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P Fleckman

Publications and source records attributed to P Fleckman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Differentiation of cultured keratinocytes promotes the adherence of Streptococcus pyogenes.

Based on a consideration of the histopathology of nonbullous impetigo that shows localization of Streptococcus pyogenes to highly differentiated, subcorneal keratinocytes, we hypothesized that adherence of an impetigo strain of S. pyogenes would be promoted by terminal differentiation of keratinocytes. An assay was developed in which S. pyogenes adhered via pilus-like projections from the cell wall to the surface of cultured human keratinocytes in a time- and inoculum-dependent manner suggestive of a receptor-mediated process. Terminal differentiation of keratinocytes was induced by increasing the calcium concentration in the growth medium, and was confirmed by morphologic analysis using electron microscopy. Adherence of S. pyogenes was three and fourfold greater to keratinocytes differentiated in 1.0 and 1.5 mM calcium, respectively, compared with undifferentiated keratinocytes in 0.15 mM calcium. The presence of calcium during the adherence assay further enhanced adherence nearly twofold. Adherence occurred preferentially to sites of contact between adjacent keratinocytes, suggesting that the keratinocyte receptor may be a molecule involved in cell-to-cell adhesion. In contrast, nonpathogenic Streptococcus gordonii adhered poorly to keratinocytes regardless of their state of terminal differentiation, and adherence of a pharyngeal strain of S. pyogenes was twofold greater to undifferentiated than differentiated keratinocytes. This is the first report of in vitro adherence of S. pyogenes to keratinocytes in a manner that emulates human impetigo. Adherence of only the impetigo strain, and not the pharyngeal strain of S. pyogenes or the nonpathogenic S. gorgonii isolate, was promoted by keratinocyte differentiation. This result provides a model system for investigating the molecular pathogenesis of streptococcal skin infections.

Bacterial Adhesion↗
PubMed1998

Reduced stability and bi-allelic, coequal expression of profilaggrin mRNA in keratinocytes cultured from subjects with ichthyosis vulgaris.

Ichthyosis vulgaris (IV) is an inherited scaling skin disorder in which expression of profilaggrin is reduced. Previous studies have indicated that the reduction is caused by defective post-transcriptional control of gene expression. Here we present evidence that profilaggrin mRNA in keratinocytes cultured from subjects with IV is intrinsically unstable and has a shorter half-life compared with that in normal cells. When IV-affected keratinocytes were treated with the protein synthesis inhibitor cycloheximide, the steady-state level of profilaggrin mRNA was increased due to stabilization of the transcript. In addition, the number of filaggrin repeats within the profilaggrin gene was studied. The number of filaggrin repeats (10-12) in individuals with IV did not differ from that of unaffected subjects. Expression of the gene was bi-allelic and coequal in both control and affected individuals. Our results suggest a model in which a labile ribonuclease and a stabilizing factor may modulate the profilaggrin mRNA steady-state level in normal cells, whereas the stabilizing factor may be absent or functionally inactive in IV-affected keratinocytes.

Alleles↗
PubMed1997

Harlequin ichthyosis keratinocytes in lifted culture differentiate poorly by morphologic and biochemical criteria.

Harlequin ichthyosis (HI) is a severe congenital ichthyosis in which massively thickened stratum corneum with abnormal barrier function often results in death of affected newborns. Survivors evolve into a severe nonbullous ichthyosiform erythroderma. Previously we have ascertained three biochemical phenotypes of HI, based on abnormal profilaggrin and K6 and K16 expression in epidermis. Submerged cultures of HI keratinocytes differentiated abnormally, but the three phenotypes were indistinguishable in vitro. We hypothesized that differentiation in submerged culture was insufficient to reflect in vivo biochemical abnormalities or that dermal components might be necessary for expression. To test these hypotheses HI keratinocytes and fibroblasts (n = 3) were grown on collagen gels at the air-medium interface in a cross-over design with normal keratinocytes and fibroblasts. Epithelia derived from lifted cultures were studied by light microscopy and immunocytochemistry and extracted for western blot analysis. In contrast to our prediction, lifted cultures of HI keratinocytes formed a poorly differentiated epithelium, and normal keratinocytes formed an epidermal-like tissue with expression of K1 and expression and processing of profilaggrin to filaggrin. In addition, the presence of HI fibroblasts consistently altered differentiation of both HI and normal keratinocytes, resulting in less complete morphologic differentiation. The findings suggest that both epithelial and mesenchymal elements of the skin from HI are affected but that the primary abnormality lies in the keratinocytes.

Adult↗
PubMed1997

Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity.

