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PubMed1995

Rapid mRNA degradation mediated by the c-fos 3' AU-rich element and that mediated by the granulocyte-macrophage colony-stimulating factor 3' AU-rich element occur through similar polysome-associated mechanisms.

The different 3' noncoding AU-rich elements (ARE) that mediate the degradation of many short-lived mRNAs may function through distinct decay pathways; translation-dependent and -independent mechanisms have been proposed. To investigate the cotranslational model, we designed an expression system that exploits the properties of the ferritin iron-responsive element to shuttle chimeric mRNAs from ribonucleoproteins to polyribosomes. The iron-responsive element was introduced in the 5' untranslated regions of alpha-globin mRNAs that harbored in their 3' untranslated regions either the c-fos ARE or the granulocyte-macrophage colony-stimulating factor ARE as prototypes of the different ARE subsets. The cytoplasmic location of the transcripts was controlled by intracellular iron availability and monitored by polysomal profile analysis. We report that these two mRNA subsets behaved identically in this system. Iron deprivation by desferrioxamine treatment stabilized both transcripts by sequestering them away from polyribosomes. Sequential treatments with desferrioxamine, followed by hemin to concentrate the mRNAs in the ribonucleoprotein pool prior to translation, showed that rapid degradation occurred only upon redistribution of the transcripts to polyribosomes. Deletion of a critical cytosine in the iron-responsive element abolished targeted sequestration and restored high-level constitutive mRNA instability. These observations demonstrate that the c-fos and granulocyte-macrophage colony-stimulating factor ARE subsets mediate selective mRNA degradation through similar polysome-associated mechanisms coupled with ongoing translation.

3T3 Cells↗
PubMed1990

Neutrophil-mediated increased permeability of microcarrier-cultured endothelial monolayers: a model for the in vitro study of neutrophil-dependent mediators of vasopermeability.

Changes in the permeability of human endothelial monolayers in response to activated human neutrophils were examined in a novel, in vitro model of vasopermeability changes. Microcarrier-cultured human umbilical vein endothelial monolayers were used in a system that responds to histamine. Human neutrophils did not increase Evans Blue staining of the endothelium-covered microcarriers if added alone or if added with the neutrophil-dependent mediator of vasopermeability, formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM). In contrast, neutrophils, added to the endothelial cells in a ratio as low as 2.5:1 caused time-dependent increases in microcarrier staining if pretreated with cytochalasin B (5 micrograms/mL) before addition with FMLP. Neutrophil cell-free releasate and purified human sputum elastase also caused concentration-related increases in Evans Blue staining of the endothelial-covered microcarriers and these effects were inhibited by the elastase inhibitor methoxysuccinyl-alanyl-alanyl-prolyl-valyl chloromethyl ketone. This compound also inhibited neutrophil-mediated endothelial permeability increases. The microcarrier-cultured human endothelial monolayer system rapidly detects permeability alterations of endothelial monolayers in response to activated human neutrophils. This model is a potentially useful screening assay for the development of therapeutic agents, directed at neutrophil degranulation or degranulation products, for the control of inflammatory vasopermeability abnormalities.

Capillary Permeability↗
PubMed2002

NHE3 serves as a molecular tool for cAMP-mediated regulation of receptor-mediated endocytosis.

Receptor-mediated, clathrin-dependent endocytosis (RME) is important for macromolecular transport and regulation of cell-surface protein expression. Pharmacological studies have shown that the plasma membrane transport protein Na(+)/H(+) exchanger 3 (NHE3), which shuttles between the plasma membrane and the early endosomal compartment by means of clathrin-mediated endocytosis, contributes to endosomal pH homeostasis and endocytic fusion events. Furthermore, it is known that NHE3 is phosphorylated and inhibited by cAMP-dependent kinase (protein kinase A). Here, we show, in a cellular knockout/retransfection approach, that NHE3 supports RME and confers cAMP sensitivity to RME, using megalin/cubilin-mediated albumin uptake in opossum kidney cells. RME, but not fluid-phase endocytosis, was dependent on NHE3 activity and expression. Furthermore, NHE3 deficiency or inhibition reduced the relative surface expression of megalin without altering total expression. In wild-type cells, cAMP inhibits NHE3 activity, leads to endosomal alkalinization, and reduces RME. In NHE3-deficient cells, endosomal pH is not sensitive to NHE3 inhibition, and cAMP does not affect endosomal pH or RME. NHE3 transfection into deficient cells restores RME and the effects of cAMP. Thus our data show that NHE3 is important for cAMP sensitivity of clathrin-dependent RME.

