The development of inflammation is an important component of host defense against infection. The cellular and molecular processes underlying inflammation are well-studied, and it is known that cells of the blood vessel wall, such as endothelial cells and smooth muscle cells, play pivotal roles. Additionally, a wide variety of proinflammatory mediators have been defined, which coordinate the multicellular processes of inflammation. Knowledge of the potential role of blood vessel gap junctional intercellular communication (GJIC) in coordinating the inflammation process, however, is limited. In this study, we report that bacterial lipopolysaccharide (LPS), as well as the proinflammatory cytokines TNF-alpha and IL-1beta, selectively inhibit human myoendothelial GJIC in vitro without affecting GJIC between the respective homologous cell populations. This finding may represent a physiologically relevant component of the inflammatory response to infection. The work also provides some of the first clear evidence suggesting that a single eukaryotic cell can differentially regulate its GJIC between homologous and heterologous cell types in a simultaneous manner.
J Hu, I A Cotgreave
Previous studies in the rat have shown that antibodies to gp280, a protein > 200 kD and closely associated with the early endocytic system can induce fetal malformations. Although gp280 is thought to act as a receptor, its ligand(s) is not known. In the current study, we report that purified gp280 from rat kidney, like the intrinsic factor-Cobalamin receptor (IFCR), binds to the intrinsic factor-cobalamin (IFCbl) complex with an association constant of 0.3 x 10(9) M-1 and mediates its internalization. Furthermore, antibodies raised to purified gp280 and IFCR inhibited the binding of IF-[57Co]Cbl complex to intestinal, renal, and yolk sac apical membranes and revealed a single identically sized protein on immunoblotting of the renal membranes. Both antibodies precipitated a single radiolabeled protein > 200 kD from cellular extract from [35S]methionine-labeled yolk sac epithelial cells, and antibody to gp280 inhibited the uptake and internalization of 125IF-Cbl. Immunoelectron microscopy using the two antibodies revealed that in the kidney, both proteins were colocalized. These observations suggest that IF-Cbl complex is a ligand for gp280 and that gp280 and IFCR are identical proteins.
B Seetharam, E I Christensen, S K Moestrup, T G Hammond, P J Verroust
Recently, it has been shown that Factor XI can be activated by thrombin, and that Factor XIa significantly contributes to the generation of thrombin via the intrinsic pathway after the clot has been formed. This additional thrombin, generated inside the clot, was found to protect the clot from fibrinolysis. A plausible mechanism for this inhibitory effect of thrombin involves TAFI (thrombin-activatable fibrinolysis inhibitor, procarboxypeptidase B) which, upon activation, may inhibit fibrinolysis by removing carboxy-terminal lysines from fibrin. We studied the role of Factor XI and TAFI in fibrinolysis using a clot lysis assay. The lysis time was decreased twofold when TAFI was absent, when TAFI activation was inhibited by anti-TAFI antibodies, or when activated TAFI was inhibited by the competitive inhibitor (2-guanidinoethylmercapto)succinic acid. Inhibition of either TAFI activation or Factor XIa exhibited equivalent profibrinolytic effects. In the absence of TAFI, no additional effect of anti-Factor XI was observed on the rate of clot lysis. We conclude that the mechanism of Factor XI-dependent inhibition of fibrinolysis is through the generation of thrombin via the intrinsic pathway, and is dependent upon TAFI. This pathway may play a role in determining the fate of in vivo formed clots.
P A Von dem Borne, L Bajzar, J C Meijers, M E Nesheim, B N Bouma
The presence of the extracellular calcium-sensing receptor on human antral gastrin cells was investigated. Reverse transcription PCR using mRNA isolated from gastrin cell- enriched cell cultures identified a product with a sequence identical to part of the human parathyroid-secreting cell calcium-sensing receptor. Immunocytochemistry with an antibody to the extracellular region of the receptor immunostained all gastrin cells (but not mucin or somatostatin cells), and detected appropriate-sized bands in Western blots of whole cell lysates. Increasing extracellular calcium levels from 0.5 to 9 mM stimulated gastrin release in a concentration-dependent manner, with maximal release obtained at 7.2 mM. A known agonist of the calcium receptor, spermine also stimulated gastrin release. Microfluorimetry of identified gastrin cells demonstrated that increasing extracellular calcium resulted in an initial rapid rise in intracellular calcium followed by a plateau level that returned to basal levels immediately after removal of the elevated calcium. The traces were consistent with activation of a receptor-mediated mechanism rather than a concentration-dependent influx of calcium. In conclusion, these data indicate that G cells express the calcium-sensing receptor, and that activation of the receptor may explain the acid rebound phenomenon associated with calcium-containing antacid preparations.
J M Ray, P E Squires, S B Curtis, M R Meloche, A M Buchan
Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia during atherosclerosis and restenosis, but the endogenous cell cycle regulatory factors underlying VSMC growth in response to arterial injury are not well understood. In the present study, we report that downregulation of cyclin-dependent kinase 2 (cdk2) activity in serum-deprived VSMCs was associated with the formation of complexes between cdk2 and its inhibitory protein p27(KIP1) (p27). Ectopic overexpression of p27 in serum-stimulated VSMCs resulted in the inhibition of cdk2 activity and repression of cyclin A promoter activity. Collectively, these findings indicate that p27 may contribute to VSMC growth arrest in vitro. Using the rat carotid model of balloon angioplasty, a marked upregulation of p27 was observed in injured arteries. High levels of p27 expression in the media and neointima correlated with downregulation of cdk2 activity at 2 wk after angioplasty, and adenovirus-mediated overexpression of p27 in balloon-injured arteries attenuated neointimal lesion formation. Thus, the inhibition of cdk2 function and repression of cyclin A gene transcription through the induction of the endogenous p27 protein provides a mechanism for the inhibition of VSMC growth at late time points after angioplasty.
