Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene.

Impaired fibrinolytic activity within the lung is a common manifestation of acute and chronic inflammatory lung diseases. Because the fibrinolytic system is active during repair processes that restore injured tissues to normal, reduced fibrinolytic activity may contribute to the subsequent development of pulmonary fibrosis. To examine the relationship between the fibrinolytic system and pulmonary fibrosis, lung inflammation was induced by bleomycin in transgenic mice that either overexpressed or were completely deficient in murine plasminogen activator inhibitor-1 (PAI-1). 2 wk after 0.075 U of bleomycin, the lungs of transgenic mice overexpressing PAI-1 contained significantly more hydroxyproline (118 +/- 8 micrograms) than littermate controls (70.5 +/- 8 micrograms, P < 0.005). 3 wk after administration of a higher dose of bleomycin (0.15 U), the lung hydroxyproline content of mice completely deficient in PAI-1 (49 +/- 8 micrograms) was not significantly different (P = 0.63) than that of control animals receiving saline (37 +/- 1 micrograms), while hydroxyproline content was significantly increased in heterozygote (77 +/- 12 micrograms, P = 0.06) and wild-type (124 +/- 19 micrograms, P < 0.001) littermates. These data demonstrate a direct correlation between the genetically determined level of PAI-1 expression and the extent of collagen accumulation that follows inflammatory lung injury. These results strongly support the hypothesis that alterations in fibrinolytic activity influence the extent of pulmonary fibrosis that occurs after inflammatory injury.

Pulmonary inflammation induced by incomplete or inactivated adenoviral particles.

One of the major obstacles to pulmonary-directed gene therapy using adenoviral vectors is the induction of inflammation. We investigated whether the adenoviral particles that constitute the initial inoculum can serve as an inflammatory stimulus, independent of their ability to express genes that they contain. Viral particles were prepared that are defective in gene expression by (i) isolating particles that have incomplete genomes by selecting those that have buoyant densities on CsCl density gradients lighter than complete viruses; and (ii) cross-linking viral DNA by exposure to ultraviolet light in the presence of 8-methoxypsoralen. The defective particles retained their icosahedral appearance when viewed by electron microscopy but lost their plaque-forming ability on 293 cells. High doses of intact, incomplete, or inactivated viral particles were instilled intratracheally into CBA/J mice, and after 6 days the amount of inflammation was quantified by counting inflammatory cells contained within lung tissue. We found that the inflammatory responses induced by the incomplete or inactivated viral vectors were quantitatively similar to those caused by intact, competent viral vectors. We conclude that high doses of adenoviral vectors that are used for gene therapy can induce pulmonary inflammation, independent of expressing the genes they contain.

Expression of human interleukin-1 receptor antagonist in mouse lungs using a recombinant adenovirus: effects on vector-induced inflammation.

Pulmonary inflammation is a major obstacle to using adenovirus-based vectors for gene transfer to the lung. Since the pro-inflammatory cytokine, interleukin-1 (IL-1), is expressed early following adenovirus infection, we hypothesized that inhibition of IL-1 might block the inflammation caused by adenoviral vectors. To inhibit IL-1 activity at the site of infection continuously, we employed a recombinant adenovirus that contained the cDNA for human IL-1 receptor antagonist protein (IL-1ra) designated as Ad.RSVIL-1ra. When Ad.RSVIL-1ra was instilled intratracheally into CBA/J mice, human IL-1ra was recovered in lung tissue and bronchoalveolar lavage fluid for up to 30 days. Human IL-1ra is known to bind to murine IL-1 receptors and inhibit IL-1-mediated responses. To measure pulmonary inflammation, the number of inflammatory cells contained within suspensions of protease-digested lung tissue were counted 6 days after virus administration. Ad.RSVIL-1ra failed to reduce the number of inflammatory cells below that induced by a control vector that lacked an expression cassette (Ad.BgIII). Light microscopy showed that the lung tissue from Ad.RSVIL-1ra and Ad.BgIII-treated mice contained qualitatively similar amounts of inflammatory infiltrate. We conclude that adenovirus-based vectors can be used to induce high levels of IL-1ra expression within the lung, but such expression was unable to prevent adenoviral vector-induced inflammation.

Photosensitization associated with exposure to Pithomyces chartarum in lambs.

An epidemic of photosensitization was observed in a group of lambs on irrigated autumn pasture in western Oregon. Signs included crusting, necrosis, and sloughing of the skin over the nostrils, lips, and ears, and of the mucous membranes of the buccal regions. Microscopic examination of plant material from the pasture disclosed spores of Pithomyces chartarum. This fungus has been documented as a causal factor in photosensitization in sheep and cattle (facial eczema) in other parts of the world. An infective agent or other plant material that could have induced the clinical signs in the lambs was not evident. Weather and humidity conditions were ideal for fungal growth during the grazing period, and the fungus was detected in large numbers before and during the epidemic. Even though facial eczema has not been reported previously in northwestern United States, we feel the circumstances surrounding this epidemic warrant such a diagnosis.

Indolent presentation of pancreatic abscess. Experience with 100 cases.