Albumins↗
PubMed2006

Mediator mechanisms involved in TRPV1 and P2X receptor-mediated, ROS-evoked bradypneic reflex in anesthetized rats.

Inhalation of H2O2 is known to evoke bradypnea followed by tachypnea, which are reflexes resulting from stimulation by reactive oxygen species of vagal lung capsaicin-sensitive and myelinated afferents, respectively. This study investigated the pharmacological receptors and chemical mediators involved in triggering these responses. The ventilatory responses to 0.2% aerosolized H2O2 were studied before and after various pharmacological pretreatments in anesthetized rats. The initial bradypneic response was reduced by a transient receptor potential vanilloid 1 (TRPV1) receptor antagonist [capsazepine; change (Delta) = -53%] or a P2X purinoceptor antagonist [iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (PPADS); Delta = -47%] and was further reduced by capsazepine and iso-PPADS in combination (Delta = -78%). The initial bradypneic response was reduced by a cyclooxygenase inhibitor (indomethacin; Delta = -48%), ATP scavengers (apyrase and adenosine deaminase in combination; Delta = -50%), or capsazepine and indomethacin in combination (Delta = -47%), was further reduced by iso-PPADS and indomethacin in combination (Delta = -75%) or capsazepine and ATP scavengers in combination (Delta = -83%), but was not affected by a lipoxygenase inhibitor (nordihydroguaiaretic acid) or by any of the various vehicles. No pretreatment influenced delayed tachypnea. We concluded that 1) the initial bradypneic response to H2O2 results from activation of both TRPV1 and P2X receptors, possibly located at terminals of vagal lung capsaicin-sensitive afferent fibers; 2) the functioning of the TRPV1 and P2X receptors in triggering the initial bradypnea is, in part, mediated through the actions of cyclooxygenase metabolites and ATP, respectively; and 3) these mechanisms do not contribute to the H2O2-evoked delayed tachypnea.

Adenosine Deaminase↗
PubMed2004

Overexpression of histone deacetylase 1 confers resistance to sodium butyrate-mediated apoptosis in melanoma cells through a p53-mediated pathway.

Melanoma cells typically express wild-type p53, yet they are notoriously resistant to DNA-damaging agents. Here, we show that sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, induced apoptosis in human melanoma cells in a dose- and time-dependent manner. Apoptosis was associated with HDAC1-dependent induction of Bax and acetylation of p53. Down-regulation of HDAC1 by an antisense vector sensitized the cells to NaB-induced apoptosis, whereas its overexpression conferred resistance to this agent. Increased HDAC1 levels and activity impaired NaB-mediated activation of Bax promoter and Bax protein levels. Finally, using p53-null melanoma cell line and RNA interference in cells expressing wild-type p53 protein, we show that Bax induction and NaB-mediated apoptosis is p53 dependent.

Apoptosis↗
PubMed2005

Inhibitory anti-FLT3 antibodies are capable of mediating antibody-dependent cell-mediated cytotoxicity and reducing engraftment of acute myelogenous leukemia blasts in nonobese diabetic/severe combined immunodeficient mice.

