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A Dean Befus

Publications and source records attributed to A Dean Befus.

11 recordsLinked to original sources
PubMed2003

Tetrahydrobiopterin, a critical factor in the production and role of nitric oxide in mast cells.

Mast cells (MC) are biologically potent, ubiquitously distributed immune cells with fundamental roles in host integrity and disease. MC diversity and function is regulated by exogenous nitric oxide; however, the production and function of endogenously produced NO in MC is enigmatic. We used rat peritoneal MC (PMC) as an in vivo model to examine intracellular NO production. Live cell confocal analysis of PMC using the NO-sensitive probe diaminofluorescein showed distinct patterns of intracellular NO formation with either antigen (Ag)/IgE (short term) or interferon-gamma (IFN-gamma) (long term). Ag/IgE-induced NO production is preceded by increased intracellular Ca2+, implying constitutive nitric-oxide synthase (NOS) activity. NO formation inhibits MC degranulation. NOS has obligate requirements for tetrahydrobiopterin (BH4), a product of GTP-cyclohydrolase I (CHI), IFN-gamma-stimulated PMC increased CHI mRNA, protein, and enzymatic activity, while decreasing CHI feedback regulatory protein mRNA, causing sustained NO production. Treatment with the CHI inhibitor, 2,4-diamino-6-hydroxypyrimidine, inhibited NO in both IFN-gamma and Ag/IgE systems, increasing MC degranulation. Reconstitution with the exogenous BH4 substrate, sepiapterin, restored NO formation and inhibited exocytosis. Thus, Ag/IgE and IFN-gamma induced intracellular NO plays a key role in MC mediator release, and alterations in NOS activity via BH4 availability may be critical to the heterogeneous responsiveness of MC.

Animals↗
PubMed2003

Nitric oxide and cyclic GMP increase the expression of matrix metalloproteinase-9 in vascular smooth muscle.

Interactions and possible cross talk between inducible nitricoxide synthase (iNOS), cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9), were studied in rat aortic vascular smooth muscle cells stimulated with bacterial lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and phorbol 12-myristate13-acetate (PMA). The expression and activity of iNOS, COX-2, and MMP-9 were characterized at the transcriptional, protein, and enzyme activity levels. The NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) was used to investigate the effects of NO on COX-2 and MMP-9 at the transcriptional level. The measurements of mRNAs for these enzymes using real-time polymerase chain reaction (PCR) showed that COX-2 mRNA was up-regulated 2.3-fold, whereas MMP-9 mRNA up-regulation was 11.7-fold in the presence of LPS, IFN-gamma, and PMA. Real-time PCR results indicated that L-NAME exerted an inhibitory effect on COX-2 and MMP-9 mRNA synthesis. Both superoxide dismutase (SOD) and the SOD mimetic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) did not modify significantly the up-regulation of these enzymes, indicating that neither superoxide nor peroxynitrite are involved in this mechanism. Furthermore, NO-mediated up-regulation of MMP-9 was cGMP-dependent since 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, blocked, in a concentration-dependent manner, the increased expression of MMP-9, an effect reversed by 8-bromo-cGMP, a soluble analog of cGMP. Our findings suggest that NO and cGMP are necessary to up-regulate the expression of MMP-9.

Animals↗
PubMed2003

Modulation of neutrophil function by the tripeptide feG.

BACKGROUND: Neutrophils are critical in the defense against potentially harmful microorganisms, but their excessive and inappropriate activation can contribute significantly to tissue damage and a worsening pathology. Through the release of endocrine factors submandibular glands contribute to achieving a balance in neutrophil function by modulating the state of activation and migratory potential of circulating neutrophils. A putative hormonal candidate for these effects on neutrophils was identified as a heptapeptide named submandibular gland peptide T (SGP-T; sequence = TDIFEGG). Since the tripeptide FEG, derived from SGP-T, and its D-amino acid analogue feG had similar inhibitory effects on inflammatory reactions, we investigated the effects of feG on human and rat neutrophil function. RESULTS: With human neutrophils feG had no discernible effect on oxidative burst or phagocytosis, but in picomolar amounts it reduced PAF-induced neutrophil movement and adhesion, and the binding of CD11b by 34% and that of CD16b close to control values. In the rat feG (10-11M) reduced the binding of CD11b and CD16 antibodies to PAF-stimulated circulating neutrophils by 35% and 43%, respectively, and at 100 micrograms/kilograms intraperitoneally feG reduced neutrophil in vivo migration by 40%. With ovalbumin-sensitized rats that were challenged with antigen, feG inhibited binding of antibodies against CD16b but not CD11b, on peritoneal leukocytes. CONCLUSIONS: The inhibitory effect of feG on neutrophil movement may be mediated by alterations in the co-stimulatory molecules CD11b and CD16.

