The effect of an inhibitor of matrix metalloproteinases on colonic inflammation in a trinitrobenzenesulphonic acid rat model of inflammatory bowel disease

BACKGROUND: Recent publications have reported that matrix metalloproteinases (MMPs) are expressed in colonic tissue taken from ulcerative colitis and Crohn's disease patients. AIM: To evaluate the effects of a matrix metalloproteinase inhibitor, marimastat, on colonic inflammation in experimental colitis induced by trinitrobenzenesulphonic acid (TNBS)-ethanol in the rat. METHODS: Rats were dosed (by mouth) for 7 days (b.d.) with either sulphasalazine (50 mg/kg), marimastat (40 mg/kg) or vehicle. TNBS-ethanol was administered rectally on the 4th day of dosing. On the last day of dosing, colons were removed and assessed for inflammation using myeloperoxidase activity, production of soluble TNFalpha (tumour necrosis factor alpha), clinical score and histological assessment. In addition, the bioavailability and effect of marimastat on a range of MMPs were assessed in-vitro. RESULTS: In this study we have confirmed that marimastat is a broad spectrum MMPI with a bioavailability of 5%. TNBS rats dosed with sulphasalazine had a significantly lower (P < 0.05) myeloperoxidase activity, TNFalpha production and a markedly lower clinical score. Similarly, rats dosed with marimastat had a significantly lower (P < 0.05) myeloperoxidase activity and clinical score, but the TNFalpha production was not significantly reduced. CONCLUSIONS: Dosing rats with TNBS-induced colitis using sulphasalazine or marimastat produced a significant reduction in tissue injury and inflammation.     

Pathogenic factors in bronchopulmonary dysplasia

Serum factors related to oxygen exposure were studied in 56 full-term cord blood samples and in 69 newborn infants of varying gestational age (GA). Serum malondialdehyde (MDA), which reflects membrane lipid peroxidation, was elevated during the first 2 d of life and rose to a peak at 3-5 d of life. This peak value was unrelated to GA or to assisted ventilation. The serum antioxidant, vitamin E, showed a significant rise by 6-10 d, and came into the adult range after d 11. Vitamin E levels did not correlate with GA, assisted ventilation, or the development of bronchopulmonary dysplasia (BPD). Serum ceruloplasmin, another antioxidant, was measured both by activity assay and by protein concentration assay. Little activity was found in cord blood. Ceruloplasmin activity increased during the first 48 h of life, and both activity and protein concentration correlated with GA at that time. Infants who subsequently developed BPD had a less active protein than infants on ventilators who did not develop BPD. In addition, activity and protein levels on 3-5 d were lower in infants on ventilators than in those not requiring assisted ventilation. Serum levels of alpha-1-AP activity and protein concentration were also correlated with GA during the first 48 h of life. The less mature infants had levels of activity and protein which were significantly less than the more mature infants and significantly less than the full-term cord values. The proportion of active protein correlated with GA at 3-5 d, indicating that the less mature infants had a lower proportion of active protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of suppressor T cells for antibody production by chicken spleen cells. II. Comparison of CT8+ cells from concanavalin A-injected normal and bursa cell-injected agammaglobulinaemic chickens.

The phenotypes of two different types of suppressor T cells in the chicken, both capable of inhibiting secondary antibody responses in vitro, were determined. The first of these, induced by injection of concanavalin A (Con A) into normal chickens, was CT8+, TcR2+ (alpha beta), CT4-, TcR1- (gamma delta). These cells appeared to exhibit histamine type 2 (H2) receptors, as they adhered to cimetidine-BSA-coated dishes. Moreover, cimetidine added to the medium at 2 x 10(-4) M completely prevented the suppression induced by these suppressor cells. The second type of 'suppressor' T-cell studied, induced in agammaglobulinaemic (A gamma) chickens by injection of bursa cells, exhibited the same phenotype, but was insensitive to cimetidine and did not adhere to cimetidine-BSA-coated dishes, indicating heterogeneity with respect to H2 receptor expression on CT8+ chicken T cells with suppressor activity. The results also showed that a relatively larger proportion of CT8+ than of CT4+ cells adhered to cimetidine-BSA-coated dishes and thus appeared to be H2 receptor positive. TcR1 (gamma delta) cells did not contribute significantly to the antigen non-specific suppressor effects examined in this study.     