Aberrant FLT3 expression and/or mutation plays a significant role in leukemogenesis. This has prompted the development of selective small molecule tyrosine kinase inhibitors against FLT3. However, like most tyrosine kinase inhibitors, those against FLT3 are not completely specific and at the doses required to completely inhibit target, significant toxicities may occur. In addition, tyrosine kinase inhibitors for other kinases have been shown to select for cells that become resistant. To overcome some of these limitations we developed two fully human phage display monoclonal antibodies against FLT3 (IMC-EB10 and IMC-NC7). These antibodies inhibited ligand-mediated activation of wild-type FLT3 and constitutively activated mutant FLT3 and in most cell types affected downstream STAT5, AKT, and mitogen-activated protein kinase activation. In addition to interfering with FLT3 signaling, IMC-EB10 and, to a significantly lesser extent, IMC-NC7 initiated antibody-dependent cell-mediated cytotoxicity on FLT3-expressing cells. When IMC-EB10 was used in vivo to treat nonobese diabetic/severe combined immunodeficient mice given injections of primary FLT3/ITD acute myelogenous leukemia samples or myeloid cell lines with FLT3 expression, it significantly decreased engraftment of leukemic cells and increased survival, respectively. In contrast, IMC-EB10 treatment did not reduce engraftment of normal human CD34+ cord blood cells nor did it show any significant inhibition of normal murine hematopoiesis. Thus, these types of antibodies have the potential to be safe and effective new therapeutic agents for acute myelogenous leukemia and possibly other FLT3-expressing malignancies.

Animals↗
PubMedDate not supplied

Relaxation in different-sized rat blood vessels mediated by endothelium-derived hyperpolarizing factor: importance of processes mediating precontractions.

To clarify the mechanisms involved in relaxations mediated by endothelium-derived hyperpolarizing factor (EDHF), acetylcholine (ACh)-induced endothelium-dependent relaxations and hyperpolarizations were examined in the rat aorta, the main branch of the mesenteric artery (MBMA) and the first branch of the mesenteric aftery (FBMA). In the presence of 100 microM N(G)-nitro-L-arginine (L-NNA) and 10 microM indomethacin, ACh (1 nM to 100 microM) produced no relaxation in the phenylephrine-precontracted aorta. The L-NNA-resistant relaxations by ACh in MBMA precontracted with phenylephrine were eliminated in the presence of 1 microM nifedipine where contractions were independent of L-type Ca(2+) channel activation. In FBMA precontracted with phenylephrine, the L-NNA-resistant relaxations were only partially inhibited by nifedipine. When vessels had been contracted with 300 nM phorbol-12,13-dibutyrate in the presence of nifedipine, ACh-induced L-NNA-resistant relaxations were observed in FBMA only. Pinacidil produced relaxations in all different-sized blood vessels, although sensitivity was inversely related to vessel size. The extent of the ACh hyperpolarizing responses was much smaller than that by pinacidil in the aorta. The membrane potential changes by ACh and pinacidil were almost the same in FBMA. These results indicate that the contribution of EDHF to endothelium-dependent relaxations increases as the vessel size decreases. This may be partly explained by precontractile processes dependent on Ca(2+) entry through L-type Ca(2+) channels, because Ca(2+) channel deactivation seems to be involved as a major mechanism of EDHF-mediated vasorelaxations. However, EDHF may also generate vasorelaxations by an additional mechanism, probably a reduced Ca(2+) sensitivity of contractile elements, as proposed for ATP-sensitive K(+) channel openers.

Analysis of Variance↗
PubMed1990

Reactivity of mast-cell-bound IgE idiotypes with anti-idiotypic antibody: mediator release or inhibition of antigen-induced mediator release?

When anti-idiotypic antibodies specific for idiotopes on IgE antibodies react with mast-cell-bound IgE antibody, the reaction may be expected to lead either to mediator release or to inhibition of allergen-induced release. This study was carried out to determine which would occur. Anti-idiotype antiserum (anti-Ids) was raised in syngeneic mice by immunisation with affinity-purified DNP-specific mouse monoclonal IgE. Sera from these immunised mice and from a similarly immunised rabbit inhibited the binding of the monoclonal IgE to radio-iodinated dinitrophenylated (DNP) ovalbumin, whereas a high concentration of affinity-purified rabbit antimouse epsilon-chain-specific antibody was unable to inhibit even though it was shown to bind to the monoclonal IgE. The mouse anti-Ids did not give a positive passive cutaneous anaphylactic reaction in rats sensitised with high-titre grass-pollen-specific IgE-containing antiserum, whereas it gave a positive result in rats sensitised with the DNP-specific monoclonal IgE. Rabbit antimonoclonal antiserum and rabbit anti-epsilon-chain antibody both gave positive results in these two assays. The results indicated that anti-idiotypic antibodies specific for idiotopes on IgE antibody can react and release mediators from IgE-sensitised mast cells. The significance of this in view of the increased levels of anti-idiotypes that can occur during immunotherapy is discussed.

