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L B Taichman

Publications and source records attributed to L B Taichman.

At least 37 records · Page 2Linked to original sources
PubMed1994

Systemic delivery of secreted protein by grafts of epidermal keratinocytes: prospects for keratinocyte gene therapy.

Grafts of autologous keratinocytes genetically altered to secrete a new gene product are a potential vehicle for gene therapy. To consider the feasibility of such an approach, we have examined the ability of keratinocytes to secrete and deliver apolipoprotein E (apoE) to the circulation of mice bearing grafts of human keratinocytes. The grafted keratinocytes secreted two forms of apoE, an endogenous apoE encoded in the genome and a recombinant apoE encoded in a transfected gene construct. In vitro studies showed that endogenous apoE was secreted from basal keratinocytes whereas recombinant apoE was secreted from basal as well as suprabasal cells. On the basis of amounts of recombinant apoE present in the serum of grafted mice, we estimate that a graft occupying 2% of the surface area of an adult human would deliver 6.5-8.3 mg of recombinant apoE protein per day.

Animals↗
PubMed1994

Epidermis as a secretory tissue: an in vitro tissue model to study keratinocyte secretion.

In addition to protective functions, keratinocytes secrete a variety of effector molecules that may have local or distant effects. To explore the secretory activity of keratinocytes we have developed a two-chamber culture model in which a fully differentiated stratified epithelium is present in the upper chamber and secreted protein is collected in a lower chamber. A collagen matrix is not used and during collection of secreted protein, 3T3 feeder cells are absent. Keratinocytes secrete protein at the rate of 0.67 microgram/h/10(6) cells, encompassing a molecular weight range from several kD to over 180 kD. Secretion is an unexplored area of keratinocyte biology and this model will allow investigation of this activity.

Adult↗
PubMed1994

Secretion of apolipoprotein E by basal cells in cultures of epidermal keratinocytes.

Recently it has been shown that apolipoprotein E (apoE) secreted by keratinocytes in transplanted epidermal grafts reaches the systemic circulation. In this study we ask which cells in cultures of epidermal keratinocytes, basal or suprabasal, are the source of apoE. By fractionating disaggregated cultures in gradients of Ficoll400, the small nondifferentiated cells derived from the basal compartment were shown to be the source of apoE. The larger more differentiated cells derived from suprabasal layers could not be shown to contain or secrete apoE, although they did contain the apoE mRNA. Basal cells are the primary site for replication. However, analysis during growth in culture indicated that secretion did not correlate with cell replication but appeared to be linked to specific changes in metabolic activity of the basal cell compartment. Localization of apoE secretion to the basal compartment may provide a mechanism for lipid uptake and redistribution within the epidermis and may be viewed within the larger context of keratinocyte differentiation.

Adult↗
PubMed1994

Effect of TGF-beta 1 on re-epithelialization of human keratinocytes in vitro: an organotypic model.

Transforming growth factor beta-1 (TGF-beta 1) has been shown to inhibit keratinocyte proliferation in vitro yet and migration was investigated in organotypic cultures after incisional wounding. Organotypic cultures provide a more in vivo-like epidermal tissue and may therefore respond in a different manner than previous culture models in which epidermal differentiation is incomplete. Without TGF-beta 1, keratinocytes were hyperproliferative in response to wounding. At doses of 2.5 ng/ml or greater, a delay in re-epithelialization was seen at 24 h post-wounding along with a reduction in hyperproliferation. By 48 h, however, re-epithelialization was complete in all cultures treated with TGF-beta 1. In particular, 7 ng/ml TGF-beta 1 inhibited proliferation yet had no effect on re-epithelialization by 48 h. These studies demonstrate that i) TGF-beta 1 induced a delay in re-epithelialization, ii) proliferation of wounded keratinocytes was not inhibited at 2.5 ng/ml doses of TGF-beta 1, and iii) at 7 ng/ml TGF-beta 1, re-epithelialization was complete by 48 h in spite of the profound inhibition of cell proliferation. In the organotypic model, TGF-beta 1 appears to alter re-epithelialization.

Cell Division↗
PubMed1994

Fate of human keratinocytes during reepithelialization in an organotypic culture model.

