Biomedical subjects

M M Brysk

Publications and source records attributed to M M Brysk.

At least 37 records · Page 2Linked to original sources

PubMed1996

Induction of iNOS mRNA by interferon-gamma in epithelial cells is associated with growth arrest and differentiation.

Induction of inducible nitric oxide synthase (iNOS) mRNA by interferon-gamma (IFNgamma) is well established in various cell types. We observed earlier that this induction is differentiation-dependent in human keratinocytes. Since IFNgamma-mediated growth arrest and differentiation are separable but interrelated processes in keratinocytes, iNOS might play a role in their regulation. We conducted a series of in vitro experiments on normal and transformed epithelial cells with different tissue origins. We found that immortalization and transformation can influence the IFNgamma-mediated iNOS inducibility, but this process is tissue-specific as the induction correlates with the ability of the cells to differentiate.

Antineoplastic Agents↗

PubMed1996

Induction of blister-causing antibodies by a recombinant full-length, but not the extracellular, domain of the pemphigus vulgaris antigen (desmoglein 3).

Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.

Amino Acid Sequence↗

PubMed1996

Regulation of inducible nitric oxide synthase mRNA levels by differentiation and cytokines in human keratinocytes.

Inducible nitric oxide synthase (iNOS) transcription is regulated by cytokines and immune stimuli in various cell types. However, there are few data available on iNOS expression and regulation in keratinocytes, even though it can play a role in keratinocyte proliferation during inflammation following wound healing. Our studies demonstrated a differentiation-dependent regulation of iNOS transcription in human keratinocytes from epidermis but not in buccal mucosal cells, which are less differentiated. Different interferon (IFN) types (alpha and gamma) regulated skin keratinocyte iNOS mRNA levels in different ways, predominantly in a differentiation-dependent manner. TGFbeta1 and IFNalpha can antagonize IFNgamma related iNOS induction. However, the antagonistic effects of IFNalpha on IFNgamma differ from those of TGFbeta1. Our studies demonstrate: (a) the presence of iNOS mRNA in human keratinocytes; (b) inducibility of iNOS mRNA by different cytokines; and (c) antagonism between certain cytokines in induction of iNOS mRNA. Further studies are needed to determine biological and therapeutical relevance of our findings.

Cell Differentiation↗

PubMed1996

Response to interferon treatment decreases with epidermal dedifferentiation in condylomas.

After interferon (IFN) treatment of patients with condyloma acuminatum, groups clinically proven to be responders or nonresponders were selected, and cellular parameters that might influence the clinical response were studied in pretreatment biopsies by reverse transcription polymerase chain reaction (RT-PCR). The nonresponders were found to express higher amounts of cellular proliferative markers, such as proliferating cell nuclear antigen (PCNA), cyclin A, and cdc 2 kinase, but lower levels of growth suppressor genes (TGF-beta 1, TGF-beta 2 and p53) before IFN treatment. The responders retained the epidermal keratinization, except for some signs of hyperproliferation (K6, K16 cytokeratins). In addition, the nonresponders showed a shift in the keratinization pattern to a mucosal or fetal type, as evidenced by high expression of the K18, K6, K16 and K13 cytokeratins but decreased K5, K14 and K10 levels before treatment. The expression of the human papillomavirus (HPV) genes is consistent with these differentiation patterns. The crucial conclusion to be drawn from this study is that those condylomas whose pretreatment phenotype most closely resembles that of normal epidermis respond to IFN treatment, whereas those more akin to nonkeratinizing epithelia fail to respond, i.e. the resistance of condylomas to IFN treatment is correlated with dedifferentiation.

Base Sequence↗

PubMed1995

Gene expression of markers associated with proliferation and differentiation in human keratinocytes cultured from epidermis and from buccal mucosa.

Normal keratinocytes from epidermis and from buccal mucosa underwent dissimilar stages of differentiation in the same culture medium and responded differently to changes in the composition of the medium. Manifestations of these variations were examined in terms of the expression at the mRNA level (as measured by reverse transcriptase-polymerase chain reaction) of three regulatory genes (cdc2, c-myc, and p53) and five that encode structural proteins (keratins K5, K10 and K13, involucrin, and filaggrin), in three growth-medium formulations. The culture conditions enhanced or retarded maturation; the observed alterations in gene expression correlated with these changes. Except for the proliferation genes, the non-keratinizing buccal mucosa generally responded more weakly than the orthokeratotic epidermis to culture-medium supplementation favouring differentiation. Gene expression in cultured keratinocytes reflected their ability to differentiate in vivo; genes were expressed even when the corresponding protein was not seen in vitro. Although keratin K10 is not prevalent in the buccal mucosa nor keratin K13 in the epidermis, the genes for both were found to be expressed in both tissues.