Animals↗
PubMed2003

Dual functionality of cyclooxygenase-2 as a regulator of tumor necrosis factor-mediated G1 shortening and nitric oxide-mediated inhibition of vascular smooth muscle cell proliferation.

BACKGROUND: Cyclooxygenase (COX)-2 contributes to vascular smooth muscle cell (VSMC) proliferation induced by tumor necrosis factor (TNF) and angiotensin II. The present study demonstrates, however, that depending on prevailing conditions, COX-2-derived prostanoids may also inhibit VSMC proliferation. METHODS AND RESULTS: TNF-alpha stimulated proliferation of VSMCs by shortening the G1 phase of the cell cycle. This effect was abolished by NS-398, a selective COX-2 inhibitor. Addition of TNF did not affect the protein-to-DNA ratio, measured by flow cytometry, suggesting that TNF does not induce VSMC hypertrophy. Inhibition of nitric oxide synthase (NOS) activity attenuated TNF-mediated increases in prostaglandin (PG) I2 synthesis, whereas thromboxane (TX) A2 production and COX-2 protein expression were unaffected. Moreover, inhibition of NOS activity increased TNF-mediated proliferation by approximately 23%. Thus, NO preferentially stimulates PGI2 production, suggesting that production of NO by VSMCs challenged with TNF limits the ability of the cytokine to increase proliferation. NO donors increased COX-2 protein expression and PGI2 synthesis, had no effect on TXA2 production, and decreased cell numbers by 50%, indicating that expression of COX-2 per se might not be sufficient to support proliferation. The effects of NO donors were prevented when COX-2 activity was inhibited with NS-398. CONCLUSIONS: The COX-2-dependent proliferative response of VSMCs to TNF was modulated in an NO-dependent manner, and PGI2 derived from COX-2 might contribute to the antiproliferative effect of NO donors.

Animals↗
PubMed1996

Coronary artery spasm does not depend on the intracellular calcium store but is substantially mediated by the protein kinase C-mediated pathway in a swine model with interleukin-1 beta in vivo.

BACKGROUND: The intracellular mechanism for coronary artery spasm is still unknown. Since the protein kinase C (PKC)-mediated pathway and Ca2+ release from sarcoplasmic reticulum (SR) are important intracellular mechanisms of vascular smooth muscle contraction, we examined the possible role of these two mechanisms in the pathogenesis of coronary spasm in our swine model in vivo. METHODS AND RESULTS: In 25 pigs, interleukin-1 beta (IL-1 beta) was applied chronically to the coronary arteries from the adventitia to induce an inflammatory/proliferative lesion. Two weeks after the operation, either intracoronary serotonin or histamine repeatedly induced coronary spasm at the IL-1 beta-treated site. At those spastic sites, phorbol-12, 13-dibutyrate, a PKC-activating phorbol ester, also induced coronary spasm, which was blocked by pretreatment with the PKC inhibitors staurosporine and sphingosine. Serotonin- and histamine-induced coronary spasm was also significantly inhibited by pretreatment with staurosporine, sphingosine, or nifedipine (an L-type Ca2+ channel antagonist) but not by ryanodine (an inhibitor of Ca(2+)-induced Ca2+ release from SR) or thapsigargin (an inhibitor of Ca(2+)-ATPase of SR). Bay K 8644 (an L-type Ca2+ channel agonist) also induced coronary spasm at the IL-1 beta-treated site, which was significantly inhibited by pretreatment with staurosporine, sphingosine, and nifedipine. In contrast, coronary vasoconstriction induced by prostaglandin F2 alpha was not affected by pretreatment with staurosporine or sphingosine but was significantly inhibited by pretreatment with ryanodine, thapsigargin, or nifedipine. CONCLUSIONS: These results suggest that (1) PKC activation largely accounts for the serotonin- and histamine-induced coronary spasm; (2) at the spastic site, the calcium influx through L-type Ca2+ channels may be augmented via the PKC-mediated pathway; and (3) the Ca2+ release from the SR into the cytosol may not play a primary role in coronary spasm.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