Filaggrin is an intermediate filament-associated protein (IFAP) that aggregates epidermal keratin filaments in vitro and is thought to perform a similar function during terminal differentiation in vivo. To test this function in living cells, we transiently expressed constructs encoding human filaggrin in both simple epithelial cells (COS-7) and rat keratinocytes. Scanning laser confocal microscopy showed that filaggrin-positive cells had collapsed keratin and vimentin intermediate filament (IF) networks, and that filaggrin partially co-localized with the IF networks. Filaggrin was also detected diffusely in the cytoplasm and nucleus. In contrast, when profilaggrin-like constructs, containing five filaggrin domains separated by the linker sequences, were expressed in cultured cells, immunoreactive granules formed. This finding is reminiscent of the insoluble nature of native profilaggrin that accumulates in keratohyalin granules in vivo, suggesting that the linker peptides (present in profilaggrin but not filaggrin) are important for granule formation. Cells expressing filaggrin also displayed disruption of the nucleus and the nuclear envelope; they rounded up and lost attachment to the substratum, in contrast to control cells over-expressing beta-galactosidase. This functional test of filaggrin in living cells supports its role in the reorganization and packing of keratin IF in epidermal differentiation. Moreover, the observed effects on cell morphology and nuclear integrity suggest that filaggrin may contribute to the form of apoptosis associated with terminal differentiation in epidermis.

Animals↗
PubMed1997

Nail-Tutor: an image-based personal computer program that teaches the anatomy, patterns of pathology, and disorders of the nails.

Despite the clinical relevance of the nails, the interpretation of the appearance of the nails is often neglected by clinicians and in medical training programs. To help overcome this we developed Nail-Tutor(TM), a personal computer program based on approximately 150 images that systematically teaches the anatomy, patterns of pathology, and diseases of the nails and tests the user's understanding of the material in the program. Nail-Tutor(TM) is intended for medical students, residents, other healthcare trainees, general practitioners, and dermatologists and requires approximately 3 hours to use. The program has a number of advantages over textbook-based instruction and supervised instruction. It does not require an instructor or textbook. In addition it uses animation to show an important anatomic relationship, graphic overlays to unambiguously point out key features of images, it can show key images side by side for comparison, and an examination to evaluate learning objectives. The program can be used as a stand-alone teaching module, a supplement to traditional instruction, a source for continuing education, or as a reference.

Computer-Assisted Instruction↗
PubMed1997

Protein phosphatase activity in human keratinocytes cultured from normal epidermis and epidermis from patients with harlequin ichthyosis.

We investigated serine/threonine protein phosphatase (PP) activity and the expression of PP2A during growth and differentiation of epidermal keratinocytes in culture. Keratinocyte PP activity was strongly inhibited by calyculin A and okadaic acid, indicating that the activity was mainly due to PP2A and PP1. The phosphatase activity decreased to about 20% of the initial (day 1) level by the time of confluence and to about 10% at day 7 postconfluence. In contrast to activity, the level of expression of the PP2A catalytic subunit protein and the mRNA for the two isoforms increased slightly over the period of growth. Keratinocyte differentiation was shown by a significant increase in profilaggrin expression after confluence. Keratinocytes were also cultured from individuals affected with harlequin ichthyosis. This severe hyperkeratotic skin disorder has abnormal lipid structures and is blocked in the PP2A-dependent conversion of phosphorylated profilggrin to the non-phosphorylated filaggrin. The PP activity in harlequin cultures was lower than in normal cultures (about 20% of the subconfluent normal control value) and decreased even further in confluent cultures. In contrast, the level of expression of the PP2A catalytic subunit protein and mRNA for the two isoforms was similar to that of normal keratinocytes and increased with confluence. These results suggest that PP activity in keratinocytes is regulated in a post-translational manner; they also support the possibility of impaired or reduced function of PPs in harlequin ichthyosis.

Blotting, Northern↗
PubMed1995

Decreased profilaggrin expression in ichthyosis vulgaris is a result of selectively impaired posttranscriptional control.

Ichthyosis vulgaris is an autosomal dominant disorder of keratinization characterized by mild hyperkeratosis and reduced or absent keratohyalin granules in the epidermis. Profilaggrin, a major component of keratohyalin granules, is reduced or absent from the skin of individuals with ichthyosis vulgaris. In this report, we have further characterized the molecular basis of low profilaggrin expression, which occurs in this disease. In situ hybridization revealed little profilaggrin mRNA in ichthyosis vulgaris-affected epidermis. In keratinocytes cultured from the epidermis of affected individuals, the abundance of profilaggrin was reduced to less than 10% of normal controls, while the mRNA level was decreased to 30-60% of controls. Expression of K1 and loricrin, other markers of epidermal differentiation, were not affected. Nuclear run-on assays indicated that the decrease in mRNA levels was not caused by aberrant transcription. Nucleotide sequencing of 5'-upstream, 3'-non-coding, and flanking regions of the profilaggrin gene from ichthyosis vulgaris-affected individuals revealed only minor changes, probably due to genetic polymorphisms. Our results indicate that defective profilaggrin expression in ichthyosis vulgaris is a result of selectively impaired posttranscriptional control.

Adult↗
PubMed1995

Retinoic acid regulates oral epithelial differentiation by two mechanisms.