One hundred cases of pancreatic abscess were identified at five hospitals affiliated with UCLA between 1973 and 1985. Patients were included if a pancreatic mass or phlegmon followed an episode of pancreatitis, if the clinical impression was pancreatic abscess, and if drainage cultures were positive. Less than three Ranson's signs were present on admission in 72% of patients. The admission temperature was less than 38.3 degrees C in 71% of patients, and 27% of patients never had a fever. Abdominal tenderness was absent in 40% of patients. The admission amylase concentrations and white blood cell counts were normal in 36% and 23% of patients, respectively. Extensive débridement, external drainage, and a low threshold for reoperation were the mainstays of surgical therapy. Twenty patients (20%) died, but Ranson's signs did not predict outcome. pancreatic abscess may have an insidious presentation. A high index of suspicion, early computed tomographic scanning, and diagnostic needle aspiration may be necessary to establish this diagnosis.

Angiographic treatment of gastrointestinal hemorrhage: comparison of vasopressin infusion and embolization.

The results of selective intraarterial vasopressin-infusion therapy and embolization therapy were compared in two groups of patients with major gastrointestinal hemorrhage. The site of bleeding, clinical course, complications, and transfusion requirements were evaluated in each group. Intraarterial vasopressin infusion therapy resulted in successful control of hemorrhage in 16 (70%) of 23 patients. Four patients, however, rebled and an operation was necessary, reducing the overall success rate to 52% (12 of 23). In the group treated with embolization therapy, primary success was achieved in 17 (71%) of 24 patients. Four patients in whom initial embolization failed to control bleeding underwent repeat embolization and in all four permanent control of hemorrhage was obtained, producing an overall success rate of 21 (88%) of 24. Analysis of our results according to site of hemorrhage suggests that at certain sites embolization is a preferred method of treatment; embolization allows earlier control of gastrointestinal hemorrhage and a reduction in transfusion requirements.

CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the biosynthesis of polysialosyl units in neural cell adhesion molecules.

Prokaryotic derived probes that specifically recognize alpha-2,8-ketosidically linked polysialosyl units were developed to identify and study the temporal expression of these unique carbohydrate moieties in developing neural tissue (Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C., and Troy, F. A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1971-1975). These polysialosyl units cap N-linked oligosaccharides of the complex-type on neural cell adhesion molecules (N-CAM). A Golgi-enriched fraction from 20-day-old fetal rat brain contains a membrane-associated sialyltransferase that catalyzes the incorporation of [14C]N-acetylneuraminic acid [( 14C]NeuNAc) from CMP-[14C] NeuNAc into polymeric products. At pH 6.0, 84 pmol of NeuNAc mg of protein-1 h-1 were incorporated. In sodium dodecyl sulfate-polyacrylamide gels, the major radiolabeled species migrated with a mobility expected for N-CAM. A bacteriophage-derived endoneuraminidase specific for polysialic acid was used to demonstrate that at least 20-30% of the [14C]NeuNAc was incorporated into alpha-2,8-linked polysialosyl units. This was confirmed by structural studies which showed that the endoneuraminidase-sensitive brain material consisted of multimers of sialic acid. The addition of a partially purified preparation of chick N-CAM to the membranous sialyltransferase stimulated sialic acid incorporation 3-fold. The product of this reaction was also sensitive to endoneuraminidase and contained alpha-2,8-linked polysialosyl chains, thus showing that N-CAM can serve as an exogenous acceptor for sialylation in vitro. Sialic acid incorporated into adult rat brain membranes was resistant to endoneuraminidase, indicating that the poly-alpha-2,8-sialosyl sialyltransferase activity is restricted to an early developmental epoch. It is recommended that the enzyme described here be designated CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the trivial name poly-alpha-2,8-sialosyl sialyltransferase be adopted.

Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes.

Three prokaryotic-derived probes to identify and study the temporal expression of polysialosyl units in neuronal tissue have been developed. A polyclonal antibody, a bacteriophage-derived endo-neuraminidase, and an Escherichia coli K1 sialyltransferase are all specific for either recognizing or synthesizing poly(sialic acid) containing alpha-2,8-ketosidic linkages. Polysialosyl immunoreactivity with apparent Mr values of 180,000-240,000 was specific for developing neuronal tissue; it was not detected in neonatal liver or kidney or in adult brain tissue. The developmentally regulated disappearance in poly(sialic acid) is consistent with the probes described here recognizing the polysialosyl carbohydrate units of a neuronal cell adhesion molecule (N-CAM). Treatment of brain extracts with a bacteriophage-derived endo-neuraminidase specific for alpha-2,8-linked polysialosyl units abolished the immunoreactivity. The material solubilized by endo-neuraminidase was isolated, reduced with borotritide, and shown to contain oligomers of sialic acid with three to six sialyl units. Treatment of the 3H-labeled oligosialic acid with exo-neuraminidase quantitatively converted the radioactivity to sialitol, establishing that the brain-derived oligomers were composed solely of sialic acid. A membranous sialytransferase from E. coli K1 that can transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid) also recognized rat brain membranes, further substantiating the presence of poly(sialic acid) in rat brain. This conclusion was confirmed by using a mutant of E. coli K1 that was defective in the synthesis of poly(sialic acid) and could only transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid). Sialyl polymer synthesis was restored in the mutant when brain membranes were added as exogenous acceptor.