IgG1-mediated acute pulmonary hypersensitivity response in the guinea pig. Involvement of specific lipid mediators.

We determined the pulmonary obstructive response to aerosolized antigen challenge, and its sensitivity to antagonists of specific lipid mediators, in IgG, passively sensitized (IgG1-PS) guinea pigs. Antiovalbumin (OA)-IgG1 was isolated by affinity chromatography from serum derived from actively immunized Hartley guinea pigs. Propranolol and pyrilamine pretreated, IgG1-PS guinea pigs were challenged with aerosolized antigen and pulmonary obstruction was quantified by measurements of excised lung gas volume (ELGV). ELGV increased between 150 and 1,035% in a dose-proportional fashion with increasing antigen exposure (0.001 to 0.1% nebulizer concentration). The leukotriene antagonists ICI-204,219 and SKF-104,353 exhibited dose-proportional inhibitions in antigen-induced elevations in ELGV, inhibiting up to 65 and 87% at the maximal concentrations examined. Similarly, the platelet-activating factor (PAF) antagonists WEB-2086 and L-659,989 inhibited antigen-induced elevations in ELGV, inhibiting up to 94 and 59% at the maximal concentrations examined. In contrast, the cyclooxygenase (CO) inhibitor piroxicam significantly enhanced (p less than 0.05) the OA-induced elevations in ELGV. Aerosolized PAF challenge produced dose-proportional elevations in ELGV that were significantly inhibited by the LTD, antagonist ICI-204,219 (38 and 43% inhibition) and the CO inhibitor piroxicam (62 and 48% inhibition) in sensitized and nonsensitized animals, respectively. We hypothesize that IgG1-dependent airway obstruction is mediated in part by LTD, produced in response to PAF generation.

Acute Disease↗
PubMed1998

Beta-adrenoceptor-mediated inhibition of IFN-gamma, IL-3, and GM-CSF mRNA accumulation in activated human T lymphocytes is solely mediated by the beta2-adrenoceptor subtype.

Cytokine gene expression in T lymphocytes is a strictly regulated process, involving both stimulatory and inhibitory signals. beta-Adrenoceptor (betaAR) agonists are widely used in the treatment of asthma and are able to induce an inhibitory signal on immunological responses after binding to their specific receptors. In this study, the characterization of betaAR subtype(s) (beta1, beta2, and beta3) involved in the regulation of interleukin (IL)-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma (IFN-gamma) mRNA accumulation was studied by using various betaAR agonists and antagonists. Concanavalin A (Con A)-induced IFN-gamma, GM-CSF, and IL-3 mRNAs are dose-dependently inhibited by the nonselective betaAR agonist isoproterenol and by the selective beta2AR agonist fenoterol. IL-4 mRNA accumulation was not susceptible to betaAR stimulation. The observed inhibition on IFN-gamma, GM-CSF, and IL-3 mRNA was blocked by the selective beta2AR antagonist ICI 118,551 (10(-6) M) and by timolol (10(-6) M), a nonselective antagonist. The selective beta1AR antagonist atenolol (0.3 x 10(-6) M) did not have any effect. Secretion of GM-CSF protein in the presence of increasing concentrations of isoproterenol followed a similar pattern as observed for GM-CSF mRNA. In addition, the betaAR-mediated inhibition of IFN-gamma, GM-CSF, and IL-3 mRNA accumulation and GM-CSF protein secretion were related to the accumulation of intracellular cyclic adenosine monophosphate (cAMP) levels. Although beta3AR mRNA was detectable in Con A-activated T lymphocytes, we could not demonstrate a functional activity in the regulation of cytokine expression: the beta3AR agonist BRL 37344 had no effect on the accumulation of the studied cytokine mRNAs, and did not significantly affect cellular cAMP levels. These data demonstrate that beta-agonist-induced inhibition of IFN-gamma, GM-CSF, and IL-3 mRNA accumulation is solely mediated by beta2-adrenoceptors.