Animals↗
PubMed2003

Inhibition of calpain is a component of nitric oxide-induced down-regulation of human mast cell adhesion.

Nitric oxide is an important messenger that regulates mast cell activity by modifications to gene expression and intracellular pathways associated with exocytosis and adhesion. Integrin interactions with extracellular matrix components modulate an array of cell activities, including mediator production and secretion. To investigate the molecular mechanisms underlying NO regulation of mast cell function, we studied its effects on adhesion of a human mast cell line (HMC-1) to fibronectin (FN). The NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine strongly down-regulated the adhesion of HMC-1 to FN. Inhibitors of soluble guanylate cyclase and protein kinase G did not alter the response of cells to NO. A peroxynitrite scavenger did not affect modulation of adhesion by NO, nor could the effect of NO be mimicked by the peroxynitrite-producing compound 3-morpholinosydnonimine. NO donors inhibited the cysteine protease, calpain, while calpain inhibitors mimicked the effect of NO and led to a decrease in the ability of HMC-1 cells to adhere to FN. Thus, NO is an effective down-regulator of human mast cell adhesion. The mechanism for this action does not involve peroxynitrite or activation of soluble guanylate cyclase. Instead, a portion of NO-induced down-regulation of adhesion may be attributed to inhibition of the cysteine protease, calpain, an enzyme that has been associated with control of integrin activation in other cell types. The inhibition of calpain is most likely mediated via nitrosylation of its active site thiol group. Calpain may represent a novel therapeutic target for the regulation of mast cell activity in inflammatory disorders.

Calpain↗
PubMed2003

The dynamic and complex role of mast cells in allergic disease.

Mast cells (MCs) are found widely distributed in tissues and contribute to regulation of inflammatory responses and ongoing modulation of the tissues. Although MCs are important in a variety of processes, including innate immunity, their role in allergic disease has received increasing attention in the past decade. MCs are located throughout the human body and, upon allergen exposure, they are stimulated via the immunoglobulin E (IgE) receptor (Fc(epsilon)RI) to release several pro-inflammatory mediators such as tumor necrosis factor (TNF), reactive oxygen species such as nitric oxide (NO), proteases, and lipid-derived mediators. However, we now recognize that MCs can be activated by a variety of mechanisms and that mediator release is a consequence of several intra- and extracellular signals. Some of these mechanisms, such as Fc receptor aggregation and proteinase-activated receptor (PAR)-mediated activation facilitate and augment local inflammatory responses. Other mechanisms, such as interferon gamma (IFN-gamma) induction of NO, may inhibit MC function and downregulate inflammatory responses. Increased understanding of these complex pathways has encouraged the development of therapies for allergic inflammation that target specific MC functions and mediators. Some novel strategies include oligonucleotides that induce or inhibit the production of specific mediators. Such approaches may yield useful therapies for allergic individuals in the near future.

Animals↗
PubMed2003

Antisense oligonucleotides to Syk kinase: a novel therapeutic approach for respiratory disorders.

The non-receptor protein tyrosine kinase Syk plays a critical role in intracellular signaling in the inflammatory response. Specific inhibition of Syk using aerosolized antisense delivered in liposome complexes can significantly decrease inflammatory responses in the airways in experimental animal models. Thus, it is tempting to examine local application of Syk antisense for the treatment of inflammatory respiratory diseases as asthma. However, evidence that Syk kinase is more widely distributed in different cell types than previously recognized, as well as its potential involvement in cell differentiation, adhesion and proliferation, dictates that the precise cellular targets for antisense therapy in the airways must be determined. Given the critical role of Syk in intracellular signaling in inflammatory responses, Syk antisense oligonucleotides (InKine Pharmaceutical Co Inc) may prove useful as anti-inflammatory therapy in disorders such as asthma.

