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Recent studies suggest that expression of cyclooxygenase-2 (Cox-2) is elevated in transitional cell carcinoma (TCC) of the urinary bladder and that inhibition of Cox-2 activity suppresses bladder cancer in experimental animal models. We have investigated the expression of Cox-2 protein in human TCCs (n = 85), in in situ carcinomas (Tis) of the urinary bladder (n = 17), and in nonneoplastic urinary bladder samples (n = 16) using immunohistochemistry. Cox-2 immunoreactivity was detected in 66% (67 of 102) of the carcinomas, whereas only 25% (4 of 16) of the nonneoplastic samples were positive (P: < 0.005). Cox-2 immunoreactivity localized to neoplastic cells in the carcinoma samples. The rate of positivity was the same in invasive (T1-3; 70%, n = 40) and in noninvasive (Tis and Ta; 65%, n = 62) carcinomas, but noninvasive tumors had a higher frequency (32%) of homogenous pattern of staining (>90% of the tumor cells positive) than the invasive carcinomas (10%) (P: < 0.05). However, several invasive TCCs exhibited the strongest intensity of Cox-2 staining in the invading cells, whereas other parts of the tumor were virtually negative. Finally, strong Cox-2 positivity was also found in nonneoplastic ulcerations (2 of 2) and in inflammatory pseudotumors (2 of 2), in which the immunoreactivity localized to the nonepithelial cells. Taken together, our data suggest that Cox-2 is highly expressed in noninvasive bladder carcinomas, whereas the highest expression of invasive tumors associated with the invading cells, and that Cox-2 may also have a pathophysiological role in nonneoplastic conditions of the urinary bladder, such as ulcerations and inflammatory pseudotumors.

Cytokines mediate many host responses to bacterial infections. We determined the inflammatory activities of five cytokines in the central nervous system: TNF-alpha, IL-1 alpha, IL-1 beta, macrophage inflammatory protein 1 (MIP-1), and macrophage inflammatory protein 2 (MIP-2). Using a rabbit model of meningeal inflammation, each cytokine (except IL-1 beta) induced enhanced blood brain barrier permeability, leukocytosis in cerebrospinal fluid, and brain edema. Homologous antibodies to each mediator inhibited leukocytosis and brain edema, and moderately decreased blood brain barrier permeability. In rabbits treated with anti-CD-18 antibody to render neutrophils dysfunctional for adhesion, each cytokine studied lost the ability to cause leukocytosis and brain edema. After intracisternal challenge with pneumococci, antibodies to TNF or IL-1 prevented inflammation, while anti-MIP-1 or anti-MIP-2 caused only a 2-h delay in the onset of inflammation. We suggest these cytokines have multiple inflammatory activities in the central nervous system and contribute to tissue damage during pneumococcal meningitis.

SummaryThe adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough but the significance of bacterial attachment to macrophages has not been determined . Adherence to cilia and macrophages is mediated by two large, nonfimbrial bacterial proteins, filamentous hemagglutinin (FHA), and pertussis toxin (PT) . PT and FHA both recognize carbohydrates on cilia and macrophages; FHA also contains an Arg-Gly-Asp (RGD) sequence which promotes bacterial association with the macrophage integrin complement receptor 3 (CR3) . We determined that virulent R pertussis enter and survive in mammalian macrophages in vitro and that CR3 is important for this uptake process . We then determined the relative contribution of CR3 versus carbohydrate-dependent interactions to in vivo pulmonary colonization using a rabbit model. R pertussis colonized the lung as two approximately equal populations, one extracellular population attached to ciliary and macrophage surface glycoconjugates and another population within pulmonary macrophages. Loss of the CR3 interaction, either by mutation of FHA or treatment with antibody to CR3, disrupted accumulation of viable intracellular bacteria but did not prevent lung pathology. In contrast, elimination of carbohydrate-bound bacteria, either by a competitive receptor analogue or an anti-receptor antibody, was sufficient to prevent pulmonary edema. We propose that CR3-dependent localization of R pertussis within macrophages promotes persistence of bacteria in the lung without pulmonary injury. On the other hand, the presence of extracellular bacteria adherent to cilia and macrophages in carbohydrate-dependent interactions is associated with pulmonary pathology. ordetelia pertussis is a Gram-negative coccobacillus that is the causative agent of whooping cough. It has long been recognized that R pertussis establishes pulmonary colonization by adhering specifically to human ciliated epithelial cells, and this interaction has been studied in detail in vitro (1) . Recently, however, in vitro studies have shown that R pertussis also adheres specifically to human macrophages (2) and can enter and survive within tissue culture cells (3-5) . The purpose of this study was to determine the importance of the interaction between R pertussis and macrophages during pulmonary infection .Bacterial adherence to cilia and macrophages is mediated by two nonfimbrial bacterial proteins, filamentous hemagglutinin (FHA)t and pertussis toxin (PT) which are expressed only by virulent cells (1, 2) . These proteins are functional either when bound to the bacterial surface or when secreted into the surrounding medium during growth and serve as bifunctional ligands bridging the bacterial surface and glycoconjugates on eukaryotic cell membranes (1, 6). Both 'Abbreviations used in this paper. FHA, filamentous hemagglutinin ; PT, pertussis toxin; RGD, Arg-Gly-Asp sequence .adhesins are unusually large molecules with multiple binding affinities, features reminiscent of eukaryotic extra...