D Chen, K Krasinski, A Sylvester, J Chen, P D Nisen, V Andrés
Human endometrium is unique since it is the only tissue in the body that bleeds at regular intervals. In addition, abnormal endometrial bleeding is one of the most common manifestations of gynecological diseases, and is a prime indication for hysterectomy. Here, we report on a novel human gene, endometrial bleeding associated factor (ebaf), whose strong expression in endometrium was associated with abnormal endometrial bleeding. In normal human endometrium, this gene was transiently expressed before and during menstrual bleeding. In situ hybridization showed that the mRNA of ebaf was expressed in the stroma without any significant mRNA expression in the endometrial glands or endothelial cells. The predicted protein sequence of ebaf showed homology with and structural features of the members of TGF-beta superfamily. Fluorescence in situ hybridization showed that the ebaf gene is located on human chromosome 1 at band q42.1. Thus, ebaf is a novel member of the TGF-beta superfamily and an endometrial tissue factor whose expression is associated with normal menstrual and abnormal endometrial bleeding.
R Kothapalli, I Buyuksal, S Q Wu, N Chegini, S Tabibzadeh
Homeostasis of body fluid is maintained by the kidneys, which contain two million glomeruli for blood filtration. A glomerulus is formed by growth of Bowman's capsule harmonized with a capillary during kidney development. The vascular endothelial growth factor (VEGF) is an essential angiogenic cytokine, and VEGF deficiency is known to be fatal in mice in early embryonic stages. As secretions of VEGF from cultured kidneys vary according to developmental stages, the role of VEGF in kidney development was studied in vivo by blocking the endogenous VEGF activity with antibody in newborn mice, in which most organs are already developed but kidneys are still developing. The antibody-treated animals showed normal growth but systemic edema. Vessel formation in the superficial renal cortex was disturbed, nephrogenic areas were diminished, and the number of developing nephrons decreased significantly. Many abnormal glomeruli, lacking capillary tufts, were observed in the antibody-treated mice, and VEGF expression in their Bowman's capsule showed a compensatory increase. These results suggest that VEGF mediates communication between the Bowman's capsule and capillary endothelial cells for developing a glomerulus as well as promoting nephrogenesis. In conclusion, VEGF is likely to be an essential molecule for kidney development, and especially for glomerulogenesis.
Y Kitamoto, H Tokunaga, K Tomita
Cigarette smoking within minutes induces leukocyte adhesion to the vascular wall and formation of intravascular leukocyte-platelet aggregates. We find this is inhibited by platelet-activating factor (PAF) receptor antagonists, and correlates with the accumulation of PAF-like mediators in the blood of cigarette smoke-exposed hamsters. These mediators were PAF-like lipids, formed by nonenzymatic oxidative modification of existing phospholipids, that were distinct from biosynthetic PAF. These PAF-like lipids induced isolated human monocytes and platelets to aggregate, which greatly increased their secretion of IL-8 and macrophage inflammatory protein-1alpha. Both events were blocked by a PAF receptor antagonist. Similarly, blocking the PAF receptor in vivo blocked smoke-induced leukocyte aggregation and pavementing along the vascular wall. Dietary supplementation with the antioxidant vitamin C prevented the accumulation of PAF-like lipids, and it prevented cigarette smoke-induced leukocyte adhesion to the vascular wall and formation of leukocyte-platelet aggregates. This is the first in vivo demonstration of inflammatory phospholipid oxidation products and it suggests a molecular mechanism coupling cigarette smoke with rapid inflammatory changes. Inhibition of PAF-like lipid formation and their intravascular sequela by vitamin C suggests a simple dietary means to reduce smoking-related cardiovascular disease.
H A Lehr, A S Weyrich, R K Saetzler, A Jurek, K E Arfors, G A Zimmerman, S M Prescott, T M McIntyre
Members of the TGFbeta family of peptides exert antiproliferative effects and induce apoptosis in epithelial cell populations. In the exocrine pancreas, these peptides not only regulate normal cell growth, but alterations in these pathways have been associated with neoplastic transformation. Therefore, the identification of molecules that regulate exocrine pancreatic cell proliferation and apoptotic cell death in response to TGFbeta peptides is necessary for a better understanding of normal morphogenesis as well as carcinogenesis of the pancreas. In this study, we have characterized the expression and function in exocrine pancreatic epithelial cells of the TGFbeta-inducible early gene (TIEG), a Krüppel-like zinc finger transcription factor encoding gene previously isolated from mesodermally derived osteoblastic cells. We demonstrate that this gene is expressed in both acinar and ductular epithelial cell populations from the exocrine pancreas. In addition, we show that the expression of TIEG is regulated by TGFbeta1 as an early response gene in pancreatic epithelial cell lines. Moreover, overexpression of TIEG in the TGFbeta-sensitive epithelial cell line PANC1 is sufficient to induce apoptosis. Together, these results support a role for TIEG in linking TGFbeta-mediated signaling cascades to the regulation of pancreatic epithelial cell growth.
