Method to analyze collagenase and gelatinase activity by fibroblasts in culture.

The net amount of collagen produced and deposited by fibroblasts in cell culture is determined by the rate of collagen synthesis as well as the rate of collagen degradation. Although collagen synthesis can be analyzed by several techniques, it is more difficult to measure collagen degradation. Breakdown of collagen depends upon the activity of a family of structurally and catalytically related mammalian enzymes termed matrix metalloproteinases (MMPs). Interstitial collagenase (MMP1) initiates the cleavage of fibrillar collagen, whereas gelatinases (MMP2 and MMP9) digest the denatured collagen fragments. A method has been developed to quantitate the activity of collagenase (MMP1) and gelatinase (MMP9) in conditioned medium from fibroblast cell cultures. The assay, which uses the fluorogenic substrate Dnp-Pro-Cha-Gly-Cys(Me)-His-AlaLys(Nma)NH2, is technically simple and amenable to high throughput analysis. Addition of specific inhibitors of the metalloproteinases allows for simultaneous measurement of both collagenase and gelatinase activity.

In vitro analysis of fetal fibroblast collagenolytic activity.

Tissue repair in the rabbit fetus is remarkably rapid and occurs without significant inflammation or excessive collagen deposition. The objective of this study was to compare the ability of rabbit fetal and adult fibroblasts to express the matrix metalloproteinases which are thought to be critical to scar tissue remodeling. In vitro, both fetal and adult rabbit fibroblasts express procollagenase messenger RNA in a constitutive manner. Mechanical disruption of fetal fibroblast monolayers caused a twofold increase in procollagenase mRNA. In contrast, the adult rabbit fibroblast procollagenase mRNA remained unchanged. The mRNA data correlated well with enzyme protein levels. Quantitation by immunoprecipitation showed a 2.3-fold increase in fetal fibroblast procollagenase protein after mechanical injury, whereas the level in adult rabbit fibroblasts remained unchanged. However, it was noted that the constitutive levels of procollagenase mRNA and protein were higher in adult fibroblasts. Analysis of enzyme activity, by means of a fluorogenic substrate, showed that adult fibroblasts had 2.2 times more collagenase activity compared with fetal cells. After mechanical injury, the fetal fibroblast collagenase activity increased 1.3-fold compared with 1.7-fold in the adult fibroblasts. In contrast, fetal fibroblast gelatinase activity was 1.25 times greater than in adult cells and increased 1.4-fold after mechanical injury, whereas the adult profile remained unchanged. Immunolocalization studies indicated that 1 hour after mechanical injury, procollagenase was produced primarily by fibroblasts along the mechanical injury ridge. By 4 hours after injury, the ridge cells began to migrate out into the open area and procollagenase was noted in adjacent cells of both adult and fetal origin. By 12 and 18 hours, all cells throughout the monolayer were expressing procollagenase. These findings show that in vitro fetal fibroblasts actually had lower levels of procollagenase, but higher levels of gelatinase compared with adult fibroblasts. The increased gelatinase expression may explain why fetal wounds do not have excessive collagen accumulation and heal without a visible scar.

Comparison of the polyvinyl alcohol sponge and expanded polytetrafluoroethylene subcutaneous implants as models to evaluate wound healing potential in human beings.

Our current understanding of the complex processes involved in wound healing is based mainly on studies of animal models. Although this information has been useful, it may not totally reflect the response found in human beings. For example, human beings have a tendency to either "overheal," as seen in keloids and hypertrophic scar formation, or have deficient healing, as seen in chronic ulcer formation. No animal models are available to analyze these human clinical pathologic conditions. Therefore the objective of this study was to analyze the wound healing response in a large population (n = 40) of normal healthy human beings as a first step to begin studies of abnormal human wound healing. Simultaneously, a comparison was made between the polyvinyl alcohol implant and the expanded polytetrafluoroethylene implant model. Under sterile conditions with the use of local anesthesia, two preweighed polyvinyl alcohol implants and two standard 6 cm expanded polytetrafluoroethylene implants were placed subcutaneously in the upper arm of each subject. High-performance liquid chromatography was used to quantitate isoleucine and hydroxy-l-proline in acid hydrolysates of each implant. Isoleucine was used as an indicator of protein content in the tissue sample, whereas hydroxyproline reflected collagen content. No infectious or hemorrhagic complications were found in the 40 volunteers included in the study. No significant difference was found in isoleucine or hydroxy-l-proline content between postoperative day 7 polyvinyl alcohol implants and day 14 polyvinyl alcohol implants. In contrast, both isoleucine and hydroxy-l-proline content were significantly increased in day 14 expanded polytetrafluoroethylene implants compared with day 7 implants (p < 0.005 and p < 0.001, respectively). In addition, the ratio of hydroxy-l-proline to isoleucine was significantly increased in day 14 expanded polytetrafluoroethylene implants compared with day 7 expanded polytetrafluoroethylene and both day 7 and day 14 polyvinyl alcohol implants (p < 0.001). This observation suggests that by 14 days implantation of expanded polytetrafluoroethylene stimulated an increased deposition of collagen. No significant differences were found in the hydroxy-l-proline to isoleucine ratios among day 7 expanded polytetrafluoroethylene, day 7 polyvinyl alcohol, and day 14 polyvinyl alcohol implants. Histologic analyses correlated with the biochemical findings. These results suggest that expanded polytetrafluoroethylene may be the preferred implant for studies designed to examine pathologic processes associated with retarded wound healing. In contrast, the polyvinyl alcohol implant may be better suited for studies where a low background response is required. Moreover, the extreme variability in normal healthy volunteers seen in this study correlates clinically with the finding that, among the normal adult human population, there is a heterogeneous wound healing response.

Norfloxacin: a three-day course for the treatment of urinary tract infection.

A total of 102 patients with urinary tract infection were treated with 400 mg norfloxacin twice daily for three days. Midstream specimens of urine were cultured at the initial visit and also one and five weeks after treatment. Forty-eight out of 50 patients (96%) with significant bacteriuria were successfully treated and remained free from recurrences during the follow-up period. There was no evidence of serious toxicity and only minor adverse symptoms were recorded.