We tested if specific inhibition of recruitment of leukocytes across the blood brain barrier from the vascular compartment to the cerebrospinal fluid (CSF) space reduced tissue damage and improved the outcome of infection in a rabbit model of experimental meningitis. The CD11/CD18 complex of receptors on leukocytes promotes adhesion of these cells to endothelia, a process required for egress of cells into the extravascular space. Intravenous injection of the anti-CD18 mAb IB4 effectively blocked the development of leukocytosis in the CSF of animals challenged intracisternally with living bacteria, bacterial endotoxin, or bacterial cell wall. This effect was associated with protection from blood brain barrier injury as measured by exclusion of serum proteins from CSF in mAb-treated animals. The densities of bacteria in CSF and the degrees of bacterial killing due to ampicillin were not affected by the antibody. Animals receiving the antibody experienced a delay in the development of bacteremia and a significantly reduced inflammatory response during ampicillin-induced bacterial killing. Therapy with mAb IB4 prevented development of brain edema and death in animals challenged with lethal doses of Streptococcus pneumoniae. These studies indicate that the major mechanism of leukocyte migration across the blood brain barrier involves the CD11/CD18 receptors and that inflammatory leukocytes recruited by this mechanism are a major cause of blood brain barrier injury and cerebral edema during meningitis.

Bordetela peinussis is bound to glycocoijugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite >80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.During the clinical course of whooping cough, Bordetella pertussis specifically attaches to the cilia of the respiratory epithelium (1), establishes an intracellular state within alveolar macrophages (2), and produces systemic disease by elaborating several toxins (3). At least two adhesins, filamentous hemagglutinin and pertussis toxin (PT), are known to mediate bacterial attachment to human cells (4, 5). Both adhesins are required for attachment to cilia, but each can act independently in the adherence of the organism to macrophages. These adhesins have several unusual features of interest. They are large, nonfimbrial molecules that contain multiple binding domains (1, 5) and, when shed from the cell surface, retain the ability to mediate adherence even for other bacterial species (6).PT is a major virulence determinant of B. pertussis, which induces metabolic changes in the host (7), alters immune responses (8), and is the only toxin known to bind whole bacteria to eukaryotic cells (4). The toxin is a hexameric protein with an A-B architecture (9). The A protomer is composed of a single S1 subunit (Mr, 26,026) containing the catalytic site for toxic ADP-ribosylation of cellular signaltransducing guanine nucleotide regulatory proteins. The B oligomer possesses the cellular recognition domains; it is a complex pentamer containing subunits S2 (Me,21,925), S3 (Mr,21,873), S4 (Mr,12,059), and S5 (Mr, 11,013) in a respective molar ratio of 1:1:2:1 (9). By interacting with glycoproteins and glycolipids on many types of eukaryotic cells (4,(10)(11)(12)(13)(14)(15)(16), the B oligomer serves several distinct and independent functions (4, 15): mitogenicity for T lymphocytes, adherence of the bacteria to eukaryotic cells, and delivery of the toxic S1 subunit to its target. Several findings suggest that the B oligomer contains at least two distinct binding specificit...

The prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with a reduced risk of gastric cancer. The best-known target of these drugs is cyclooxygenase (COX); the COX-2 isoform is frequently up-regulated in gastric adenocarcinomas. Using the post-gastrectomy stomach as a model, the expression of COX-2 mRNA and protein has been investigated during tumour progression in the human stomach. COX-2 expression was comparable in gastric stump carcinomas and conventional gastric carcinomas and localized primarily to the cytoplasm of the neoplastic cells. COX-2 mRNA was elevated in biopsies containing intestinal metaplasia, as determined by reverse transcriptase polymerase chain reaction (RT-PCR). COX-2 immunopositivity became more frequent during progression from reactive epithelium to high-grade dysplasia, both in the epithelial and in the stromal cell compartment. Co-localization of COX-2-positive stromal cells was seen with CD68, alpha-smooth muscle actin (alpha-SMA), vimentin, and HLA-DR, but an as yet unidentified subpopulation of stromal cells remained. Co-localization with the macrophage marker CD68 was only observed in a minority of COX-2-positive cells. These data show that COX-2 expression is a relatively early event during carcinogenesis in the stomach. COX-2 expression increases during tumour progression in the stomach, suggesting a role for COX-2 expression in gastric tumourigenesis.

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