Faculty Bibliography

Rabbit corneal epithelial cells cultured in the presence of 3T3 feeder cells undergo biochemical differentiation, as evidenced by their initial expression of K5 and K14 keratins characteristic of basal keratinocytes, followed by the subsequent expression of K3 and K12 keratin markers of corneal epithelial differentiation. Previous data established that mutations of an Sp1 site in a DNA element, E, that contains overlapping Sp1 and AP-2 motifs reduce K3 gene promoter activity by 70% in transfection assays. We show here that Sp1 activates while AP-2 represses the K3 promoter. Although undifferentiated corneal epithelial basal cells express equal amounts of Sp1 and AP-2 DNA-binding activities, the differentiated cells down-regulate their Sp1 activity slightly but their AP-2 activity drastically, thus resulting in a six- to sevenfold increase in the Sp1/AP-2 ratio. This change coincides with the activation and suppression of the differentiation-related K3 gene and the basal cell-related K14 keratin gene, respectively. In addition, we show that polyamines, which are present in a high concentration in proliferating basal keratinocytes, can inhibit the binding of Sp1 to its cognate binding motif but not that of AP-2. These results suggest that the relatively low Sp1/AP-2 ratio as well as the polyamine-mediated inhibition of Sp1 binding to the E motif may account, in part, for the suppression of the K3 gene in corneal epithelial basal cells, while the elevated Sp1/AP-2 ratio may be involved in activating the K3 gene in differentiated corneal epithelial cells. Coupled with the previous demonstration that AP-2 activates the K14 gene in basal cells, the switch of the Sp1/AP-2 ratio during corneal epithelial differentiation may play a role in the reciprocal expression of the K3 and K14 genes in the basal and suprabasal cell layers

PURPOSE: This study investigated the in vivo differentiation of conjunctival keratinocytes. METHODS: Keratinocytes from the fornical region of the conjunctival epithelium were isolated and plated at low density (5 x 102 per 100-mm dish) in Dulbecco's minimum essential medium containing 20% fetal bovine serum in the presence of mitomycin C-treated 3T3 feeder cells. At this density, only single, isolated cells were attached after overnight culture. Eight days later, small, well-isolated colonies separated from one another by the feeder cells were detached as a sheet from the dish and were injected subcutaneously into the flanks of BALB/c athymic mice through an 18-gauge needle. Within a day, a small firm nodule appeared at the site of injection. At different time points, the animals were killed, and the nodules were excised for morphologic, histogeometric, and cell kinetic analyses. RESULTS: Each implanted colony derived from a single cell gave rise to a single epithelial cyst lined with a reconstituted stratified epithelium. Goblet-like cells loaded with periodic acid-Schiff-positive cytoplasmic granules began to appear singularly in some of the cysts by day 8 postimplantation and were observed in approximately 85% of the cysts by day 14. CONCLUSIONS: Because the cysts formed were derived from clonal populations of epithelial cells and the majority of cysts had a mixed keratinocyte-goblet cell phenotype, these results suggest strongly the existence of a bipotent precursor cell in conjunctival epithelium that can give rise to both goblet and nongoblet cells. This system can be used to study factors that can influence the commitment of pluripotent epithelial stem cells to divergent pathways of differentiation

Urinary tract infections, caused mainly by Escherichia coli, are among the most common infectious diseases. Most isolates of the uropathogenic E.coli can express type 1 and P fimbriae containing adhesins that recognize cell receptors. While P fimbriae recognize kidney glycolipid receptors and are involved in peyelonephritis, the urothelial for type 1 fimbriae were not identified. We show that type 1-fimbriated E. coli recognize uroplakins Ia and Ib, two major glycoproteins of urothelial apical plaques. Anchorage of E. coli to urothelial surface via type 1 fimbriae-uroplakin I interactions may play a role in its bladder colonization and eventual ascent through the ureters, against urine flow, to invade the kidneys

BACKGROUND: Increasing incidence and mortality rates from cutaneous melanoma are a major public health concern. As part of a national effort to enhance early detection of melanoma/skin cancer, the American Academy of Dermatology (AAD) has sponsored an annual education and early detection program that couples provision of skin cancer information to the general public with almost 750,000 free skin cancer examinations (1985-1994). OBJECTIVE: To begin to evaluate the impact of this effort, we determined the final pathology diagnosis of persons attending the 1992-1994 programs who had a suspected melanoma at the time of examination. METHODS: We directly contacted all such persons by telephone or mail and received pathology reports from those who had a subsequent biopsy. RESULTS: We contacted 96% of the 4458 persons with such lesions among the 282,555 screenings in the 1992-1994 programs. We obtained a final diagnosis for 72%, and the positive predictive value for melanoma was 17%. Three hundred seventy-one melanomas were found in 364 persons. More than 98% had localized disease. More than 90% of the confirmed melanomas with known histology were in situ or 'thin' lesions (< or = 1.50 mm thick). The median thickness of all melanomas was 0.30 mm. The 8.3% of AAD cases with advanced melanoma (metastatic disease, regional disease, or lesions > or = 1.51 mm) is a lower proportion than that reported by the 1990 Surveillance, Epidemiology and End Result Registry. The rate of thickest lesions (> or = 4 mm) and late-stage melanomas among all participants was 2.83 per 100,000 population. Of persons with a confirmed melanoma, 39% indicated (before their examination) that without the free program, they would not have considered having a physician examine their skin. CONCLUSION: The 1992-1994 free AAD programs disseminated broad skin cancer educational messages, enabled thousands to obtain a free expert skin cancer examination, and found mostly thin, localized stage 1 melanomas (usually associated with a high projected 5-year survival rate). Because biases impose possible limitations, future studies with long-term follow-up and formal control groups should determine the impact of early detection programs on melanoma mortality

