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Hemolymph coagulation is vital for larval hemostasis and important in immunity, yet the molecular basis of coagulation is not well understood in insects. Of the larval clotting factors identified in Drosophila, Fondue is not conserved in other insects, but is notable for its effects on the clot's physical properties, a possible function in the cuticle, and for being a substrate of transglutaminase. Transglutaminase is the only mammalian clotting factor found in Drosophila, and as it acts in coagulation in other invertebrates, it is also likely to be important in clotting in Drosophila. Here we describe a Fondue-GFP fusion construct that labels the cuticle and clot, and show that chemical inhibition and RNAi knockdown of the Drosophila transglutaminase gene affect clot properties and composition in ways similar to knockdown of the fon gene. Thus, Fondue appears to be incorporated into the cuticle and is a key transglutaminase substrate in the clot. This is also the first direct functional confirmation that transglutaminase acts in coagulation in Drosophila.

Matrix metalloproteinase-13 (MMP13) is a Zn(2+)-dependent protease that catalyzes the cleavage of type II collagen, the main structural protein in articular cartilage. Excess MMP13 activity causes cartilage degradation in osteoarthritis, making this protease an attractive therapeutic target. However, clinically tested MMP inhibitors have been associated with a painful, joint-stiffening musculoskeletal side effect that may be due to their lack of selectivity. In our efforts to develop a disease-modifying osteoarthritis drug, we have discovered MMP13 inhibitors that differ greatly from previous MMP inhibitors; they do not bind to the catalytic zinc ion, they are noncompetitive with respect to substrate binding, and they show extreme selectivity for inhibiting MMP13. By structure-based drug design, we generated an orally active MMP13 inhibitor that effectively reduces cartilage damage in vivo and does not induce joint fibroplasias in a rat model of musculoskeletal syndrome side effects. Thus, highly selective inhibition of MMP13 in patients may overcome the major safety and efficacy challenges that have limited previously tested non-selective MMP inhibitors. MMP13 inhibitors such as the ones described here will help further define the role of this protease in arthritis and other diseases and may soon lead to drugs that safely halt cartilage damage in patients.

Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution.

Hemolymph coagulation is a first response to injury, impeding infection, and ending bleeding. Little is known about its molecular basis in insects, but clotting factors have been identified in the fruit fly Drosophila melanogaster. Here, we have begun to study coagulation in the aquatic larvae of the malaria vector mosquito Anopheles gambiae using methods developed for Drosophila. A delicate clot was seen by light microscopy, and pullout and proteomic analysis identified phenoloxidase and apolipophorin-I as major candidate clotting factors. Electron microscopic analysis confirmed clot formation and revealed it contains fine molecular sheets, most likely a result of lipophorin assembly. Phenoloxidase appears to be more critical in clot formation in Anopheles than in Drosophila. The Anopheles larval clot thus differs in formation, structure, and composition from the clot in Drosophila, confirming the need to study coagulation in different insect species to learn more about its evolution and adaptation to different lifestyles.

Hemolectin has been identified as a candidate clotting factor in Drosophila. We reassessed the domain structure of Hemolectin (Hml) and propose that instead of C-type lectin domains, the two discoidin domains are most likely responsible for the protein's lectin activity. We also tested Hml's role in coagulation and immunity in Drosophila. Here we describe the isolation of a new hml allele in a forward screen for coagulation mutants, and our characterization of this and two other hml alleles, one of which is a functional null. While loss of Hml had strong effects on larval hemolymph coagulation ex vivo, mutant larvae survived wounding. Drosophila thus possesses redundant hemostatic mechanisms. We also found that loss of Hml in immune-handicapped adults rendered them more sensitive to Gram(-) bacterial infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.