Concanavalin A↗
PubMed1999

Restoration of cyclic adenosine monophosphate-stimulated chloride channel activity in human cystic fibrosis tracheobronchial submucosal gland cells by adenovirus-mediated and cationic lipid-mediated gene transfer.

In human airways, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is predominantly expressed in serous cells of the tracheobronchial glands. Despite considerable evidence that submucosal glands are important contributors to the pathophysiology of CF lung disease, most attempts at CFTR gene transfer have primarily targeted airway surface epithelial cells. In this study, we systematically evaluated CFTR gene transfer into cultures of immortalized CF human tracheobronchial submucosal gland (6CFSMEO) cells using adenovirus and cationic lipid vectors. We found that the efficiency of adenovirus-mediated gene transfer was comparable in 6CFSMEO and CFT1 cells (a surface airway epithelial cell line isolated from a subject with CF). So was the ranking order of adenovirus vectors containing different enhancers/promoters (CMV >> E1a approximately phosphoglycerokinase), as determined by both X-Gal staining and quantitative measurement of beta-galactosidase activity. Further, we provide the first demonstration that cationic lipids mediate efficient gene transfer into 6CFSMEO cells in vitro. The transfection efficiency at optimal conditions was higher in 6CFSMEO than in CFT1 cells. Finally, either infection with adenoviral vectors or transfection with cationic lipid:plasmid DNA complexes encoding CFTR significantly increased chloride (Cl-) permeability, as assessed using the 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorescence assay, indicating restoration of functional CFTR Cl- channel activity. These data show that although the mechanisms of transfection may be different between the two cell types, 6CFSMEO cells are as susceptible as CFT1 cells to transfection by adenoviral and cationic-lipid gene transfer vectors.

Adenoviridae↗
PubMed2006

Reactive nitrogen species augment fibroblast-mediated collagen gel contraction, mediator production, and chemotaxis.

Reactive nitrogen species (RNS) such as peroxynitrite cause cellular injury and tissue inflammation. Excessive production of nitrotyrosine, which is a footprint of RNS, has been observed in the airways of patients with asthma and chronic obstructive pulmonary disease, disorders characterized by tissue remodeling. The aim of this study was to evaluate whether RNS can affect tissue remodeling through direct effects on fibroblasts, and to determine if these effects depend on production of transforming growth factor-beta (TGF-beta). To accomplish this, human fetal lung fibroblasts (HFL-1) were used to assess fibroblast-mediated contraction of floating gels and chemotaxis toward fibronectin. In addition, the ability of fibroblasts to release TGF-beta1, fibronectin, and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay. Authentic peroxynitrite significantly augmented gel contraction (P < 0.01) and chemotaxis (P < 0.01) compared with control in a concentration-dependent manner. Similarly, the peroxynitrite donor 3-morpholynosidenonimine hydrochloride (SIN-1) also augmented gel contraction (P < 0.01). RNS also significantly increased TGF-beta1 (P < 0.01), fibronectin (P < 0.01), and VEGF (P < 0.01) release into the media in both 3D gel and monolayer culture. Anti-TGF-beta antibody reversed RNS-augmented gel contraction (P < 0.01) and mediator production (P < 0.01). Anti-TGF-beta antibody also partially, but significantly, reversed RNS-augmented chemotaxis toward fibronectin (P < 0.01). Finally, peroxynitrite enhanced expression of alpha5beta1 integrin, which is a receptor for fibronectin (P < 0.01), and neutralizing anti-TGF-beta antibody suppressed peroxynitrite-augmented alpha5beta1 expression (P < 0.01). These results suggest that RNS can affect the tissue repair process by modulating TGF-beta1.

