung Chang, Agnes Boullier, Simone R. Green, Andrew Li, Christopher K. Glass, Oswald Quehenberger
Abstract
Although oligodendrocytes (OLGs) are thought to be vulnerable to hypoxia and ischemia, little is known about the detailed mechanism by which these insults induce OLG death. From the clinical viewpoint, it is imperative to protect OLGs as well as neurons against ischemic injury (stroke), because they are the only myelin-forming cells of the central nervous system. Using the Cre/loxP system, we have established a transgenic mouse line that selectively expresses p35, a broad-spectrum caspase inhibitor, in OLGs. After hypoxia, cultured OLGs derived from wild-type mice exhibited significant upregulation of caspase-11 and substantial activation of caspase-3, which led to cell loss. Expression of p35 or elimination of caspase-11 suppressed the caspase-3 activation and conferred significant protection against hypoxic injury. Expression of p35 in OLGs in vivo resulted in significant protection from ischemia-induced cell injury, thus indicating that caspases are involved in the ischemia-induced cell death of OLGs. Furthermore, the induction of caspase-11 was evident in the ischemic brains of wild-type mice, and OLGs exhibited resistance to brain ischemia in mice deficient in caspase-11, suggesting that caspase-11 is critically implicated in the mechanism(s) underlying ischemia-induced OLG death. Caspases may therefore offer a good therapeutic target for reducing ischemia-induced damage to OLGs.
Authors
Mamoru Shibata, Shin Hisahara, Hideaki Hara, Takemori Yamawaki, Yasuo Fukuuchi, Junying Yuan, Hideyuki Okano, Masayuki Miura
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