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In the present work we have demonstrated that the low NOS activity of circulating blood neutrophils is related to an endogenous inhibitory factor. This factor inhibits constitutive NOS (cNOS) of cerebellum and inducible NOS (iNOS) of macrophages in a concentration-dependent manner. Boiling only partially diminished its activity. The inhibition of cNOS was specific, since to some degree NADPH (0.5-4 mM) and more effectively L-arginine (0.1-1 mM), but not D-arginine, reversed the inhibition.

Long-term exposure of platelets to prostacyclin or iloprost (100nM, 3hr) results in receptor desensitization measured as decrease in 3H-iloprost binding sites by 47 +/- 14%. Desensitized platelets respond with an increased adhesion to endothelial cells. The mechanism of increased adhesiveness was studied by measuring the expression of the adhesion molecule CD62p (p-selectin; GMP140) on washed human platelets by flowcytometry. In thrombin stimulated platelets CD62p expression was dose-dependently reduced by iloprost. In receptor desensitized platelets IC50 for iloprost inhibition of thrombin-induced CD62p expression increased from 0.48 +/- 0.10 to 2.4 +/- 0.7 nM.

During the past two years, the enzyme responsible for production of endothelium-derived nitric oxide, the endothelial cell NO synthase (ecNOS) has been cloned and the gene encoding this enzyme isolated, cloned and its structure characterized. This research has provided direction for a variety of studies of regulation of the ecNOS. Several features of the ecNOS are compatible with a constitutively expressed, poorly regulated gene, including absence of a TATA box and numerous SP-1 sites. The promoter also contains a number of putative binding domains which suggest that it may be regulated by a variety of transcription factor mediated signals. In this review we will discuss evidence to support the concept that the ecNOS is a constitutively expressed gene subject to a modest degree of regulation by important physiological influences.

Using gene targeting in mouse embryonic stem cells, it is possible to introduce diverse mutations into specific genes. Using these methods, various laboratories have reported mutations for a variety of inflammatory cell adhesion molecules including CD18, alpha 5 integrin, ICAM-1, P-selectin, and L-selectin; preliminary reports of other mutations are also available. Mutations in CD18 and ICAM-1 cause impaired inflammatory and immune responses, mutations in P-selectin and L-selectin cause decreased leukocyte rolling and emigration, and a mutation in alpha 5 integrin causes embryonic lethality. Gene targeting complements other approaches for analyzing the function of inflammatory cell adhesion molecules.

The distinct pattern of transcriptional responses of cells to different extracellular signals requires a signal transduction pathway that provides rapid, accurate, and faithful transmission of information from the cell surface to the nucleus. One mechanism exploited by many cytokines, exemplified by interferons (IFN) but also used by many interleukins and growth factors, uses a family of cytoplasmic transcription factors that are activated by tyrosine phosphorylation. Once phosphorylated by receptor-associated tyrosine kinases, these proteins assemble into multimeric transcription factors, translocate to the nucleus, and bind specific DNA sequence elements in the promoters of target genes.

The main target of non-steroidal anti-inflammatory drugs (NSAIDs) is prostaglandin G/H synthase (PGHS), also known as cyclooxygenase (COX), which exists as two isoforms. In order to evaluate the contributions of PGHS isoforms to physiological and pathological conditions and their sensitivity to inhibition by non-steroidal anti-inflammatory drugs, we have established high level expression systems of recombinant human PGHS isoforms. The inducible form of PGHS, termed PGHS-2, has been purified and characterized with respect to substrate specificity, product formation, enzymatic activity, glycosylation, heme content, quaternary structure, and modification by aspirin. Pharmacological profiles of the recombinant PGHS isoforms indicate that conventional NSAIDs show little selectivity for either enzyme, however, the recently described NSAID, NS-398, exhibits a high degree of specificity for PGHS-2 through a time dependent mechanism.

We have performed a detailed structure-function analysis of human interleukin 5 (hIL5) and its receptor. By testing a hIL5 mutant panel in a solid phase binding assay and a proliferation assay using hIL5 dependent cell-lines, areas on hIL5 involved in either the receptor alpha-subunit interaction or in receptor activation were identified. Epitope mapping data of a neutralizing and a non-neutralizing monoclonal antibody were in agreement with the mutant analysis. hIL5 binding areas on the IL5R alpha-subunit were identified by interspecies chimaera analysis. Finally, hIL5 mutants with reduced receptor activation potential have antagonistic properties.

The past several years have produced a dramatic increase in our understanding of the steps involved in the infiltration of leukocytes into inflamed tissues. At least four major families of adhesion molecules: the selectins, sialomucins, integrins, and Ig supergene family CAMs have been identified; and their interactions are being elucidated. The role of the leukocyte beta 2 and alpha 4 integrins and the selective use of these integrins by leukocytes for migration into inflamed tissues in various organs is presented.