Animals↗
PubMed2003

FGF-2 antagonizes the TGF-beta1-mediated induction of pericyte alpha-smooth muscle actin expression: a role for myf-5 and Smad-mediated signaling pathways.

PURPOSE: Although the FGF and TGF-beta families are known to play an important role in regulating vascular endothelial and smooth muscle cell behavior, the influence of these matrix-binding growth factors on microvascular pericyte morphogenesis is not well understood. The current study was undertaken to examine the molecular mechanisms that mediate the effects of the endothelium-produced growth regulators FGF-2 and TGF-beta1 on retinal pericyte proliferation and contractile phenotype. METHODS: Using purified retinal pericytes, a series of assays were implemented, including RT-PCR, DNA binding, immunoprecipitation, electrophoretic mobility shift, and indirect immunofluorescence, in an attempt to elucidate the FGF/TGF-beta1 signaling cascades that mediate retinal microvascular cell growth and contractile phenotype. RESULTS: Treatment of retinal pericytes with FGF-2 and heparin stimulated nearly a log order increase in proliferation, whereas removal of FGF-2 or addition of TGF-beta1 caused withdrawal from the growth cycle, inducing a smooth-muscle-like contractile phenotype, as indicated by upregulation of alpha-smooth muscle actin (alpha-SMA). This switch from a growth-potentiated to a growth-arrested state followed induction of the transcriptional regulator myf-5, as well as the nuclear translocation of myf-5 and Smad2. CONCLUSIONS: Several critical features of the endothelial cell-extracellular matrix-pericyte molecular signaling axis were elucidated in the study that are likely to be responsible for regulating retinal microvascular morphogenesis during normal development, as well as the pathologic angiogenesis accompanying several ocular disorders, including diabetic retinopathy and age-related macular degeneration.

Actins↗
PubMed1984

New avian model of experimental glomerulonephritis consistent with mediation by cellular immunity. Nonhumorally mediated glomerulonephritis in chickens.

The present study examined the role of cell-mediated immunity (CMI) in the production of experimental autoimmune glomerulonephritis (EAG) in chickens deficient in humorally mediated immunity (HMI). Cyclophosphamide bursectomized (Bsx) and normal control chickens were used. Bsx chickens were used only if they had severe depression of HMI, which was evidenced by marked reduction in bursal weights (0.89 +/- 0.23 vs. 2.92 +/- 0.9 g), decreased serum IgG to less than or equal to 10% of normal, and total lack of HMI to immunization with sheep red blood cells. EAG was produced by immunizing chickens with bovine glomerular basement membrane (GBM) in complete Freund's adjuvant. CMI manifested by wattle thickness increments to PPD was not different, 3.89 +/- 0.45 mm for Bsx compared with 3.73 +/- 0.75 mm for controls. No circulating antibodies to GBM developed in 68% of Bsx chickens, and the anti-GBM titers were less than 1:312 in those Bsx chickens positive for antibody compared with greater than 2,000 for controls. GBM deposits of IgG by fluorescence were much decreased, 0.53 +/- 0.16 compared with 2.19 +/- 0.32 for controls, and were absent in 64% of Bsx chickens. Nonetheless, proliferative nephritis with crescents was present and was even more severe in Bsx chickens than in controls, with glomerular sizes of 20.8 +/- 0.6 U for Bsx-GBM, 19.8 +/- 1.2 for control-GBM, 14.9 +/- 1.5 for Bsx, and 13.6 +/- 0.8 for normal chickens. Nephritic eluates did not produce disease in normal chickens, while administration of sensitized cells with [H3]thymidine to naive birds was associated with increased mesangial grain counts by autoradiography. These findings suggest that CMI plays a major role in the pathogenesis of EAG in chickens in the absence of HMI. By implication, CMI may be crucial in the development of other types of glomerulonephritis as well.

