Chronic leg wounds are characterized by defective remodeling of the extracellular matrix, failure of reepithelialization, and prolonged inflammation. The hypothesis that this defective extracellular matrix remodeling is associated with phenotypic differences in the activity of the matrix metalloproteinases and tissue inhibitors of metalloproteinases was studied in chronic wound and patient-matched normal fibroblasts in three-dimensional collagen lattice systems. Chronic wound fibroblasts exhibited no differences in morphology or proliferation (p > 0.1) compared with patient-matched uninvolved dermal fibroblasts. The ability of chronic wound fibroblasts to reorganize extracellular matrix was significantly impaired, however, in comparison to the uninvolved dermal fibroblasts (p < 0.01). This difference in extracellular matrix reorganization was not related to differences in proliferation within the collagen lattices (p > 0.05) or attachment to type I collagen (p > 0.1). Marked differences were evident in matrix metalloproteinase-2 activity between chronic wound and patient-matched normal fibroblasts. Whereas levels of pro-matrix metalloproteinase-2 were similar between the two fibroblast populations (p > 0.1), the chronic wound fibroblasts exhibited significantly decreased levels of the 62 kDa active form of matrix metalloproteinase-2 (p < 0.01). Reverse zymography and enzyme-linked immunosorbent assay demonstrated that the decreased matrix metalloproteinase-2 activity was associated with increased production of tissue inhibitors of metalloproteinase-1 and -2 by the chronic wound fibroblasts (p < 0.05). Increased production of tissue inhibitors of metalloproteinases in chronic wound fibroblasts was also reflected in decreased levels of matrix metalloproteinase-1 (p < 0.005). These data suggest that the impaired ability of chronic wound fibroblasts to reorganize extracellular matrix in vitro is related to decreased levels of active matrix metalloproteinase-2 and matrix metalloproteinase-1 resulting from increased production of tissue inhibitors of metalloproteinase-1 and -2 by chronic wound fibroblasts. These findings provide a mechanism to explain the impaired cellular responses and extracellular matrix reorganization observed in chronic leg wounds in vivo.
Intra-oral wounds, like wounds in children, demonstrate privileged healing when compared with adult wounds at extra-oral sites. This study investigated whether this preferential healing is related to an increased ability of oral mucosal fibroblasts to reorganize extracellular matrix (ECM) when compared with their dermal counterparts. ECM reorganization was investigated by means of a fibroblast-populated collagen lattice (FPCL) system. The effect of donor age was also investigated in this system. Differences in ECM reorganization and FPCL contraction were evident: FPCL contraction was more rapid by oral mucosal fibroblasts than dermal fibroblasts (p < 0.01). FPCL contraction was also greater in child (donor < 10 years) than adult (donor > 18 years) oral mucosal fibroblasts (p < 0.01). These differences were not related to phenotypic differences in cell viability (p > 0.5), DNA synthesis (p > 0.05), and cell number (p > 0.5) within the FPCLs, or cellular attachment to collagen (p > 0.07). FPCL contraction was not stimulated by the addition of conditioned medium from oral mucosal or dermal fibroblasts (p > 0.05). These data show that the significantly increased ability of oral mucosal fibroblasts to reorganize ECM in vitro, when compared with dermal fibroblasts, represents a distinct phenotypic contractile difference, rather than differences in their production of soluble mediators or cell attachment to ECM.
Denitrification by Paracoccus denitrificans and Pseudomonas aeruginosa was studied using quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously dissolved gases. Evidence was provided for aerobic denitrification by both species: in the presence of O2, N2O production increased in Pa. denitrificans, while that of N2 decreased; with Ps. aeruginosa, the concentrations of both N2 and N2O increased on introducing O2 into the gas phase. Disappearance of NO-3 was monitored in anaerobically and aerobically grown cells which were maintained either anaerobically or aerobically: the rate and extent of NO-3 utilization by both species depended on growth and maintenance conditions. The initial rate of disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred when cells were grown anaerobically and maintained aerobically. In nitrogen balance experiments both species converted over 87% of the added NO-3 to N2 and N2O under both anaerobic and aerobic maintenance conditions.
Bacterial decomposition of nitrate to dinitrogen and oxides of nitrogen, essential steps in the nitrogen cycle, are regarded as predominantly anaerobic processes. However, here we have shown, using simultaneous mass spectrometric monitoring of dissolved N2, NOx and O2, that in the laboratory a number of different bacteria can denitrify, even when O2 concentrations approach or exceed air saturation values. The proportions of gaseous end products vary from one organism to another and depend on the level of dissolved O2. We, therefore, suggest that aerobic bacterial denitrification, with the production of N2 and/or NOx, may, contrary to the beliefs of many, be as widespread and ecologically important as its anaerobic counterpart.
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