BACKGROUND: Reepithelialization of an incisional wound in a stratified squamous epithelium is accomplished by mobilizing keratinocytes from the wound margins. In vitro models to study this phenomenon have been limited by incomplete differentiation of the cultured epithelium. In addition, it has been difficult to follow fate of recruited keratinocytes, since techniques for marking cells have not been available. We have adapted an organotypic culture model in which keratinocytes are fully differentiated and have utilized a genetic marking protocol with retroviral vectors to study reepithelialization after an incisional wound. EXPERIMENTAL DESIGN: The fully differentiated epithelium of an organotypic culture model was incised, supported on a collagen matrix, and allowed to reepithelialize. At various times after wounding, healing cultures were monitored for migration, differentiation, and proliferation by immunohistochemical staining. Histochemical staining specific for the genetically marked cells assisted in the determination of how these cells behaved during reepithelialization. RESULTS: The first event observed (at 8 hours) was migration of suprabasal keratinocytes into the wound followed by a transient proliferative burst at the wound margin. Reepithelialization was complete by 24 hours. Proliferation in the wound epithelium persisted during stratification and terminal differentiation. Genetically marked cells in the wound epithelium were present in clusters demonstrating that proliferation and displacement of cells occurred near the edge of the epithelial tongue. Individual genetically marked cells were also found in the wound epithelium, indicating that individual cells had migrated a considerable distance from the wound edge without having undergone replication. CONCLUSIONS: This is the first report of an organotypic model for reepithelialization, and we demonstrate that migration, proliferation, and differentiation occur during this process. The proliferative response which follows initial cell migration at the wound margins suggests that these events are temporally coordinated as phenotypically different populations of cells are sequentially activated. By following the distribution of genetically marked cells in the wound, it is evident that at least two types of cells repopulate a wound-proliferative and migratory cells.

Cell Division↗
PubMed1993

Tissue- and stratum-specific expression of the human involucrin promoter in transgenic mice.

Involucrin is a marker of keratinocyte terminal differentiation and is expressed only in the suprabasal layers of stratified squamous epithelium. In a previous study with various cell types in culture, we noted that expression of the putative human involucrin promoter was keratinocyte specific. To determine if this promoter is sufficient to direct expression to the suprabasal cells of stratified squamous epithelia in vivo, we have now generated transgenic mouse lines harboring the involucrin promoter sequences linked to a beta-galactosidase reporter gene. In the resulting lines, beta-galactosidase was expressed in the suprabasal compartment of stratified squamous epithelia and in hair follicles in a tissue-specific manner. In the palate, distinct vertical stacks of beta-galactosidase-expressing cells were present, suggesting movement of clonally derived cells through the epithelium. The involucrin gene has a single intron upstream of the translational start site, and removal of this intron did not affect tissue- or stratum-specific expression. These results show that the 3.7-kb involucrin upstream sequences contain all the information necessary for a high level of tissue- and stratum-specific expression.

Animals↗
PubMed1993

The fate of genetically marked human oral keratinocytes in vitro.

The fate of the progeny of human oral gingival keratinocytes was mapped in stratified epithelial tissues in vitro by following the expression of a marker gene in genetically related clones. Oral epithelial progenitor cells were genetically marked at high efficiency by transducing them with a retrovirus vector that carried the gene for a histochemically detectable product, Escherichia coli beta-galactosidase (beta-gal). These cells were then grown in submerged cultures and on collagen rafts at the air-liquid interface to demonstrate the distribution of genetically marked cells in a differentiating tissue in vitro. The dynamics of transduced cells showed that clonally related cells were arranged in discrete units of labelled cells and these clusters were defined as 'clonal proliferation units'. The size and configuration of these units were related to the proliferative potential and differentiating capacity of the cell that was initially transduced. This model demonstrates the relation between clonally related cells and tissue architecture for oral keratinocytes in vitro.

Cell Differentiation↗
PubMed1992

Replication and persistence of HPV DNA in cultured cells derived from laryngeal papillomas.

We have investigated the replication and persistence of human papillomavirus (HPV) type 6 and 11 DNA in cultured cells derived from laryngeal papillomas, with paradoxical findings. Measured by bromodeoxyuridine incorporation into heavy/light DNA separated on a cesium chloride gradient, viral DNA replicates in both primary and secondary cells. The ratio of the fraction of replicated viral to replicated cellular DNA was equal to or greater than 1 in all but one case and was closer to 2 in primary cells. Despite this efficient replication, HPV DNA is rapidly lost from the cells with passage. We propose that infected cells, or those with a high HPV copy number, show a selective decrease in plating efficiency compared to uninfected cells or those with a low copy number, which explains the loss of HPV DNA with repeated passage.

