Quantitative immunocytochemical staining for recombinant tissue-type plasminogen activator in transfected Chinese hamster ovary cells.

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction.

Leukotriene D4 (LTD4) induces mucus secretion from goblet cells in the guinea pig respiratory epithelium.

To study the potential role of leukotriene (LTD4) as a mucus secretagogue, anesthetized and spontaneously breathing guinea pigs were intubated and challenged with various concentrations of an LTD4 aerosol. The resulting changes in airway resistance and compliance were then observed for 20 min, after which the animals were euthanized and the lower respiratory tract airways fixed for morphometric evaluation. Sections for these airways were stained with alcian blue-periodic acid Schiff (AB-PAS), photographed, and the content of AB-PAS positive granules in the epithelium of the extrapulmonary bronchi quantified.

Internalization of recombinant tissue-type plasminogen activator by isolated rat hepatocytes is via coated pits.

The uptake and internalization of tissue-type plasminogen activator (t-PA) by freshly isolated rat hepatocytes was investigated. Electron microscopic examination of the uptake of t-PA-colloidal gold conjugates (t-PA-gold) by isolated rat hepatocytes showed that t-PA-gold was internalized via coated pits. This was inhibited with excess t-PA.

Inhibition of inflammatory cell infiltration by bicyclic imidazoles, SK&F 86002 and SK&F 104493.

The mode of action of the dual inhibitors of eicosanoid metabolism, SK&F 86002 and SK&F 104493 was evaluated on inflammatory cell infiltration induced in mice by carrageenan, monosodium urate crystals, and arachidonic acid. The results were compared to those seen with standard antiinflammatory compounds. Inflammatory cell infiltration was inhibited by SK&F 86002.

A new cytochemical method for ultrastructural detection of liposomes in tissues in vivo.

Multilamellar vesicles (MLVs) have been used as drug carriers to increase efficacy or decrease toxicity of a variety of therapeutic agents, including antineoplastics, antibiotics, and immunomodulators. Although analysis of the disposition of encapsulated materials is relatively simple using radiolabels or single enzymes, determining the cellular and subcellular disposition of intact MLVs, i.e.

Interleukin-1 stimulation of phospholipase activity in rat synovial fibroblasts. Possible regulation by cyclooxygenase products.

Tritiated arachidonic acid (3H-AA)-labeled rat synovial fibroblasts stimulated with human recombinant interleukin-1 beta (rIL-1 beta) released incorporated radiolabel in a time-dependent and dose-dependent manner, with labeled prostaglandins representing 29% of the released radiolabel. Treatment of the cells with dibutyryl cAMP or prostaglandin E2 enhanced both spontaneous and rIL-1 beta-induced 3H-AA release; treatment with indomethacin or naproxen inhibited the response. The effects of these cyclooxygenase inhibitors on 3H-AA release were not reversed by the addition of prostaglandin E2.

FMLP-induced arachidonic acid release, phospholipid metabolism, and calcium mobilization in human monocytes. Regulation by cyclic AMP.

Radiolabeled human peripheral blood monocytes released [3H]arachidonic acid upon challenge with the calcium ionophore A23187 (10 microM), or f-Met-Leu-Phe (FMLP, 1 microM). Chromatographic analysis of [3H]arachidonic acid labeled phospholipids showed that stimulation by FMLP reduced the amount of labeled phosphatidylcholine exclusively. Treatment of the monocytes with 10(-3) M dibutyryl cyclic AMP (d-cAMP) or 5 X 10(-4) M isobutylmethylxanthine (IBMX) substantially inhibited [3H]arachidonic acid release (30%) and depletion from labeled phosphatidylcholine (PC) in FMLP--but not calcium ionophore--stimulated cells.

Biological characterization and oncogene expression in human colorectal carcinoma cell lines.

To establish well-characterized cellular reagents for the study of colon carcinoma, we have examined 19 human colorectal carcinoma cell lines with regard to morphology, ultrastructure, expression of tumor-associated antigens, proliferative capacity in vitro, anchorage-independent growth, oncogene expression, tumorigenicity and malignant potential. Cell lines examined were cultured under identical conditions, and in vitro and in vivo analyses were performed in parallel on replicate cultures. Three classes of colorectal cell lines were defined according to their tumorigenicity in nude mice.

Cytochemical demonstration of constitutive H2O2 production by macrophages in synovial tissue from rats with adjuvant arthritis.

Generation of toxic oxygen metabolites by inflammatory cells is considered to be one of the mechanisms by which inflammation produces tissue injury. This concept is based on in vitro studies of purified leukocyte populations because it has not been possible to assess production of these metabolites in tissues. In order to determine whether or not inflammatory cells in tissue generate H2O2, the authors modified an earlier cytochemical method for the localization of H2O2.