Calcium↗

PubMed1995

Induction of apoptotic nuclei by interferon-gamma and by predesquamin in cultured keratinocytes.

Predesquamin is a glycoprotein found in the transition layer and the lower stratum corneum of human epidermis. Interferon-gamma (IFN-gamma) induces the synthesis of predesquamin by keratinocytes in culture. We now show ultrastructurally that exogenous addition of either predesquamin or IFN-gamma to cultured keratinocytes induces apoptotic nuclei with condensed chromatin. Degradation of cellular DNA is also evident as a ladder pattern in an agarose gel. After incubation with both predesquamin and IFN-gamma (but not either alone), the mobility of plasmid DNA in a gel shows retardation specific for guanine residues. This binding to the DNA may impart to it a conformational change that facilitates access by endogenous cellular nucleases. In epidermal cells cultured with IFN-gamma supplementation, we also show by RT-PCR that there is an upregulation of the genes c-myc, p53, gadd45, dsRNA-activated protein kinase, and 2'-5'-oligo(A)-dependent RNase, which have all been implicated in apoptosis in other cell types. These results are pertinent to the mechanism of occurrence of apoptosis in the epidermis in vivo, where predesquamin and IFN-gamma are endogenous. Programmed cell death is an inherent step in the terminal differentiation and desquamation of the epidermis.

Apoptosis↗

PubMed1994

Cytokines modulate terminal differentiation and the expression of predesquamin in cultured keratinocytes.

Predesquamin is a glycoprotein that we have isolated from human epidermis. Its monospecific polyclonal antibody immunolocalizes to the lower stratum corneum. We have previously found that it is not expressed in primary keratinocyte cultures unless both interferon-gamma and serum are added to the culture media. We now show similar results with interleukin-1 alpha substituted for interferon-gamma (but not with retinoic acid). Predesquamin is expressed in media supplemented with both cytokines (with or without serum), accompanied by the observation of larger outer squames and a more mature pattern of desquamation. As both cytokines are naturally present in the epidermis, this suggests that they contribute to desquamation in vivo. The predesquamin expression is demonstrated by Western blots of cell extracts with the specific antibody. Corresponding morphological changes are illustrated by phase-contrast microscopy.

Blotting, Western↗

PubMed1994

Predesquamin extends maturation of cultured keratinocytes.

Predesquamin is a glycoprotein that we have isolated from human epidermis. Its monospecific polyclonal antibody immunolocalizes to the lower stratum corneum. We have previously found that it is not expressed ordinarily in primary keratinocyte cultures, but that it is expressed in a serum-containing medium that is supplemented with interferon-gamma. Under these conditions, we observed larger outer squames and a more mature pattern of desquamation. We now demonstrate, by adding predesquamin directly to the culture medium without the cytokine, that the morphological changes are caused by predesquamin (and the role of IFN-gamma in the previous experiment was to stimulate the expression of predesquamin).

Antibodies↗

PubMed1994

Enzymatic activity of desquamin.

We have previously described the properties of desquamin, a cell adhesion molecule in the stratum corneum with lectin-like properties specific for amino sugars. We report here that desquamin is also a trypsin-like serine proteinase. It degrades several chromogenic peptides with arginine in the P1 position, with greatest activity for the tissue plasminogen activator peptide; it has no chymotrypsin-like activity. The enzymatic activity of desquamin is inhibited by aprotinin, leupeptin, and soybean trypsin inhibitor. The Km for all active substrates is in the millimole range and the pH for optimal activity is near 10. The enzymatic activity is stable in the temperature range from 37 to 80 degrees C, peaking near the upper end; it is only partially inhibited at 100 degrees C. Using zymogels with immobilized substrates, we show that desquamin degrades both casein and human keratins. Because desquamin is localized to the lipid envelopes of the stratum corneum and can function as an enzyme (and is extremely resistant to chemical and thermal degradation), it is in a position to play a crucial role in desquamation.

Amino Acid Sequence↗

PubMed1993

Predesquamin inhibits desquamation.