I Tachibana, M Imoto, P N Adjei, G J Gores, M Subramaniam, T C Spelsberg, R Urrutia
Angiotensin-converting enzyme (ACE) generates the vasoconstrictor angiotensin II, which plays a critical role in maintenance of blood pressure in mammals. Although significant ACE activity is found in plasma, the majority of the enzyme is bound to tissues such as the vascular endothelium. We used targeted homologous recombination to create mice expressing a form of ACE that lacks the COOH-terminal half of the molecule. This modified ACE protein is catalytically active but entirely secreted from cells. Mice that express only this modified ACE have significant plasma ACE activity but no tissue-bound enzyme. These animals have low blood pressure, renal vascular thickening, and a urine concentrating defect. The phenotype is very similar to that of completely ACE-deficient mice previously reported, except that the renal pathology is less severe. These studies strongly support the concept that the tissue-bound ACE is essential to the control of blood pressure and the structure and function of the kidney.
C R Esther, E M Marino, T E Howard, A Machaud, P Corvol, M R Capecchi, K E Bernstein
Farmers lung disease is a common form of hypersensitivity pneumonitis (HP) and is characterized by inflammation and granuloma formation in the lung. Interferon-gamma is important for the expression of granulomatous diseases caused by infectious agents; however, the role this mediator in regulating expression of the granulomatous response to inhaled antigen is not known. To evaluate this, we compared the response to inhaled antigen of mice that do not express the gene coding for interferon-gamma (GKO) with that of their normal littermates (WT). GKO and WT mice on a BALB/c background were exposed to 150 microg of the thermophilic bacteria Saccharopolyspora rectivirgula or saline alone, for three consecutive days a week, for 3 wk. After exposure to antigen, WT mice developed a marked granulomatous inflammation associated with an increase in lung weight and numbers of cells in bronchoalveolar lavage fluid (BAL). Although GKO mice also exhibited an increase in lung weight and numbers of cells in BAL fluid, they developed minimal inflammation and no granulomas after a similar exposure to antigen. To further evaluate if the lack of a response to antigen in GKO mice was due to lack of IFN-gamma, we replaced this mediator via intraperitoneal injections. When given replacement IFN-gamma, the GKO mice developed granulomatous inflammation in the lung. These studies show that IFN-gamma is essential for the expression of hypersensitivity pneumonitis.
G Gudmundsson, G W Hunninghake
Hereditary xanthinuria is classified into three categories. Classical xanthinuria type I lacks only xanthine dehydrogenase activity, while type II and molybdenum cofactor deficiency also lack one or two additional enzyme activities. In the present study, we examined four individuals with classical xanthinuria to discover the cause of the enzyme deficiency at the molecular level. One subject had a C to T base substitution at nucleotide 682 that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 228. The duodenal mucosa from the subject had no xanthine dehydrogenase protein while the mRNA level was not reduced. The two subjects who were siblings with type I xanthinuria were homozygous concerning this mutation, while another subject was found to contain the same mutation in a heterozygous state. The last subject who was also with type I xanthinuria had a deletion of C at nucleotide 2567 in cDNA that should generate a termination codon from nucleotide 2783. This subject was homozygous for the mutation and the level of mRNA in the duodenal mucosa from the subject was not reduced. Thus, in three subjects with type I xanthinuria, the primary genetic defects were confirmed to be in the xanthine dehydrogenase gene.
K Ichida, Y Amaya, N Kamatani, T Nishino, T Hosoya, O Sakai
The role of expression and secretion of the ob gene product, leptin, for the regulation of plasma leptin levels has been investigated in vitro using abdominal subcutaneous adipose tissue of 20 obese, otherwise healthy, and 11 nonobese women. Body mass index (BMI, mean+/-SEM; kg/m2) in the two groups was 41+/-2 and 23+/-1, respectively. Fat cell volume was 815+/-55 pl in the obese and 320+/-46 pl in the nonobese group. In the obese group, plasma leptin concentrations and adipose leptin mRNA (relative to gamma actin) were increased five and two times, respectively. Moreover, adipose tissue secretion rates per gram lipid weight or per fat cell number were also increased two and seven times, respectively, in the obese group. There were strong linear correlations (r = 0.6-0.8) between plasma leptin, leptin secretion, and leptin mRNA. All of these leptin measurements correlated strongly with BMI and fat cell volume (r = 0.7- 0.9). About 60% of the variation in plasma leptin could be attributed to variations in leptin secretion rate, BMI, or fat cell volume. We conclude that elevated circulating levels of leptin in obese women above all result from accelerated secretion rates of the peptide from adipose tissue because of increased ob gene expression. However, leptin mRNA, leptin secretion, and circulating leptin levels are all more closely related to the stored amount of lipids in the fat cells of adipose tissue than they are to an arbitrary division into obese versus nonobese.