Effects of the protein kinase A inhibitor H-89 on Ca2+ regulation in isolated ferret ventricular myocytes

 We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca²⁺ regulation in Fura-2-loaded ferret myocytes. H-89 (10 μmol/l) decreased the amplitude of the Fura-2 transient to 28.2±4.3% (P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca²⁺ to diastolic levels: t _1/2 increased from 311±35 ms to 547±43 ms (P<0.001, n=7). Reduced Ca²⁺ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca²⁺ content, as assessed with caffeine. The apparent slowing of the SR Ca²⁺-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca²⁺ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca²⁺-ATPase. In electrophysiological experiments, 5.0 μmol/l H-89 decreased the L-type Ca²⁺ current (I _Ca) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the resting membrane potential. In summary, data show that [Ca²⁺ ]_i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of β-agonists. However, the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the SR Ca²⁺-ATPase. Received: 4 September 1998 / Received after revision: 19 October 1998 / Accepted: 20 October 1998     

Production of mycoplasma-specific antisera in rabbits immunologically tolerized at birth to mycoplasma medium constituents

Neonatal rabbits were injected intraperitoneally (i.p.) within 12 h of birth followed by similar injections every day for 10 consecutive days and then every second day for a further 8 weeks, with mycoplasma broth medium (tolerogen), to induce immune tolerance. The rabbits were then immunized with the porcine mycoplasmas, M. hyopneumoniae or M. hyorhinis at 9 weeks of age. Immune sera obtained from these rabbits and from normal control rabbits were tested for antibodies against both mycoplasma antigens and for antibodies to medium components by double immunodiffusion in agarose and by enzyme-linked immunosorbent assay (ELISA). Antisera obtained from the tolerized rabbits contained no antibodies to medium components as evidenced by lack of reactivity in both assays. In immunofluorescence tests the antisera obtained from tolerized rabbits permitted specific staining of colonies of the homologous mycoplasma grown on mycoplasma agarose medium. In contrast the antisera obtained from normal rabbits produced strong reactions in all of the tests and non-specific background fluorescence due to reactions with components of the culture medium.     

Characterization and vaccine potential of a novel recombinant coccidial antigen.

A cDNA clone derived from sporulated oocysts of Eimeria tenella and encoding the expression product GX3262 was identified using a monoclonal antibody raised against Eimeria acervulina sporozoites. The cDNA fragment containing the coccidial antigen gene was cloned in bacteriophage lambda gt11, transferred to a plasmid, and introduced into Escherichia coli for analysis of the gene products. The strain carrying the plasmid produced GX3262 as part of a fusion protein consisting of the first 1,006 amino acids of E. coli beta-galactosidase and 112 amino acids of the E. tenella protein of approximately 12 kilodaltons. Partially purified antigen, heat-killed recombinant bacterin, and live E. coli containing the recombinant coccidial antigen were used to immunize 1-week-old or newly hatched broiler chicks. Several immunization protocols were utilized, including boosts with partially purified beta-galactosidase-GX3262, bacterin, or small numbers of live E. tenella oocysts. After challenge with an experimental E. tenella infection, the birds were evaluated by scoring cecal lesions to determine the level of protection. The greatest degree of protection was seen after only a single immunization of 2-day-old birds with a live recombinant E. coli preparation. The results presented here identify GX3262 as a potential candidate coccidial vaccine antigen and provide evidence for the first time that newly hatched chickens can be successfully vaccinated with a recombinant antigen.     

The role of macrophages in the removal of apoptotic B-cells in the sheep ileal Peyer's patch

In the process of generating the cells that populate the sheep's B-cell pool, the ileal Peyer's patch (PP) produces an immense number of B-cells and then destroys most of them by apoptosis. Rapid clearance of these apoptotic cells is essential for tissue homeostasis and for preventing pathology. Macrophages comprise a small percentage of cells in the follicles. They resemble macrophages found in other tissues and can be identified by the expression of MHC Class II and CD14. In this study, enriched macrophages co-cultured with apoptotic ileal PP cells showed increased DNA content as they ingested apoptotic cells. The higher the proportion of apoptotic cells in culture the greater the increase in DNA content of the macrophages. This occurred when B-cell apoptosis was initiated by a period in culture or in response to treating the animals with steroids. Thus, macrophages resident in the ileal PP follicle mediate the phagocytosis and removal of discarded B-cells.