Predesquamin is a Con A binding glycoprotein from human epidermis, with a molecular weight of 650 kDa. Its isolation and characterization are detailed, as well as the preparation of a polyclonal antibody to it. The antibody labels primarily the lower stratum corneum in fluorescent and ultrastructural immunolocalization, both in normal skin and plantar callus. A desquamation model system, in which plantar callus is incubated with the chelating agent ethylenediaminetetraacetic acid (EDTA) is refined by substituting for EDTA the Ca(2+)-specific ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Moreover, the shedding activity can be suppressed by raising the Ca2+ concentration. Within this model, it is demonstrated that predesquamin at nanomolar concentrations inhibits squame shedding in a dose-dependent manner. We conclude that predesquamin plays an important role in blocking premature desquamation of the lower stratum corneum.

Bony Callus↗

PubMed1992

Cohesion and desquamation of epidermal stratum corneum.

This article attempts to provide a comprehensive review on the roles of various classes of molecules in the cohesion and desquamation of the stratum corneum. In the first part of this monograph we review the field of epidermal differentiation in vivo and vitro, describing the expression and functions of a number of key structural molecules that characterize the process. In the second part we emphasize terminal differentiation and the biogenesis of the stratum corneum. The stratum corneum is a cell layer unique to fully differentiated squamous epithelia such as skin. While it is a dead stratum, it nevertheless is in a homeostatic process of continual shedding and renewal in synchrony with basal cell replication. It is also a degradative layer containing many proteinases and glycosidases in which a variety of intracellular and intercellular macromolecules are degraded. We highlight the molecules localized within the intercorneal matrix that are most likely to play a role in cohesion and desquamation, including: glycoproteins, lipids and enzymes. Because it is difficult to study the stratum corneum and desquamation in the native tissue, we discuss a number of model systems that have been used. The stratum corneum can be dispersed into single squames in different ways; these include mechanical dispersion as well as agents such as detergents and enzymes. The solubilized molecules and the structures remaining can then be studied as to their specific roles in desquamation. Using this approach it is possible to reconstitute multilayered structures that resemble a real stratum corneum. We have shown that glycoproteins play a key role in squame reaggregation and that this process can be modulated with amino sugars in a lectin-like fashion. Cohesion and desquamation can also be studied in tissue culture. Depending on the culture system, the extent of terminal differentiation and squame accumulation varies. Yet desquamation does not normally occur. It can be induced however by the inclusion of exogenous agents such as IFN-gamma which are found in the native epidermis but are absent in vitro. Modulation of desquamation by other exogenous agents is likely to yield further knowledge of how shedding occurs in vivo. Insight has also come from studies of scaling skin disorders. The glycoprotein and lipid profiles are altered in the stratum corneum in many diseases of aberrant terminal differentiation. A number of abnormalities in the levels of cytokines and growth factors have also been reported in the lesional tissue of such diseases.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Culture of basal cell carcinoma.

Studies of basal cell carcinoma have been hindered by a lack of a suitable and reproducible tissue-culture model system. We have succeeded in growing this tumor in primary culture from eight different patients. We can separate and grow the tumor cells and the stromal cell component. We also culture normal keratinocytes and fibroblasts from the same patient for comparative studies. All the cell types have been subcultured four to five times and cryopreserved. The normal keratinocytes were indistinguishable from the tumor cells in ploidy, in rate of growth, and in the failure to express ICAM-1. Both cell types also fail to synthesize the matrix proteins: types I and IV collagens. Differences were noted in the expression of fibronectin and the bullous pemphigoid antigen, with the normal cells expressing the antigens although the tumor cells did not. Interferons exogenously added to the culture media preferentially killed the basal cell carcinoma cells, as compared to normal keratinocytes from the same patients. We believe that our culture system opens possibilities for biochemical and molecular studies of this disease, and for in vitro testing of antitumor agents for clinical therapy.

Basal Cell Carcinoma↗

PubMed1991

Interferon-gamma modulates terminal differentiation and the expression of desquamin in cultured keratinocytes.

Interferon (IFN)-gamma has been shown to modulate cell differentiation and the expression of cell surface molecules of cultured human keratinocytes; it also induces cell shedding. We have previously described the properties of desquamin, a cell surface adhesion molecule from the stratum corneum. We report here on the impact of IFN-gamma on the expression of desquamin. We document the related morphological changes in terminal differentiation. We cultured human keratinocytes in three different culture systems: in serum-free medium at low Ca2+ (0.1 mM), at high Ca2+ (1.5 mM), and at high Ca2+ with 10% serum. IFN-gamma (100 U/ml) was added to each culture system after overnight incubation. In all cases, IFN-gamma induced an altered phenotype, as shown by phase contrast and electron microscopy. We exposed cultured cells to antibodies to the desquamins (glycoproteins from the stratum corneum). Immunoflurescent localization and Western blotting showed that the desquamins were expressed only under culture conditions where both serum and IFN-gamma were present. The induction of desquamin expression by IFN-gamma coupled with an increase in cell shedding, suggests that we have developed a suitable culture system for the study of desquamation in vitro.