F Lönnqvist, L Nordfors, M Jansson, A Thörne, M Schalling, P Arner
Glutamic acid decarboxylase (GAD) has been defined as a major target antigen in insulin-dependent diabetes mellitus (IDDM). To identify the molecular ligands triggering a T cell response to GAD, a panel of human GAD65-specific T lymphocyte lines was generated from peripheral blood of three recent onset IDDM patients. All lines derived from a patient expressing the high-risk-conferring HLA-DR*0301/ *0401 haplotypes recognized a single epitope localized between amino acid positions 270 and 283 of GAD65, a stretch that is located in close proximity to the homology region shared with Coxsackie virus P2-C protein. All lines with this specificity were restricted to the DRA, B1*0401 product of the DR4 haplotype. Analysis of the GAD-specific T cell response in a second patient homozygous for DR4 haplotypes demonstrated that the same DRA, B1*0401 allele selected T cells specific for a different determinant. The T cell response profile in a third patient showed that DR*1501/ *1601-encoding haplotypes could present at least three different epitopes to GAD65-specific T lymphocytes. One of these epitopes was presented by a DR allele associated with the resistance-conferring DRB1*1501 haplotype. GAD-specific T cell lines could not be isolated from HLA class II-matched normal individuals. Our data reveal that (a) the T cell response to GAD65 is quite heterogenous in recent onset IDDM patients; (b) HLA-DR, not DQ, seems to be the principal restriction element used by T cells present at the onset of the disease; and (c) T cells responding to epitopes containing identical sequences to Coxsackie virus P2-C protein were not detected.
J Endl, H Otto, G Jung, B Dreisbusch, F Donie, P Stahl, R Elbracht, G Schmitz, E Meinl, M Hummel, A G Ziegler, R Wank, D J Schendel
The peroxisome proliferator activated receptor (PPAR gamma) plays a key role in adipogenesis and adipocyte gene expression and is the receptor for the thiazolidinedione class of insulin-sensitizing drugs. The tissue expression and potential for regulation of human PPAR gamma gene expression in vivo are unknown. We have cloned a partial human PPAR gamma cDNA, and established an RNase protection assay that permits simultaneous measurements of both PPAR gamma1 and PPAR gamma2 splice variants. Both gamma1 and gamma2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma1 was detected at lower levels in liver and heart, whereas both gamma1 and gamma2 mRNAs were expressed at low levels in skeletal muscle. To examine the hypothesis that obesity is associated with abnormal adipose tissue expression of PPAR gamma, we quantitated PPARgamma mRNA splice variants in subcutaneous adipose tissue of 14 lean and 24 obese subjects. Adipose expression of PPARgamma 2 mRNA was increased in human obesity (14.25 attomol PPAR gamma2/18S in obese females vs 9.9 in lean, P = 0.003). This increase was observed in both male and females. In contrast, no differences were observed in PPAR gamma1/18S mRNA expression. There was a strong positive correlation (r = 0.70, P < 0.001) between the ratio of PPAR gamma2/gamma1 and the body mass index of these patients. We also observed sexually dimorphic expression with increased expression of both PPAR gamma1 and PPAR gamma2 mRNAs in the subcutaneous adipose tissue of women compared with men. To determine the effect of weight loss on PPAR gamma mRNA expression, seven additional obese subjects were fed a low calorie diet (800 Kcal) until 10% weight loss was achieved. Mean expression of adipose PPAR gamma2 mRNA fell 25% (P = 0.0250 after a 10% reduction in body weight), but then increased to pretreatment levels after 4 wk of weight maintenance. Nutritional regulation of PPAR gamma1 was not seen. In vitro experiments revealed a synergistic effect of insulin and corticosteroids to induce PPAR gamma expression in isolated human adipocytes in culture. We conclude that: (a) human PPAR gamma mRNA expression is most abundant in adipose tissue, but lower level expression of both splice variants is seen in skeletal muscle; to an extent that is unlikely to be due to adipose contamination. (b) RNA derived from adipose tissue of obese humans has increased expression of PPAR gamma 2 mRNA, as well as an increased ratio of PPAR gamma2/gamma1 splice variants that is proportional to the BMI; (c) a low calorie diet specifically down-regulates the expression of PPAR gamma2 mRNA in adipose tissue of obese humans; (d) insulin and corticosteroids synergistically induce PPAR gamma mRNA after in vitro exposure to isolated human adipocytes; and (e) the in vivo modulation of PPAR gamma2 mRNA levels is an additional level of regulation for the control of adipocyte development and function, and could provide a molecular mechanism for alterations in adipocyte number and function in obesity.
A J Vidal-Puig, R V Considine, M Jimenez-Liñan, A Werman, W J Pories, J F Caro, J S Flier
The stress response and stress proteins confer protection against diverse forms of cellular and tissue injury, including acute lung injury. The stress response can inhibit nonstress protein gene expression, therefore transcriptional inhibition of proinflammatory responses could be a mechanism of protection against acute lung injury. To explore this possibility, we determined the effects of the stress response on nuclear translocation of the transcription factor NF-kappaB, an important regulator of proinflammatory gene expression. In A549 cells induction of the stress response decreased tumor necrosis factor-alpha (TNF-alpha)-mediated NF-kappaB nuclear translocation. TNF-alpha initiates NF-kappaB nuclear translocation by causing dissociation of the inhibitory protein I-kappaBalpha from NF-kappaB and rapid degradation of I-kappaBalpha. Prior induction of the stress response inhibited TNF-alpha-mediated dissociation of I-kappaBalpha from NF-kappaB and subsequent degradation of I-kappaBalpha. Induction of the stress response also increased expression of I-kappaBalpha. We conclude that the stress response affects NFkappaB-mediated gene regulation by two independent mechanisms. The stress response stabilizes I-kappaBalpha and induces expression of I-kappaBalpha. The composite result of these two effects is to decrease NF-kappaB nuclear translocation. We speculate that the protective effect of the stress response against acute lung injury involves a similar effect on the I-kappaB/NF-kappaB pathway.
