Hemidesmosome formation in vitro.

Intact, viable sheets of adult rabbit corneal epithelium, 9 mm in diameter, were prepared by the Dispase II method (Gipson, I. K., and S. M. Grill, 1982, Invest. Ophthalmol. Vis. Sci. 23:269-273). The sheets, freed of the basal lamina, retained their desmosomes and stratified epithelial characteristics, but lacked hemidesmosomes (HD). Epithelial sheets were placed on fresh segments of corneal stroma with denuded basal laminae and incubated in serum-free media for 1, 3, 6, 18, or 24 h. Tissue was processed for electron microscopy, and the number of HD/micron membrane, the number of HDs with anchoring fibrils directly across the lamina densa from them, and the number of anchoring fibrils not associated with HDs were counted. After 6 h in culture, the number of newly formed HD was 82% of controls (normal rabbit corneas), and by 24 h the number had reached 95% of controls. At all time periods studied, 80-86% of HDs had anchoring fibrils directly across the lamina densa from them. Anchoring fibrils not associated with HDs decreased with culture time. These data indicate that the sites where anchoring fibrils insert into the lamina densa may be nucleation sites for new HD formation. Corneal epithelial sheets placed on two other ocular basal laminae, Descemet's membrane and lens capsule, had not formed HDs after 24 h in culture. These two laminae do not have anchoring fibrils associated with them. Rabbit epithelial sheets placed on the denuded epithelial basal lamina of rat and human corneas formed new HDs. Thus, at least in these mammalian species, HD formation may involve some of the same molecular components.

Isolation of corneal epithelium with Dispase II or EDTA. Effects on the basement membrane zone.

An ultrastructural and immunohistochemical comparison was made between the effects of Dispase II and EDTA on the basement membrane zone of corneal epithelium. The comparison was made on intact corneas as well as on freed epithelial sheets and remnant stromas that had been separated using either the enzyme or chelator. At the ultrastructural level, incubation with Dispase II disrupted the lamina densa allowing in situ blebbing of the basal cells. After separation of the epithelium and stroma, the epithelial sheets showed extensive blebs with extracellular matrix trapped between the blebs. The remnant stromas completely lacked lamina densa, but anchoring fibrils remained. Immunofluorescent studies with antibodies against laminin and BM-1 antigen (an antibody to the protein core of heparan sulfate proteoglycan) revealed that laminin antibody binding was present on the freed epithelial sheets and absent from the remnant stromas, whereas BM-1 antibody binding was absent from both the freed sheets and the remnant stromas after incubation with Dispase II. Incubation with EDTA did not disrupt the basal lamina. There was no in situ blebbing of the basal cells, but immediately after epithelial sheet removal, extensive blebbing occurred. Freed sheets lacked attached segments of extracellular matrix as seen with Dispase II treatment. Remnant stromas showed intact basal laminae and anchoring fibrils. Immunofluorescent studies revealed binding of both the laminin and BM-1 antibodies to the remnant stromas, but not to the freed epithelial sheets. Although EDTA removal of epithelial sheets gave a cleaner separation between basal cell membranes and the basal laminae, more basal cells were disrupted than after separation with Dispase II.

Transplant of corneal epithelium to rabbit corneal wounds in vivo.

Sheets of corneal epithelium removed from 9-mm buttons of adult rabbit corneas using Dispase II were placed on abraded (basement membrane intact) or keratectomized corneas of anesthetized rabbits. Both types of wounds extended from limbus to limbus. The host animals were maintained under deep anesthesia for 3 hr, during which time culture medium was dripped onto the surface of the transplant. A soft contact lens then was placed over the cornea and the eye bandaged shut. Short-term experiments indicated that after 24 hr the transplanted epithelium was adherent to both abraded and keratectomized corneas (n = 4). Hemidesmosomes had formed between basal cells of donor epithelium and denuded host basement membrane, and cytoplasmic blebs had extended from donor epithelium into host keratectomized stroma. Seven transplants to abraded corneas and 17 transplants to keratectomized corneas were followed for longer time periods. Six of the seven transplants to abraded corneas were maintained until termination of the experiment (four at 4 weeks, one at 2 weeks, one at 1 week). Three of the 17 transplants to keratectomized corneas were maintained until termination (one at 4 weeks, one at 2 weeks, and one at 6 days). The remaining 14 sloughed between days 2 and 6. These data indicate that it is feasible to transplant corneal epithelial sheets and that they can be maintained most successfully if the host basement membrane is present.

Hyperalimentation-associated neonatal cholestasis: effect of oral gentamicin.

The effect of oral gentamicin on the incidence of parenteral nutrition-associated cholestasis in preterm infants less than 1500 g birth weight was assessed retrospectively. Of 24 patients on parenteral nutrition for more than 10 days, 12 infants who received oral gentamicin (group I) for prophylaxis against neonatal necrotizing enterocolitis were compared to 12 infants who did not (group II). Both mean and peak direct bilirubin levels were significantly higher in group II. The increase in both mean and peak direct bilirubin levels after initiation of total parenteral nutrition (TPN) was significant in group II only. The incidence of cholestasis was significantly higher in group II than in group I. These results suggest that oral gentamicin may have a protective effect against parenteral nutrition-associated cholestasis in the newborn preterm infant.