Antibodies, Monoclonal↗

PubMed1991

Grafting of leg ulcers with undifferentiated keratinocytes.

A procedure for transplanting cultured human keratinocytes has been developed and used successfully to treat patients with persistent leg ulcers. Shave excisions of donor skin of 1 cm2 or less were expanded in culture in 1 week to cover wounds as large as 90 cm2. The uniqueness of this system is the use of a biochemically modified surgical dressing that permits the transplantation of undifferentiated cells. Several hours after inoculation, trypsinized single cells become attached to the dressing and are ready for grafting. Seven of eight persistent stasis ulcers completely reepithelialized after application of the modified surgical dressing with undifferentiated keratinocytes.

Bandages↗

PubMed1991

Sensitivity of desquamin to proteolytic degradation.

The stratum corneum is both a desquamating and degradative tissue; cell contacts become weaker in the outer lamellae and a variety of proteolytic and glycolytic enzymes cleave many macromolecules into free amino acids and saccharides. We have reported on a 40-kilodalton glycoprotein from human stratum corneum; because of its unique lectin-like properties, we believe that it plays a major role in desquamation and have named it desquamin. If desquamin is to function as a lectin during desquamation, it must survive enzymatic degradation in the outer stratum corneum. Accordingly, we treated this glycoprotein with a number of proteinases and found that desquamin is resistant in vitro to most of the types of proteinases that have been reported in the stratum corneum as well as several others that have not been identified as present in the tissue. In the stratum corneum, the lipids of the intercorneal mortar shield the glycoproteins; hence, desquamin is even less likely to be susceptible to endogenous proteolysis in vivo than in vitro.

Blotting, Western↗

PubMed1989

A surgical treatment for vitiligo: autologous cultured-epithelial grafts.

Medical treatment of vitiligo has been disappointing; therefore surgical treatments using autologous grafts have been tried. We investigated a new technique in which normally pigmented epidermal cells from a vitiligo patient were cultured on a collagen-coated membrane in MCDB-153, a media that supports melanocytes and keratinocytes. The cell/collagen-coated membrane was used to cover a superficially dermabraded vitiliginous area. Clinical repigmentation, though incomplete, was evident in three of four patients in 4 weeks. Immunofluorescence and electron microscopy were performed on the grafted areas (involved and uninvolved controls). These studies demonstrate the reappearance of melanocytes and melanosomes within the grafted area. All three patients who were successfully repigmented retained their pigment at 1 year follow-up. Further investigation of this promising treatment for vitiligo is warranted.

Adult↗

PubMed1989

Cohesive properties of terminally differentiated keratinocytes.

The stratum corneum can be dissociated into single squames by several extraction methods: mechanically, with organic solvents, and with detergents. We have performed studied of reaggregation from single squames prepared by these methods. Mechanically dispersed corneocytes recombined into a lamellar-like structure closely resembling intact stratum corneum. Squames obtained by dissociation in ether formed a multilayered structure which differed from the intact tissue in the collapse of the bilayered lamellar structure into a single lucent band with osmiophilic deposits. Squames obtained after extraction with detergent failed to reaggregate.

Cell Adhesion↗

PubMed1988

Glycoproteins modulate adhesion in terminally differentiated keratinocytes.

The stratum corneum can be dissociated into single squames by homogenization in ether. We have reaggregated the free corneocytes into a multilayered lamellar structure resembling an intact stratum corneum. The reconstituted stratum corneum reacts with fluorescein-conjugated lectins, unlike the intact tissue. We infer that the lack of binding in the intact tissue is due to masking of saccharide sites by lipids (which are extracted by the ether). In an extension of the procedure, the ether is removed and replaced by acetone. This system permits us to modulate corneocyte reaggregation by the addition of appropriate agents. We have used this system to corroborate our hypothesis that a 40 kD cell-surface glycoprotein (an endogenous lectin specific for amino sugars), which we have isolated from the stratum corneum, is instrumental in adhesion of corneocytes by cross-linking with amino sugar sites on adjacent cells. The reaggregation is inhibited by the antibody to the 40 kD glycoprotein. It is also inhibited by either the addition of amino sugars which bind to the endogenous lectin, or the addition of exogenous lectins specific for amino sugars which bind to the ligand.

Antibodies↗