Objective: To characterize the clinical and histopathologic changes in a rat model of broad-spectrum matrix metalloproteinase (MMP)-induced musculoskeletal syndrome (MSS), and to facilitate research into the causes and treatments of MSS in humans., Methods: Male Lewis rats weighing 150-180 gm were administered 10-30 mg of the broad-spectrum MMP inhibitor marimastat over a 2-week period via surgically implanted subcutaneous osmotic pumps. The animals were monitored and scored for the onset and severity of MSS, using clinical and histologic parameters., Results: Marimastat-treated rats exhibited various clinical signs, including compromised ability to rest on their hind feet, high-stepping gait, reluctance or inability to move, and hind paw swelling. Histologically, marimastat-treated rat joints were characterized by soft tissue and bone changes, such as increased epiphyseal growth plate, synovial hyperplasia, and increased cellularity in the joint capsule and extracapsular ligaments. The severity of MSS, as judged by clinical criteria (2 blinded observers using 3 clinical parameters), paw volume, and histologic score, was nearly identical. The observed changes were indistinguishable from those reported for primate models and mimic MSS in humans., Conclusion: This simple and sensitive model of MSS is an attractive alternative for studying the pathology of MSS.

Expression of the COX-2 enzyme has been reported in animal models of inflammatory bowel disease (IBD) as well as in patients affected by ulcerative colitis and Crohn's disease. Recently, selective inhibitors of COX-2 have become available. In this study we have evaluated three highly selective COX-2 inhibitors, NS-398, SC-58125 and PD-138387, on the trinitro-benzene sulfonic acid (TNBS) model of colitis in rats. Daily oral administration of the three compounds evaluated up to a dose of 100 mg/kg failed to significantly modify any of the parameters evaluated. Our data show that despite their potent extraintestinal antiinflammatory activity, COX-2 inhibitors do not seem to have any beneficial effect in TNBS colitis and raise the question whether this therapeutic approach would be beneficial in patients with IBD.

Nonsteroidal anti-inflammatory drugs often cause development of significant GI lesions. Selective inhibitors of prostaglandin G/H synthase/cyclooxygenase-2 (PGHS-2) enzyme and some dual inhibitors of PGHS/5-lipoxygenase (5-LO) enzymes have been reported to be potent anti-inflammatory compounds that carry a much lower risk of having GI irritating effects. We have evaluated the anti-inflammatory effect and the GI safety profile of three new anti-inflammatory compounds: the selective PGHS-2 inhibitors NS-398 and PD 138387 and the PGHS/5-LO dual inhibitor PD 137968. All the compounds tested showed an anti-inflammatory activity in the carragenan footpad edema test in rats. None of these compounds caused either gastric damage 4 h after p.o. administration of 100 mg/kg in rats or inhibition of PGE2 synthesis in the stomach. However, when administered p.o. at an effective anti-inflammatory dose to rats with pre-existing acetic acid-induced gastric ulcer, NS-398 caused a statistically significant delay of ulcer healing. No impairment of the ulcer healing was observed with the other compounds evaluated. Derivatives of 2,6-di-tert-butylphenol, whose members may act as PGHS-1/PGHS-2 inhibitors, selective PGHS-2 inhibitors or PGHS/5-LO dual inhibitors, are novel anti-inflammatory compounds that are devoid of GI irritating effects and do not affect the rate of pre-existing gastric ulcer healing.

Background & Aims: The mechanism by which gastric mucosa becomes more resistant to damage by repeated aspirin administration is not known. Transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) prevent drug-induced gastric injury. The aim of this study was to determine whether gastroduodenal tissue levels of TGF-alpha and EGF protein were altered during adaptation to aspirin-induced injury in monkeys and rats in vivo., Methods: Animals were given aspirin daily for up to 28 days. Gross mucosal injury was assessed by computerized image analysis in rats and by endoscopy in monkeys. Mucosal concentrations of TGF-alpha and EGF were quantitated by radioimmunoassays from endoscopic biopsy samples in monkeys and from scraped mucosa in rats., Results: Long-term administration of aspirin caused a significant increase in gastric and duodenal tissue levels of TGF-alpha in monkeys and rats; the increased levels of TGF-alpha significantly correlated with the decrease in aspirin-induced injury. No change in the gastroduodenal tissue levels of EGF was observed. Adaptation was not associated with any significant change in basal gastric acid secretion in monkeys and occurred despite a significant decrease in gastric mucin in rats., Conclusions: Adaptation of the gastric mucosa to the damaging effect of aspirin is associated with a significant and specific increase in TGF-alpha protein in the gastroduodenum.