Bowel function of long-term tube-fed patients consuming formulae with and without dietary fiber.

Diarrhea, constipation and subsequent laxative use are chronic problems in long-term enterally fed patients. We have conducted a double-blind randomized crossover study to evaluate the effects of two enteral formulae (Enrich, 12.8 g of dietary fiber per 1000 kcal and Ensure, fiber-free) on stool frequency, fecal weight, laxative use, gastrointestinal tolerance and bowel function in chronic care tube-fed patients. Twenty-eight subjects (24 male, 4 female) completed the study. Mean daily number of stools and mean daily fecal wet weight in Enrich-fed patients were not significantly different from those of patients receiving Ensure. Ensure-fed patients required significantly more laxatives (p = 0.02) than those receiving Enrich. There were 26 reports of diarrhea in the Ensure-fed group as compared to 6 in the Enrich-fed group, and this difference was significant (p = 0.006). Reporting rates for constipation were not significantly different in the two groups. At the end of the study, the bowel function of 57.1% of patients receiving Enrich was improved when compared with that of 14.3% of Ensure-fed patients, and this difference was significant (p = 0.005). These results suggest that the addition of dietary fiber to enteral formulae improves gastrointestinal tolerance and bowel function, and reduces laxative use in long-term enterally fed patients.

Alcoholic beverage server liability and the reduction of alcohol-involved problems.

Legal liability of alcoholic beverage servers has been suggested as a means to stimulate preventive serving practices and thus reduce alcohol-involved problems. A number of variables contribute (both negatively and positively) to the potential of such liability and a conceptual model that links these variables was developed. In this project, an expert legal panel was used to identify and rate the major legal factors contributing to server liability. As a result each state was ranked according to its relative level of liability exposure. States that ranked highest in server liability were found to have more publicity about such liability, greater awareness and higher concern among licensed establishment owners/managers and different serving practices compared to states with lowest liability exposure. As a result we conclude that server liability has a real potential for reducing alcohol-involved problems but additional research is needed.

The proposed market for human organs.

During the last decade there have been enormous advances in the transplantation of vital human organs--in particular, the kidney, lung, heart, liver, pancreas, and small intestine. Unfortunately, efforts to provide the benefits of these operations to patients have been severely hindered by limitations in the supply of organs--limitations that are a consequence of regulation prohibiting the use of market incentives to increase the supply. Markets for organs could take various forms: sales by living donors; sales of future interests in organs, to be removed on the death of the donor; and sales of organs of a recently deceased person by the family of the deceased. Two additional issues relate to the design of a market: the geographic scope of the market and rules of liability for the sale of diseased or defective organs.

Medical malpractice in Michigan: an economic analysis.

We analyzed the factors determining the amount of the recovery on claims based on medical malpractice. Our data set, which previously was not explored, consists of 20,428 claims arising within Michigan that were closed between 1978 and 1990. During this period, major changes were made in the law governing malpractice litigation. We determine the effect of these changes and of various other factors affecting medical malpractice claims. We analyze the effect of a statute that was designed to curtail "forum shopping" by attorneys for plaintiffs. This statute was initially successful in curbing such activity, but its effect seems to be diminishing. In addition, we compare mediation awards with settlement payments, and settlement payments with the expected value of claims in litigation. Evidence suggests that a mediation award is the mediation panel's estimate of a settlement payment appropriate for the case, and that cases are settled for substantially less than their expected value at trial.

Lectin binding to cell surfaces: comparisons between normal and migrating corneal epithelium.

Comparisons were made between cell surfaces of normal and migrating corneal epithelium of the rat by localizing and/or quantifying concanavalin A (Con A) and wheat germ agglutinin (WGA) binding. Our results indicate that apical cell surfaces of the leading edge of a migrating sheet of epithelium differ from those of normal epithelium and that the various cell layers within the stratified normal epithelium have different lectin-binding characteristics. Three methods of monitoring lectin binding to cell surfaces were employed. Based on ferritin-conjugated Con A, ferritin-conjugated WGA, and [3H]Con A binding, apical cell membranes of migrating epithelia bind more Con A and WGA than do apical membranes of superficial cells of normal stratified epithelia. With both fluorescein isothiocyanate (FITC)-Con A and -WGA, membranes of all the cells of the leading edge of the migrating sheet fluoresce intensely. FITC-Con A binding of normal stratified epithelium is relatively uniform through all cell layers with no discernible staining of the apical membrane of superficial cells. With FITC-WGA, however, fluorescence is present only on basal cell layers but not on superficial cells. These data demonstrate that apical cell surface sugars on a sheet of epithelium migrating to cover a wound differ from the apical cell surface sugars of normal epithelium. As indicated by FITC-WGA binding, cells of the migrating sheet have cell surface characteristics similar to basal cells of normal epithelia. Perhaps, upon wounding, the leading edge of the migrating sheet is derived from the basal cell population of the normal stratified epithelium, or perhaps there is an alteration in cell surface glycoproteins as the cells become migratory.