H R Wong, M Ryan, J R Wispé
The present study was designed to test the hypothesis that estrogen receptors (ER) in the blood vessel wall play a role in the modulation of the release of endothelium-derived nitric oxide (EDNO). Both basal and stimulated release of EDNO were determined in aortic rings isolated from female and male wild-type and male homozygous estrogen receptor knock-out (ERKO) mice. 125I-17beta-estradiol binding in aortic tissue showed significantly more high affinity cytosolic- nuclear-binding sites in male compared with female wildtype mice. Estrogen receptor transcripts were present in the aorta of male wild-type mice, but they were absent in male ERKO animals. Basal release of EDNO (determined by endothelium-dependent contraction caused by NG-nitro-arginine) was significantly higher in aorta of wild-type male mice compared with wild-type female mice, and significantly lower in the aorta of male ERKO compared with male wild-type mice. Acetylcholine-induced endothelium-dependent relaxation was similar in all groups studied. No difference was observed in the activity of calcium-dependent nitric oxide synthase in homogenates of lungs and brain taken from male wild-type and ERKO mice. These studies show a significant association between the number of estrogen receptors and basal release of EDNO in the aorta of mice, and suggest that decreased vascular estrogen receptor number may represent a novel risk factor for cardiovascular diseases.
G M Rubanyi, A D Freay, K Kauser, D Sukovich, G Burton, D B Lubahn, J F Couse, S W Curtis, K S Korach
Chylomicrons have been shown to protect mice and rats against a lethal dose of lipopolysaccharide and may serve as a therapeutic means to protect against endotoxemia. However, the requisite of isolation from human lymph hampers pharmaceutical application. Recently, we developed recombinant chylomicrons from commercially available lipids and human recombinant apolipoprotein E. The current study explored the effectiveness of these apoE-enriched emulsions in redirecting LPS from Kupffer cells to liver parenchymal cells. Upon injection into rats, 125I-LPS rapidly and specifically associated with the liver (64.3+/-3.1% of the injected dose) and spleen (4.1+/-0.7%). The uptake of LPS by the spleen was four- to fivefold reduced upon incubation with the apoE-enriched emulsion or free apoE (P < 0.0001), but not with emulsion alone or Lipofundin. Within the liver, 125I-LPS mainly associated with Kupffer cells. The uptake by Kupffer cells was eight- to ninefold reduced by the apoE-enriched emulsion or apoE alone (P < 0.01), and a 19.6-fold increased uptake ratio by liver parenchymal cells over Kupffer cells was observed. The emulsion without apoE had no effect on the in vivo kinetics of LPS. LPS interacted selectively with the apoE moiety of the recombinant chylomicron. Emulsion-associated and free apoE bound approximately two molecules of LPS, possibly by its exposed hydrophilic domain involving arginine residues. We anticipate that the protecting effect of endogenous chylomicrons against LPS-induced endotoxemia may result from the apoE moiety and that human recombinant apoE may serve as a therapeuticum to protect against endotoxemia.
P C Rensen, M Oosten, E Bilt, M Eck, J Kuiper, T J Berkel
Coagulation proteases were tested in a rat model of acute inflammation. Subplantar injection of Factor Xa (10-30 microg) produced a time- and dose-dependent edema in the rat paw, and potentiated carrageenin-induced edema. In contrast, the homologous protease Factor IXa was ineffective. This inflammatory response was recapitulated by the Factor Xa sequence L83FTRKL88(G), which mediates ligand binding to effector cell protease receptor-1 (EPR-1), while a control scrambled peptide did not induce edema in vivo. Conversely, injection of the EPR-1-derived peptide S123PGKPGNQNSKNEPP137 (corresponding to the receptor binding site for Factor Xa) inhibited carrageenin-induced rat paw edema, while the adjacent EPR-1 sequence P136PKKRERERSSHCYP150 was without effect. EPR-1-Factor Xa-induced inflammation was characterized by fast onset and prominent perivascular accumulation of activated and degranulated mast cells, was inhibited by the histamine/serotonin antagonists cyproheptadine and methysergide, but was unaffected by the thrombin-specific inhibitor, Hirulog. These findings suggest that through its interaction with EPR-1, Factor Xa may function as a mediator of acute inflammation in vivo. This pathway may amplify both coagulation and inflammatory cascades, thus contributing to the pathogenesis of tissue injury in vivo.
G Cirino, C Cicala, M Bucci, L Sorrentino, G Ambrosini, G DeDominicis, D C Altieri
Galectin-9, a beta-galactoside binding lectin, has recently been isolated from murine embryonic kidney. In this study, its biological functions and expression in embryonic, newborn, and adult mice tissues were investigated. By Northern blot analyses, it was found widely distributed and its expression was developmentally regulated. In situ hybridization studies revealed an accentuated expression of galectin-9 in liver and thymus of embryonic mice. In postnatal mice, antigalectin-9 immunoreactivity was observed in various tissues, including thymic epithelial cells. The high expression of galectin-9 in the thymus led us to investigate its role in the clonal deletion of thymocytes. Fusion proteins were generated, which retained lactose-binding activity like the endogenous galectin-9. Galectin-9, at 2.5 microM concentration, induced apoptosis in approximately 30% of the thymocytes, as assessed by terminal deoxytransferase-mediated dUTP nick end labeling method. The apoptotic effect was dose dependent and lactose inhibitable. At higher concentrations, it induced homotypic aggregation of the thymocytes. Electron microscopy revealed approximately 60% of the thymocytes undergoing apoptosis in the presence of galectin-9. By immunofluorescence microscopy, some of the thymocytes undergoing apoptosis had plasmalemmal bound galectin-9. Galectin-9 failed to induce apoptosis in hepatocytes. Taken together, these findings indicate that galectin-9, a developmentally regulated lectin, plays a role in thymocyte-epithelial interactions relevant to the biology of the thymus.