The effect of transforming growth factor-alpha was studied in histamine-stimulated gastric acid secretion in monkeys and in pylorus-ligated rats. In monkeys, transforming growth factor-alpha given intravenously in a stepwise manner gradually reduced gastric acid secretion. A dose of 0.1 mmol/kg induced about a 50% inhibition of gastric acid output, while about 100% inhibition of the same parameter, was observed after a dose of 1 nmol/kg. Following a single administration of 1 nmol/kg intravenously in monkey, the maximum inhibitory effect on gastric acid output was reached 60 min after the administration of the drug, when a 91.8% inhibition was observed. Pretreatment values were reached 210-240 min after transforming growth factor-alpha administration. In pylorus-ligated rats, transforming growth factor-alpha, at doses of 9 and 18 nmol/kg produced an almost complete inhibition of gastric acid secretion. In this model, the ED50 was calculated to be 4.5 nmol/kg. Comparison with epidermal growth factor on the same models of gastric acid secretion showed that these two compounds have similar inhibitory potency. However, the effect of transforming growth factor-alpha on gastric acid secretion in monkeys was shorter lasting than that of epidermal growth factor.

The effect of human epidermal growth factor (h-EGF) and its derivative human epidermal growth factor 1-48 (h-EGF 1-48) on histamine-stimulated gastric acid secretion was studied in monkeys and rats. In monkeys, both h-EGF and h-EGF 1-48 given intravenously (i.v.) in a stepwise manner and in doses ranging from 0.01 to 10 nmol/kg, gradually suppressed gastric acid secretion. At the highest dose tested, both compounds essentially abolished gastric acid output. In the same animals, i.v. administration of 1 nmol/kg of h-EGF or h-EGF 1-48 caused an inhibition of gastric acid output that reached a peak at 90 and 60 min after the administration of h-EGF or h-EGF 1-48 respectively. After this maximum gastric inhibitory effect, a gradual return toward pre-injection values was observed. In rats, after subcutaneous (sc) administration, both h-EGF and h-EGF 1-48 dose-dependently inhibited histamine-stimulated gastric acid output as measured 60 min after the administration of the compounds. The maximum inhibitory activity on gastric acid output, observed at a dose of 100 nmol/kg, was 74.4% and 76.0% for h-EGF and h-EGF 1-48 respectively. The same dose of both compounds, however, failed to significantly inhibit gastric acid secretion when administered orally. In all the studies h-EGF 1-48 showed activity and potency comparable to h-EGF.(ABSTRACT TRUNCATED AT 250 WORDS)

The permeability of skin and oral mucosa to various compounds has been measured but the actual pathways substances take in traversing the epithelia have not been identified. In this study, radiolabelled cholesterol, ethanol or water were placed on the surface of porcine skin, keratinized gingiva, or nonkeratinized floor of mouth mucosa, and incubated at 37 degrees C for 2 h. The tissue was then snap-frozen, and sectioned in a cryostat, picked up on precoated slides and exposed at -20 degrees C for 40 days for light microscopic autoradiography. Some tissues were freeze-dried and directly embedded in low viscosity resin and prepared for electron microscopic autoradiography. Examination of autoradiographs revealed silver grains over, or adjacent to, intercellular spaces. Counts of the grains over the extra- and intracellular compartments were made in random light and electron microscope fields. For all compounds and tissue regions, there were significantly more (p less than 0.05) grains over the intercellular spaces than over the cells. The results indicate that the intercellular compartment is the predominant route for compounds moving across the superficial barrier layer of epidermis and oral epithelia. The nature of the intercellular material is, thus, a primary determinant of epithelial permeability.