Animals↗
PubMed2002

GM-CSF, via PU.1, regulates alveolar macrophage Fcgamma R-mediated phagocytosis and the IL-18/IFN-gamma -mediated molecular connection between innate and adaptive immunity in the lung.

Severely impaired pulmonary microbial clearance was observed in granulocyte-macrophage colony-stimulating factor (GM-CSF)-deficient mice. To determine mechanisms by which GM-CSF mediates lung host defense, FcgammaR-mediated phagocytosis (opsonophagocytosis) by alveolar macrophages (AMs) was assessed in GM-CSF-sufficient (GM(+/+)) and -deficient (GM(-/-)) mice and in GM(-/-) mice expressing GM-CSF only in the lungs from a surfactant protein C (SPC) promoter (SPC-GM(+/+)/GM(-/-)). Opsonophagocytosis by GM(-/-) AMs was severely impaired and was restored by pulmonary GM-CSF expression in vivo or by PU.1 expression in vitro. Defective opsonophagocytosis by GM(-/-) AMs was associated with decreased FcgammaR expression. Because interferon-gamma (IFN-gamma) augments macrophage FcgammaR levels, the role of GM-CSF/PU.1 in the regulation of AM FcgammaR expression by IFN-gamma was assessed during adenoviral lung infection. Adenoviral infection stimulated IFN-gamma production and augmented FcgammaR levels on AMs in GM-CSF-expressing but not GM(-/-) mice. However, IFN-gamma exposure ex vivo stimulated FcgammaR expression on GM(-/-) AMs. Because interleukin-18 (IL-18) and IL-12 stimulate IFN-gamma production during adenoviral infection, their role in GM-CSF/PU.1 regulation of IFN-gamma-augmented FcgammaR expression on AMs was assessed. Adenoviral infection stimulated IL-18 and IL-12 production in GM-CSF-expressing mice, but both were markedly reduced or absent in GM(-/-) mice. IL-18 expression by GM(-/-) AMs was severely impaired and was restored by pulmonary GM-CSF expression in vivo or by PU.1 expression in vitro. Pulmonary administration of IL-18 in GM(-/-) mice stimulated IFN-gamma production and restored FcgammaR expression on AMs. These results show that GM-CSF, via PU.1, regulates constitutive AM FcgammaR expression and opsonophagocytosis and is required for the IFN-gamma-dependent regulation of AM FcgammaR expression, enabling AMs to release IL-18/IL-12 during lung infection.

Adenoviridae Infections↗
PubMed2006

Mdm2 inhibitor Nutlin-3a induces p53-mediated apoptosis by transcription-dependent and transcription-independent mechanisms and may overcome Atm-mediated resistance to fludarabine in chronic lymphocytic leukemia.

Although TP53 mutations are rare in B-cell chronic lymphocytic leukemia (CLL), Mdm2 overexpression has been reported as an alternative cause of p53 dysfunction. We investigated the potential therapeutic use of nongenotoxic p53 activation by a small-molecule antagonist of Mdm2, Nutlin-3a, in CLL. Nutlin-3a induced significant apoptosis in 30 (91%) of 33 samples from previously untreated patients with CLL; all resistant samples had TP53 mutations. Low levels of Atm (ataxia telangiectasia mutated) or high levels of Mdm2 (murine double minute 2) did not prevent Nutlin-3a from inducing apoptosis. Nutlin-3a used transcription-dependent and transcription-independent pathways to induce p53-mediated apoptosis. Predominant activation of the transcription-independent pathway induced more pronounced apoptosis than that of the transcription-dependent pathway, suggesting that activation of the transcription-independent pathway is sufficient to initiate p53-mediated apoptosis in CLL. Combination treatment of Nutlin-3a and fludarabine synergistically increased p53 levels, and induced conformational change of Bax and apoptosis in wild-type p53 cells but not in cells with mutant p53. The synergistic apoptotic effect was maintained in samples with low Atm that were fludarabine resistant. Results suggest that the nongenotoxic activation of p53 by targeting the Mdm2-p53 interaction provides a novel therapeutic strategy for CLL.

Apoptosis↗