J Wada, K Ota, A Kumar, E I Wallner, Y S Kanwar
Leukemia inhibitory factor (LIF) and LIF receptors are expressed in adenohypophyseal cells and LIF regulates pituitary hormone transcription and cell replication in vitro. Therefore, transgenic mice expressing pituitary-directed LIF driven by the rat growth hormone (GH) promoter were generated to evaluate the impact of LIF on pituitary development. Three founders were established with diminished linear growth and body weight (57-65% of wild type [WT]), and intense anterior pituitary LIF immunoreactivity. Cystic cavities observed in pituitary anterior lobes were lined by cuboidal, ciliated epithelial cells, focally immunopositive for cytokeratin and S-100 protein and immunonegative for adenohypophyseal hormones. Transgenic pituitaries showed decreased GH (40%) and prolactin (PRL) (26%) cells, and decreased GH and PRL mRNAs by in situ hybridization. ACTH cells increased 2.2-fold, whereas gonadotrophs and thyrotrophs were unchanged. Serum GH was undetectable (< 0.78 ng/ml), PRL levels were one third of WT (P < 0.05), IGF-I levels were 30% of WT (P < 0. 001), and T4 was normal. 10 human pituitary Rathke's cysts studied all showed conclusive LIF immunoreactivity in cyst-lining cells. Thus, intrapituitary murine LIF overexpression causes cystic invaginations from the anterior wall of Rathke's cleft, suggesting failed differentiation of Rathke's epithelium to hormone-secreting cells. Arrested murine pituitary maturation with formation of pituitary Rathke's cleft cysts, GH deficiency, and short stature provide a model to study human Rathke's cyst pathogenesis.
S Akita, C Readhead, L Stefaneanu, J Fine, A Tampanaru-Sarmesiu, K Kovacs, S Melmed
Normal glomerular capillaries filter plasma through a basement membrane (GBM) rich in alpha3(IV), alpha4(IV), and alpha5(IV) chains of type IV collagen. We now show that these latter isoforms are absent biochemically from the glomeruli in patients with X-linked Alport syndrome (XAS). Their GBM instead retain a fetal distribution of alpha1(IV) and alpha2(IV) isoforms because they fail to developmentally switch their alpha-chain use. The anomalous persistence of these fetal isoforms of type IV collagen in the GBM in XAS also confers an unexpected increase in susceptibility to proteolytic attack by collagenases and cathepsins. The incorporation of cysteine-rich alpha3(IV), alpha4(IV), and alpha5(IV) chains into specialized basement membranes like the GBM may have normally evolved to protectively enhance their resistance to proteolytic degradation at the site of glomerular filtration. The relative absence of these potentially protective collagen IV isoforms in GBM from XAS may explain the progressive basement membrane splitting and increased damage as these kidneys deteriorate.
R Kalluri, C F Shield, P Todd, B G Hudson, E G Neilson
Clinical experience suggests nephrotic patients are at risk for malnutrition. To determine if nephrotic patients can adapt successfully to a protein-restricted diet, nephrotic (glomerular filtration rate, 52+/-15 ml/min; urinary protein [Uprot.], 7.2+/-2.2 grams/d) and control subjects completed a crossover comparison of diets providing 0.8 or 1.6 grams protein (plus 1 gram protein/gram Uprot.) and 35 kcal per kg per day. Nitrogen balance (BN) was determined and whole body protein turnover measured during fasting and feeding using intravenous -[1-13C]leucine and intragastric -[5,5, 5- 2H3]leucine. BN was positive in both nephrotic and control subjects consuming either diet and rates of whole-body protein synthesis, protein degradation, and leucine oxidation did not differ between groups. In both nephrotic and control subjects anabolism was due to a suppression of whole-body protein degradation and stimulation of protein synthesis during feeding. The principal compensatory response to dietary protein restriction was a decrease in amino acid oxidation and this response was the same in both groups. With the low protein diet leucine oxidation rates during feeding correlated inversely with Uprot. losses (r = -0.83; P < 0. 05). Conclusions: (a) a diet providing 0.8 gram protein (plus 1 gram protein/gram Uprot.) and 35 kcal per kg per day maintains BN in nephrotic patients; (b) nephrotic patients activate normal anabolic responses to dietary protein restriction (suppression of amino acid oxidation) and feeding (stimulation of protein synthesis and inhibition of protein degradation); (c) the inverse correlation between leucine oxidation and Uprot. losses suggests that proteinuria is a stimulus to conserve dietary essential amino acids.