Animals↗
PubMed2002

Probing for submandibular gland peptide-T receptors on leukocytes with biotinylated-Lys-[Gly](6)-SGP-T.

Submandibular gland peptide-T (SGP-T) is a potent anti-chemotactic agent for human neutrophils possessing anti-inflammatory properties. Biologically active analogues of SGP-T have been synthesized and a biotinylated form (KG(6)-SGP-T; Bio-KG(6)-SGP-T) was utilized to identify binding sites on isolated human neutrophils. Neutrophils incubated with Bio-KG(6)-SGP-T followed by phycoerythrin (PE)-avidin secondary reagent were fixed and visualized using histochemistry and flow cytometry. At doses of 10(-8) and 10(-9) M, Bio-KG(6)-SGP-T was shown to bind to neutrophils. The binding of Bio-KG(6)-SGP-T, at doses of 10(-8) and 10(-9) M, to neutrophils was abolished by a 100-fold excess of non-biotinylated peptide (KG(6)-SGP-T), but not by 100-fold excess of SGP-T. However, all peptides, dose-dependently reduced the binding of a CD16b antibody (LNK16 clone) to isolated human neutrophils. This discrepancy probably results from different preferred conformations for Bio-KG(6)-SGP-T, KG(6)-SGP-T and SGP-T, since exhaustive conformational searches revealed a high degree of overlap between alpha-Bio-KG(6)-SGP-T and KG(6)-SGP-T that was not seen with SGP-T.

Antibodies↗
PubMed2002

Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase.

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of Fc gamma R and Fc epsilon R, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of beta(2) integrins, alpha(4) integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.

Administration, Inhalation↗
PubMed2002

Proteinase-activated receptor (PAR)-1 and -2 agonists induce mediator release from mast cells by pathways distinct from PAR-1 and PAR-2.

Because thrombin-induced inflammation is partially mast cell-dependent and involves proteinase-activated receptors (PARs), we hypothesized that mast cells express PAR and can be stimulated with PAR-activating peptides (PAR-AP). We demonstrated that rat peritoneal mast cells expressed PAR-1 and PAR-2 mRNA, and that PAR-2AP (tc-LIGRLO-NH(2), 1 microm) induced 64.2 +/- 4.4% specific beta-hexosaminidase release from peritoneal mast cells, whereas another PAR-2AP (SLIGRL-NH(2), 10 microM), trypsin (40 U/ml), and mast cell tryptase (1.5 microg/ml) did not. PAR-1AP (ApfFRChaCitY-NH(2), 10 microM) (Cit) induced 11.7 +/- 3.7% specific beta-hexosaminidase release, whereas another PAR-1AP (TFLLR-NH(2), 40 microM) and human thrombin (10 U/ml) did not. PAR-AP, tc-LIGRLO-NH(2), and Cit increased the free intracellular Ca(2+) concentration, whereas trypsin, tryptase, thrombin, and other PAR-APs did not. Desensitization of Ca(2+) flux with different agonists suggests that although tc-LIGRLO-NH(2), Cit, and compound 48/80 have similar mechanisms of action, tc-LIGRLO-NH(2) also activates mast cells by a mechanism distinct from that of 48/80. Using benzalkonium chloride, which antagonizes the actions of 48/80 by competing for the same G(i) protein, we determined that benzalkonium chloride suppressed tc-LIGRLO-NH(2)-mediated (0.1 microM) beta-hexosaminidase release by 62%. Moreover, removal of sialic acid from peritoneal mast cells, using neuraminidase (2 U/ml), inhibited Cit- (10 microM, 52%) and tc-LIGRLO-NH(2) (0.5 microM, 29%)-mediated beta-hexosaminidase release. Thus, tc-LIGRLO-NH(2) and Cit have at least partially similar mechanisms of action as 48/80. PAR-AP may therefore activate mast cells via multiple mechanisms that are distinct from those of classical PAR-1 and PAR-2. The responsiveness of mast cells to PAR-AP via a non-PAR-1/non-PAR-2 mechanism complicates the interpretation of in vivo studies using these peptides.

Animals↗