Blotting, Southern↗
PubMed1992

A model to study the fate of genetically-marked keratinocytes in culture.

In this study we demonstrate a method for analyzing the spatial distribution or fate of progeny keratinocytes derived from single progenitor cells. The method relies upon the use of retroviral vectors to introduce a reporter gene into replicating cells and to effect integration and expression of that new gene. All progeny cells from that initial cell inherit and express the transferred gene. The reporter gene is the E. coli beta-galactosidase gene (B-gal), which encodes a histochemically-detectable product in the cytoplasm. Using this method, we show that foci of genetically marked, B-gal positive cells can be readily identified in submerged cultures and we term this grouping of cells a "clonal proliferation unit". Analysis of B-gal stained whole mounts and paraffin sections allows visualization of the proliferative potential and differentiating capacity of clonogenic cells. This model will allow exploration of how agents known to alter epidermal proliferation and differentiation affect lineage relationships.

Cells, Cultured↗
PubMed1992

Replication of plasmid-derived human papillomavirus type 11 DNA in cultured keratinocytes.

Plasmid-derived human papillomavirus type 11 (HPV11) DNA has been shown to replicate episomally and semiconservatively following transfection of primary human foreskin keratinocytes. HPV11 DNA was excised from the bacterial plasmid, religated to form circular molecules, and cotransfected along with pSV2Neo. Transfectants were selected and shown to contain replicated episomal HPV DNA. Once selected, HPV11 DNA persists in cells through at least two additional passages.

Cells, Cultured↗
PubMed1992

Characterization of the human involucrin promoter using a transient beta-galactosidase assay.

Involucrin, a component of the cornified cell envelope, is expressed specifically in differentiating keratinocytes of stratified squamous epithelia. To explore the regulation of involucrin expression, 3.7 kb of upstream sequences of the human involucrin gene was cloned into a plasmid containing a beta-galactosidase reporter gene and transfected into early passage keratinocytes and a variety of human cell types. The full-length construct gave maximal and tissue-specific expression. Deletion analysis showed that sequences between 900 and 2500 bp upstream of the transcriptional start site and the intron located between the transcriptional and translational start sites were required for maximal expression. Further analysis of the intron indicated that its effects on expression were independent of it being present in nascent RNA and suggested that sequences within the intron have regulatory activity. These results suggest that the involucrin intron operates in vivo to regulate expression in the epidermis.

Breast Neoplasms↗
PubMed1991

Human papillomavirus infection of the oral mucosa.

This article reviews the lesions of oral mucosa that contain human papillomavirus (HPV). These HPV-associated lesions can be classified into two broad types on the basis of their biologic behavior, benign lesions and premalignant malignant or malignant lesions. Benign oral lesions include squamous cell papilloma (SCP), verruca vulgaris (VV), condyloma acuminatum (CA), and focal epithelial hyperplasia (FEH). Of these entities, VV, CA, and FEH demonstrate characteristic HPV-induced cytopathic effects, whereas SCP infrequently shows such changes. All of these lesions show a clear association with HPV. Premalignant and malignant oral lesions include leukoplakia and squamous cell carcinoma. The etiologic role of HPV in these lesions is still unclear. Koilocytosis is the most common cytopathic effect seen in both groups of lesions. Even though it is sometimes difficult to distinguish between hyperplastic lesions such as SCP, VV, and CA, clinical and certain histologic features can facilitate the diagnosis. Although exceptions do exist, each of the two classes of lesions is most commonly associated with particular HPV types. The benign oral lesions are associated with HPV 2, 4, 6, 11, 13, and 32; the malignant oral lesions are associated with HPV 16 and 18. No preferential association has been demonstrated between specific HPV types and a particular oral lesion.

Humans↗
PubMed1991

Retrovirus-mediated transduction of cultured epidermal keratinocytes.

