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Thirty hours after infection, the medium surrounding amnions infected with AdMMP-9 contained a prominent proMMP-9 signal detected as a 92-kDa lysis band by zymog-raphy and an 83-kDa band beneath it, reflecting processed proenzyme (Fig. 4), results that are consistent with transduction of cultured cells with this vector. MMP-2 activity was present in the media from cultures of both AdLacZ- and AdMMP-9-infected amnions at approximately equivalent levels. Amnions infected with AdLacZ stained uniformly for p-galactosidase activity, reflecting efficient gene transfer, whereas amnions infected with AdMMP-9 did not stain for the presence of p-galactosidase. ampicillin antibiotic
The AdMMP-9-infected amnions were characterized by a ragged appearance and detachment of cells from the membranes, histological features that are found in amnions collected on Days 20 and 21 of pregnancy (Fig. 5). AdLacZ-infected amnions were relatively intact, with normal cellularity and without evidence of cytopathic effect. Evidence for increased collagen breakdown in AdMMP-9-infected amnions was obtained from measurement of hy-droxyproline in the conditioned medium. Medium from cultures of amnions infected with AdMMP-9 contained greater than 4-fold more hydroxyproline than did medium from cultures of AdLacZ-infected amnions (p < 0.01 by Student’s f-test) (Fig. 4).
AdMMP-9 infection resulted in a marked increase in apoptosis, assessed by immunohistochemical staining for nuclear DNA fragmentation (Fig. 4). In the AdMMP-9-in-fected tissues, 6.3 ± 1.1% of the cells were undergoing apoptosis (mean ± SD, n = 3 cultures per group) in the experiment shown in Figure 5, a finding that is similar to what we have observed in amnions collected on Day 21 of pregnancy. In contrast, in the AdLacZ-infected amnions, 0.4 ± 0.2% of the cells were apoptotic (significantly different from cells in AdMMP-9-infected amnions; p < 0.01 by Student’s £-test).
The effects of AdMMP-9 infection on amnion structure and amnion cell apoptosis were blocked by the MMP inhibitor, batimastat. Batimastat treatment retained amnion membrane integrity, reduced the release of hydroxyproline-containing peptides into the culture fluid, and completely prevented amnion cell apoptosis (Fig. 5).
FIG. 4. Infection of rat amnions in organ culture with recombinant adenoviruses. A) p-Galactosidase activity was detected in amnions infected with AdLacZ but not AdMMP-9. B) High levels of MMP-9 were detected in the media (1 ^l/lane) surrounding amnions infected with AdMMP-9 but not AdLacZ. C) AdMMP-9 infection increased release of collagen breakdown products measured as hydroxyproline. Relative hydroxyproline levels, expressed as absorbance at 550 nm for a 50-^l aliquot of unconcentrated conditioned medium, are presented. Values are means ± SD for three infections. Histological appearance of AdLacZ- (D) and AdMMP-9-infected (E) amnions (X400). AdMMP-9-infected amnions had a ragged appearance and marked cell detachment. Amnion cells of AdLacZ-infected amnions (D) were intact and did not show extensive nuclear DNA fragmentation (F). AdMMP-9-infected amnions (G) showed extensive apoptosis detected by immunohistochemical methods. Brown-stained nuclei (arrowheads) contained fragmented DNA. (Original photographs at X630).
FIG. 5. Amnion cell apoptosis in AdMMP-9-infected amnion cultures was inhibited by batimastat. Some cultures were treated with batimastat (3 ^M) or the DMSO vehicle. Zymographic studies on the culture fluid documented expression of MMP-9 in all AdMMP-9-infected cultures and the absence of MMP-9 in cultures infected with AdLacZ. Values presented are means ± SD for triplicate organ cultures with the exception of the hydroxyproline measurements for the AdLacZ + DMSO and AdLacZ + batimastat groups, for which the means of duplicate determinations are shown. The percentage of apoptotic cells in the AdMMP-9 + DMSO treatment group is significantly different from that of all other treatment groups (p < 0.01, Student-Neuman-Keuls test).
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