Uroplakins are the major integral membrane proteins synthesized in terminally differentiated, superficial urothelial cells. Alteration of cell differentiation during rat urinary bladder carcinogenesis was analyzed immunohistochemically for the expression of uroplakins. Expression of uroplakins was compared in N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT)-, uracil-, sodium saccharin- or sodium ascorbate-induced urothelial simple hyperplasia, papillary-nodular hyperplasia, papilloma and carcinoma. In controls, uroplakins were located only in superficial cells, especially the luminal surface membrane. In FANFT-induced hyperplasia, including simple hyperplasia, intermediate cells also stained and the staining pattern was disorderly and intermittent. In uracil-induced simple hyperplasia, intermediate cells were stained but in an orderly fashion. In sodium saccharin- or sodium ascorbate-induced simple hyperplasia, superficial cells were swollen but alterations were not observed in the staining pattern. In carcinoma induced by FANFT and uracil, uroplakin expression was very disorderly and focal, usually with no expression on surface cells. It appears that disorderly differentiation is an index of bladder malignancy and is an early event in FANFT-induced lesions but a late event in uracil-, sodium saccharin- and sodium ascorbate-induced lesions

PURPOSE: This study investigated rabbit conjunctival and corneal epithelial cells to determine if they belong to two separate lineages. METHODS: Rabbit corneal, limbal, and conjunctival epithelial cells were isolated and grown in Dulbecco's minimum essential media and 20% fetal bovine serum in the presence of mitomycin-treated 3T3 feeder cells. After reaching 80% confluence, 3T3 feeder cells and any contaminating fibroblasts were removed, and epithelial cells were resuspended in fresh Dulbecco's minimum essential media. Aliquots containing 5x10(6) cells were placed subcutaneously into the flanks of athymic mice, which subsequently formed small nodules. At 2, 4, 6, 8, 14, 21, and 28 days, athymic mice were killed and the nodules (epithelial cyst) were excised for light and transmission electron microscopic examination and histochemical and cell kinetic analyses. RESULTS: Within 2 days after injection of single-cell suspensions, cells aggregated to form cysts lined with a stratified squamous epithelium, the structure of which resembled the original in vivo donor sites by 8 days. Limbal- and corneal-derived cysts were comprised only of glycogen-rich stratified epithelial cells. In contrast, only cysts arising from cultured conjunctival cells contained periodic acid-Schiff-positive cells with a goblet cell structure interspersed among stratified epithelial cells. Furthermore, cystic epithelium of conjunctival origin did not accumulate glycogen. CONCLUSIONS: To determine whether distinct phenotypes are caused by intrinsic divergence or by environmental modulation, the behavior of cells can be monitored in an identical in vivo growth environment. The athymic mouse provides such a permissive growth environment for cultured corneal, limbal, and conjunctival epithelial cells. All these cells reproduced their in vivo phenotype when placed in the athymic mouse. Thus, these findings provide the strongest evidence to date that the corneal-limbal lineage is distinct from the conjunctival lineage. These data also support the idea that the progenitor of goblet cells does not reside in the corneal-limbal epithelial compartment

BACKGROUND: Recently, in the filiform papillae epithelium of mouse dorsal tongue, we showed the presence of hybrid granules in which filaggrin and trichohyalin were both present, but physically segregated. Further, trichohyalin was also detected in scattered granular cells of a number of hyperplastic skin diseases. METHODS: The epidermis infected with molluscum contagiosum virus (MCV) was studied by conventional electron microscopy in conjunction with light and electron-microscopic immunohistochemistry, using both antifilaggrin and antitrichohyalin antibodies as probes. RESULTS: We found that the granular cells of MCV-infected epidermis contained both filaggrin and trichohyalin. Subsequent electron-microscopic examination showed that the granular cells contained morphologically heterogeneous granules that appeared to be composed of discrete areas of distinct electron densities. Double-labeling, using antibodies to filaggrin and trichohyalin, clearly indicated that filaggrin and trichohyalin were both present in the hybrid granules and that the electron-dense regions contained trichohyalin while the more electron-lucent regions contained filaggrin. CONCLUSIONS: The expression of trichohyalin was a common feature observed in the epidermis from a heterogenous group of hyperplastic conditions, including MCV infection. This finding has led us to speculate that trichohyalin may be specifically or preferentially involved in interacting with the hyperproliferation-related keratin pair (K6/K16), whereas the function of filaggrin is more closely linked to the skin-type keratin pair (K1/K10) that are normal keratins found in the differentiated epidermis