B J Maroni, C Staffeld, V R Young, A Manatunga, K Tom
To analyze the immunoglobulin repertoire of human IgM+ B cells and the CD5(+) and CD5(-) subsets, individual CD19(+)/ IgM+/CD5(+) or CD5(-) B cells were sorted and non-productive as well as productive VH gene rearrangements were amplified from genomic DNA and sequenced. In both subsets, the VH3 family was overrepresented largely as a result of preferential usage of a small number of specific individual family members. In the CD5(+) B cell subset, all other VH families were found at a frequency expected from random usage, whereas in the CD5(-) population, VH4 appeared to be overrepresented in the nonproductive repertoire, and also negatively selected since it was found significantly less often in the productive compared to the nonproductive repertoire; the VH1 family was significantly diminished in the productive rearrangements of CD5(-) B cells. 3-23/DP-47 was the most frequently used VH gene segment and was found significantly more often than expected from random usage in productive rearrangements of both CD5(+) and CD5(-) B cells. Evidence for selection based on the D segment and the JH gene usage was noted in CD5(+) B cells. No differences were found between the B cell subsets in CDR3 length, the number of N-nucleotides or evidence of exonuclease activity. Somatically hypermutated VHDJH rearrangements were significantly more frequent and extensive in CD5(-) compared to CD5(+) IgM+ B cells, indicating that IgM+ memory B cells were more frequent in the CD5(-) B cell population. Of note, the frequency of specific VH genes in the mutated population differed from that in the nonmutated population, suggesting that antigen stimulation imposed additional biases on the repertoire of IgM+ B cells. These results indicate that the expressed repertoire of IgM+ B cell subsets is shaped by recombinational bias, as well as selection before and after antigen exposure. Moreover, the influences on the repertoires of CD5(+) and CD5(-) B cells are significantly different, suggesting that human peripheral blood CD5(+) and CD5(-) B cells represent different B cell lineages, with similarities to murine B-1a and B-2 subsets, respectively.
H P Brezinschek, S J Foster, R I Brezinschek, T Dörner, R Domiati-Saad, P E Lipsky
The mechanisms that maintain relative uterine quiescence during pregnancy remain largely unknown. A possible role for nitric oxide has recently emerged, however, the expression of nitric oxide synthase within human myometrium at midgestation, a time when the uterus is normally quiescent, has not been investigated. The purpose of this study was to identify cell types in human myometrium that contain inducible nitric oxide synthase (iNOS), and to examine changes in its expression during pregnancy and labor. We found that iNOS is expressed in smooth muscle cells of pregnant myometrium. Expression of iNOS was highest in myometrium of preterm not-in-labor patients. At term, iNOS expression fell by 75%, and was barely detectable in preterm in-labor or term in-labor specimens. There was no staining in the myocytes of nonpregnant myometrium. Western blotting also revealed a similar pattern of changes in iNOS expression. In summary, iNOS expression in the myocytes of human myometrium is increased greatly during pregnancy, and declines towards term or with labor. Significantly, preterm inlabor patients also had a large decline in iNOS expression. These data suggest that changes in myometrial iNOS expression may participate in the regulation of uterine activity during human pregnancy.
R K Bansal, P C Goldsmith, Y He, C J Zaloudek, J L Ecker, R K Riemer
Multiple steps are involved in the metastasis of cancer cells from primary sites to distant organs. These steps should be considered in the design of pharmacologic approaches to prevent or inhibit the metastatic process. In the present study, we have compared the effects of inhibiting several steps involved in the bone metastatic process individually with inhibition of both together. The steps we chose were matrix metalloproteinase (MMP) secretion, likely involved in tumor cell invasion, and osteoclastic bone resorption, the final step in the process. We used an experimental model in which inoculation of human estrogen-independent breast cancer MDA-231 cells into the left cardiac ventricle of female nude mice causes osteolytic lesions in bone. To inhibit cancer invasiveness, the tissue inhibitor of the MMP-2 (TIMP-2), which is a natural inhibitor of MMPs, was overexpressed in MDA-231 cells. To inhibit bone resorption, a potent bisphosphonate, ibandronate (4 microg/mouse) was daily administered subcutaneously. Nude mice received either; (a) nontransfected MDA-231 cells; (b) nontransfected MDA231 cells and ibandronate; (c) TIMP-2-transfected MDA-231 cells; or (d) TIMP-2-transfected MDA-231 cells and ibandronate. In mice from group a, radiographs revealed multiple osteolytic lesions. However, in mice from group b or group c, osteolytic lesions were markedly decreased. Of particular note, in animals from group d receiving both ibandronate and TIMP-2-transfected MDA-231 cells, there were no radiologically detectable osteolytic lesions. Survival rate was increased in mice of groups c and d. There was no difference in local enlargement in the mammary fat pad between nontransfected and TIMP-2-transfected MDA-231 cells. These results suggest that inhibition of both MMPs and osteoclastic bone resorption are more efficacious treatment for prevention of osteolytic lesions than either alone, and suggest that when therapies are designed based on the uniqueness of the bone microenvironment and combined with several common steps in the metastatic process, osteolytic bone metastases can be more efficiently and selectively inhibited.