Retrovirus-mediated gene transfer is an efficient means of introducing and expressing exogenous gene(s) in many cell types including keratinocytes. However, parameters of transduction and gene expression have not been systematically analyzed for keratinocytes. To carry out such a study we have transduced cultures of newborn foreskin cells with retroviral vectors that encode the genes for neomycin resistance (neor) and for beta-galactosidase (B-gal). The neor gene is a dominant selectable marker and the B-gal gene encodes a histochemically detectable product. Our key findings are the following: 1) all keratinocytes that form colonies can be successfully transduced at a viral titer greater than 5 x 10(6) colony-forming units/ml; 2) transduction is effected by integration of a single copy of retroviral DNA; 3) transduced cells are not at a growth disadvantage and, in fact, single clones of transduced keratinocytes can be expanded to yield over 10(9) cells, suggesting that stem cells are transduced; 4) whereas most transduced colonies exhibit B-gal staining in a high percentage of constituent cells, some colonies had a mosaic or sectored staining pattern; 5) expression of the non-selectable B-gal gene was somewhat greater in differentiated cells of the culture as compared to nondifferentiated precursors. The ability to transduce stem cells at a high efficiency and to follow expression of transduced genes in clonal progeny will allow lineage mapping in stratified epithelial tissues.

Cell Differentiation↗
PubMed1991

Characterization of murine polyclonal antisera and monoclonal antibodies generated against intact and denatured human papillomavirus type 1 virions.

Human papillomavirus type 1 (HPV1) virions, both as intact virion particles (IVP) and as detergent-denatured virions (DDV), were used to prepare polyclonal antisera and monoclonal antibodies (MAbs) in BALB/c mice. Anti-IVP antiserum contained type-specific HPV1 L2-reactive antibodies and no detectable HPV1 L1-reactive antibodies. Anti-IVP MAbs recognized a linear epitope between L2 amino acids 102 and 108 (PIDVVDP). Anti-DDV antiserum contained type-specific HPV1 L1-reactive and HPV1 L2-reactive antibodies. An anti-DDV MAb recognized a linear epitope between L1 amino acids 127 and 133 (AENPTNY). HPV1a L1- and L2-encoded polypeptides expressed in Saccharomyces cerevisiae and by in vitro translation were equivalent in size to the major and minor virion capsid proteins, respectively.

Amino Acid Sequence↗
PubMed1990

Analysis of adenovirus early and late gene expression in cultured epidermal keratinocytes.

Previous studies have shown that expression of adenovirus type 2 (Ad2) is restricted in epidermal keratinocytes in what appears to be a differentiation specific manner. We have analyzed the relationship between keratinocyte differentiation and Ad2 early and late gene expression. Cultured epidermal keratinocytes infected with Ad2 were fractionated in density gradients of Ficoll 400 to enrich for populations of nondifferentiated cells and differentiated cells. Analysis of these populations revealed that both populations supported early Ad2 gene expression but restricted Ad2 late gene expression. The restriction to late gene expression differed in the two cell populations: Nondifferentiated keratinocytes did not support production of high levels of Ad2 capsid proteins, whereas differentiated keratinocytes supported synthesis of Ad2 capsid proteins but restricted Ad2 expression at a later step that normally leads to production of high titers of progeny virus. The changing restriction to Ad2 expression during keratinocyte differentiation may have resulted from changes in cellular components that play a role in cell differentiation.

Adenoviridae Infections↗
PubMed1989

The effect of trifluoperazine, a calmodulin antagonist, on the growth of normal and malignant epidermal keratinocytes in culture.

Calmodulin, a cytoplasmic calcium binding protein, is present in concentrations two- to four-fold higher in malignant cells compared to normal cells. In an effort to learn the significance of these elevated levels, we examined the effect of calmodulin blockage on the growth of normal and malignant keratinocytes in vitro. The level of calmodulin in SCC12.B2, a line of keratinocytes derived from an epidermal squamous cell carcinoma (SCC), was about 3.5 times greater than in normal, human newborn foreskin keratinocytes. When exposed to trifluoperazine (TFP), an inhibitor of calmodulin, cell growth was reduced primarily in the cultures of normal keratinocytes. This growth inhibition resulted from two changes in the replicating population of cells, namely an increase in cell cycle length and an increase in rate of cell cycle withdrawal. Cell cycle withdrawal is the irreversible arrest of the cell cycle and is an early event in keratinocyte terminal differentiation. There was no measurable effect on the cell cycle time or withdrawal rate in SCC12.B2. The increased resistance to growth arrest in SCC cells may be a consequence of the elevated level of calmodulin in these cells.

Calmodulin↗