Interleukin-1 and tumor necrosis factor are not synergistic for human synovial fibroblast PLA2 activation and PGE2 production.

Patterns of arachidonic acid release and metabolism were altered in human synovial fibroblasts following exposure to cytokines. Recombinant interleukin-1 induced an approximate 3-fold increase in [3H]-AA release, a 7-fold increase in PGE2 production and a 2-fold increase in PLA2 activity in human synovial fibroblasts. Recombinant tumor necrosis factor induced similar responses, however, the magnitude was less than that mediated by interleukin-1.

Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies.

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies.

Major histocompatibility complex class I molecules internalized via coated pits in T lymphocytes.

Endocytosis of the major histocompatibility complex (MHC)-encoded class I and class II molecules has been the subject of recent investigations. Class I molecules, which are key elements in T cell-mediated cytotoxicity, are differentially endocytosed by different cell types. Fibroblasts internalize their class I molecules via uncoated cell surface vesicles and tubular invaginations when these molecules are cross-linked with multivalent ligands.

Phospholipid and arachidonic acid metabolism in zymosan-stimulated human monocytes: modulation by cAMP.

Receptor-ligand interaction in mononuclear phagocytes is intimately linked to alterations in membrane phospholipids and release of arachidonic acid (AA). In addition, synthesis of bioactive lipids from released AA can result in further modification of cell responses. Upon challenge with opsonized zymosan, [3H]-arachidonic acid ([3H]-AA)-labeled human monocytes released 25 +/- 2% of their incorporated radiolabel within 30 min.

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils.

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation.

Recombinant tumor necrosis factor and interleukin-1 both stimulate human synovial cell arachidonic acid release and phospholipid metabolism.

Stimulation of [3H]-arachidonic acid labeled human synovial cells with 3.0 X 10(-10)M recombinant interleukin-1 or tumor necrosis factor resulted in the release of incorporated radiolabel (41.1% and 27.

Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver.

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. delta-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.

Structural requirements for chemotactic activity of leukotriene B4 (LTB4).

LTB4 (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB4 and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo-LTB4.

Neutrophils may directly synthesize both H2O2 and O2- since surface stimuli induce their release in stimulus-specific ratios.

Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O2- and H2O2, the latter being presumed to arise by spontaneous dismutation of the former. If H2O2 were indeed derived exclusively from released O2- according to the equation 2O2- + 2H+----H2O2 + O2, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils.

Surface contact inhibits neutrophil superoxide generation induced by soluble stimuli.

Human neutrophils in suspension undergo a metabolic burst and generate reactive O2- metabolites upon exposure to many soluble and particulate stimuli. They can also be stimulated to produce O2- when in contact with surfaces. We found that when neutrophils were allowed to settle into protein-coated surfaces the amount of O2- they generated varied with the nature of the protein: IgG greater than bovine serum albumin greater than plastic greater than gelatin greater than serum greater than collagen.

Biochemical and morphologic changes in rabbit lung following endotracheal instillation of zymosan particles.

In order to establish a model of lung disease in which the usefulness of potential antiinflammatory compounds can be evaluated, we have analyzed the biochemical and cellular responses of rabbits to zymosan deposition in their lungs. A suspension of zymosan particles was instilled into the lungs of rabbits using an intratracheal catheter. Because the influx of leukocytes and the transudation of plasma into affected lungs was expected to contribute to the total cellular enzyme and protein levels, lungs were homogenized and assayed after various time intervals for six cellular enzymes and for protein content.

Degranulation, membrane addition, and shape change during chemotactic factor-induced aggregation of human neutrophils.

Neutrophils stimulated by the chemotactic factor formyl-methionyl-leucyl-phenyl-alanine (FMLP) undergo a transient change in surface properties that permits the cells to adhere more readily to surfaces and to each other. This transient change can be monitored by light scattering as stimulated neutrophils form aggregates while stirred in a platelet aggregometer. Maximum change in light scattering occurs within 1 min and correlates with an increase in the percentage of cells that are in aggregates of four or more cells and a decrease in the percentage of single cells.

Superoxide anion generation by human neutrophils exposed to monosodium urate.

We studied the capacities of naked and protein-coated monosodium urate (MSU) crystals to stimulate superoxide anion (O(2)) release by human polymorphonuclear leukocytes (PMN). Uncoated MSU estimated significant O(2) production by cytochalasin B-treated PMN. Precoating MSU with IgG caused an increase in mean O(2) production, whereas precoating heated MSU with serum or plasma inhibited O(2) release.