T Yoneda, A Sasaki, C Dunstan, P J Williams, F Bauss, Y A De Clerck, G R Mundy
The human immune response to Mycobacterium tuberculosis is not well characterized. To better understand the cellular immune response to tuberculosis, a human mononuclear phagocyte culture system using a low-infecting inoculum of M. tuberculosis to mimic in vivo conditions was developed. Using this system, monocytes treated with IFNgamma/TNFalpha/ calcitriol (CytD) were permissive for the growth of virulent M. tuberculosis. In the presence of iron, however, these monocytes suppressed the growth of M. tuberculosis. The enhanced permissiveness of CytD-preincubated monocytes was found to be due to TNFalpha, however, the ability of iron to suppress M. tuberculosis growth also required preincubation with TNFalpha. Iron-mediated growth suppression was correlated with selective suppression of TNFalpha release from infected monocytes. In addition, removal of TNFalpha from CytD-treated monocytes 2 d after infection mimicked the suppressive effect of iron, suggesting that iron may also be decreasing monocyte sensitivity to exogenously added TNFalpha. In the absence of iron, permissive, CytD-treated monocytes formed large infected cellular aggregates. With iron treatment, aggregation was suppressed, suggesting that the iron-suppressive effect on M. tuberculosis growth may be related to suppression of monocyte aggregation and diminished cell-to-cell spread of M. tuberculosis. The results of this study indicate that TNFalpha preincubation is required for human monocytes to exert an iron-mediated suppressive effect on M. tuberculosis growth. In the absence of iron, however, the continued presence of TNFalpha has a growth-promoting effect on M. tuberculosis in human monocytes. Iron may be an important early modulator of M. tuberculosis growth via its effects on TNFalpha.
T F Byrd
Gaucher disease, the most prevalent lysosomal storage disease, occurs in three subtypes, all resulting from mutations in the acid beta-glucosidase gene. Molecular studies in five severely affected type 1 and two type 2 Gaucher disease patients of non-Jewish descent identified six new mutations: K74X, W179X, G195E, S271N, V352L, and a two-base deletion in exon 10 (1450del2). Two additional mutations identified in these patients (R48W and G202R) have been reported previously, but were not expressed or characterized. Heterologous expression in Sf 9 cells using the baculovirus system revealed that the missense mutations, R48W and V352L, had 14 and 7%, respectively, of the specific activity based on cross-reacting immunologic material expressed by the normal allele. In contrast, the G195E, G202R, and S271N mutant alleles were more severely compromised with only 1-2% of the normal expressed specific activity based on cross-reacting immunologic material. Structural distortion at the active site was probed by comparing the interaction of the mutant enzymes with active site-directed inhibitors (castanospermine, conduritol B epoxide and deoxynojirimycin). R48W, G202R, and S271N were normally inhibited, whereas the V352L and G195E mutant enzymes had significantly decreased binding affinity. These mutations further expand the genetic heterogeneity in the lesions causing Gaucher disease types 1 and 2, and further delineate genotype/phenotype correlations and functional domains within the acid beta-glucosidase gene.
M E Grace, R J Desnick, G M Pastores
We have investigated the metabolic actions of recombinant human IGF-1 in mice genetically deficient of insulin receptors (IR-/-). After intraperitoneal administration, IGF-1 caused a prompt and sustained decrease of plasma glucose levels in IR-/- mice. Plasma free fatty acid concentrations were unaffected. Interestingly, the effects of IGF-1 were identical in normal mice (IR+/+) and in IR-/- mice. Despite decreased glucose levels, IR-/- mice treated with IGF-1 died within 2-3 d of birth, like sham-treated IR-/- controls. In skeletal muscle, IGF-1 treatment caused phosphorylation of IGF-1 receptors and increased the levels of the phosphatidylinositol-3-kinase p85 subunit detected in antiphosphotyrosine immunoprecipitates, consistent with the possibility that IGF-1 stimulates glucose uptake in a phosphatidylinositol-3-kinase-dependent manner. IGF-1 receptor phosphorylation and coimmunoprecipitation of phosphatidylinositol3-kinase by antiphosphotyrosine antibodies was also observed in liver, and was associated with a decrease in mRNA levels of the key gluconeogenetic enzyme phosphoenolpyruvate carboxykinase. Thus, the effect of IGF-1 on plasma glucose levels may be accounted for by increased peripheral glucose use and by inhibition of hepatic gluconeogenesis. These data indicate that IGF-1 can mimic insulin's effects on glucose metabolism by acting through its own receptor. The failure of IGF-1 to rescue the lethal phenotype due to lack of insulin receptors suggests that IGF-1 receptors cannot effectively mediate all the metabolic actions of insulin receptors.
G Di Cola, M H Cool, D Accili
The increase in oral availability of felodipine and other commonly used medications when taken with grapefruit juice has been assumed to be due to inhibition of CYP3A4, a cytochrome P450 that is present in liver and intestine. To evaluate the effect of repeated grapefruit juice ingestion on CYP3A4 expression, 10 healthy men were given 8 oz of grapefruit juice three times a day for 6 d. Before and after receiving grapefruit juice, small bowel and colon mucosal biopsies were obtained endoscopically, oral felodipine kinetics were determined, and liver CYP3A4 activity was measured with the [14C N-methyl] erythromycin breath test in each subject. Grapefruit juice did not alter liver CYP3A4 activity, colon levels of CYP3A5, or small bowel concentrations of P-glycoprotein, villin, CYP1A1, and CYP2D6. In contrast, the concentration of CYP3A4 in small bowel epithelia (enterocytes) fell 62% (P = 0.0006) with no corresponding change in CYP3A4 mRNA levels. In addition, enterocyte concentrations of CYP3A4 measured before grapefruit juice consumption correlated with the increase in Cmax when felodipine was taken with either the 1st or the 16th glass of grapefruit juice relative to water (r = 0. 67, P = 0.043, and r = 0.71, P = 0.022, respectively). We conclude that a mechanism for the effect of grapefruit juice on oral felodipine kinetics is its selective downregulation of CYP3A4 in the small intestine.
K S Lown, D G Bailey, R J Fontana, S K Janardan, C H Adair, L A Fortlage, M B Brown, W Guo, P B Watkins