The effect of retinoic acid (RA) concentration on differentiation of oral keratinocytes and the influence of fibroblasts on RA-dependent regulation were investigated in a lifted culture system. Keratinocyte differentiation was assessed by morphology, immunohistochemistry and immunoblotting. Filaggrin/profilaggrin and keratin 1 were used as biochemical markers for cornified epithelium and keratins 13 and 19 as markers for noncornified epithelium. Cultured oral keratinocytes in RA-free conditions differentiated in a manner that closely resembled the differentiation pattern of gingival epithelia in vivo. Increasing RA concentrations altered the in vivo-like terminal differentiation of oral keratinocytes by disruption of organized stratification, inhibition of filaggrin/profilaggrin and K1 expression, and stimulation of K13 and K19 expression. Differentiation of keratinocytes from both cornified and noncornified regions of the oral cavity varied in a similar manner in response to added RA, with the exception of K19 expression. K19 was consistently expressed at higher levels in keratinocytes originating from noncornified epithelial as compared to those from cornified epithelia. The level of RA regulation was ultimately dependent on the type of fibroblasts underlying the epithelial cells. Homologous fibroblasts rendered the oral keratinocytes less sensitive to the effects of RA than skin fibroblasts. In addition, at a given RA concentration, fibroblasts from cornified oral mucosa potentiated keratinocyte expression of RA sensitive markers of keratinization as compared to the influence exerted by fibroblasts originating from noncornified oral mucosa. These results indicate that the RA regulation of oral epithelial differentiation is mediated by two separate mechanisms: a direct, RA concentration-dependent effect, and an indirect, fibroblast-mediated effect.

Cell Differentiation↗
PubMed1995

Retinoic acid regulates oral epithelial differentiation by two mechanisms.

The effect of retinoic acid (RA) concentration on differentiation of oral keratinocytes and the influence of fibroblasts on RA-dependent regulation were investigated in a lifted culture system. Keratinocyte differentiation was assessed by morphology, immunohistochemistry and immunoblotting. Filaggrin/profilaggrin and keratin 1 were used as biochemical markers for cornified epithelium and keratins 13 and 19 as markers for noncornified epithelium. Cultured oral keratinocytes in RA-free conditions differentiated in a manner that closely resembled the differentiation pattern of gingival epithelia in vivo. Increasing RA concentrations altered the in vivo-like terminal differentiation of oral keratinocytes by disruption of organized stratification, inhibition of filaggrin/profilaggrin and K1 expression, and stimulation of K13 and K19 expression. Differentiation of keratinocytes from both cornified and noncornified regions of the oral cavity varied in a similar manner in response to added RA, with the exception of K19 expression. K19 was consistently expressed at higher levels in keratinocytes originating from noncornified epithelia as compared to those from cornified epithelia. The level of RA regulation was ultimately dependent on the type of fibroblasts underlying the epithelial cells. Homologous fibroblasts rendered the oral keratinocytes less sensitive to the effects of RA than skin fibroblasts. In addition, at a given RA concentration, fibroblasts from cornified oral mucosa potentiated keratinocyte expression of RA sensitive markers of keratinization as compared to the influence exerted by fibroblasts originating from noncornified oral mucosa. These results indicate that the RA regulation of oral epithelial differentiation is mediated by two separate mechanisms: a direct, RA concentration-dependent effect, and an indirect, fibroblast-mediated effect.

Cell Differentiation↗
PubMed1995

Familial pityriasis rubra pilaris.

BACKGROUND: Familial pityriasis rubra pilaris is a rare autosomal dominant skin disorder. Four individuals from one family are described who demonstrate clinical features compatible with a diagnosis of familial pityriasis rubra pilaris. Results of light and electron microscopic, immunocytochemical, and biochemical analysis of skin biopsy specimens from three of these four individuals are presented. OBSERVATIONS: All affected individuals demonstrated erythematous scaly skin with follicular prominence and islands of sparing. Inheritance was consistent with an autosomal dominant trait. Light and electron microscopic findings were compatible with those reported in sporadic cases of pityriasis rubra pilaris. Immunocytochemistry showed suprabasal staining with monoclonal antibody AE1. Immunoblot analysis revealed abnormal keratins with K6/16 expression, the possibility of an abnormal K14 or K16, and a 45-kd acidic keratin not normally expressed in epidermis. Because similar biochemical analyses have not been reported previously in other cases of pityriasis rubra pilaris (familial or sporadic), comparisons cannot be made. CONCLUSIONS: The observations suggest that the cutaneous abnormality in this family extends beyond clinical and morphological alterations to abnormalities in biochemical markers of epidermal differentiation.

Adult↗
PubMed1993

Independent regulation of two cytoplasmic processing stages of the intermediate filament-associated protein filaggrin and role of Ca2+ in the second stage.

One of the final events in cornification of epidermal cells is processing of profilaggrin to the keratin-associated protein filaggrin. Processing involves several proteolytic events and occurs in two discrete proteolytic stages (Resing, K. A., Walsh, K. A., and Dale, B. A. (1984) J. Cell Biol. 99, 1372-1378; Resing, K. A., Walsh, K. A., Haugen-Scofield, J., and Dale, B. A. (1989) J. Biol. Chem. 264, 1837-1846). In a keratinocyte cell line derived from newborn rat epidermis, these two stages are independently regulated. Profilaggrin was expressed when the cells reached confluence; processing to intermediates began 24-36 h later (stage one), with filaggrin appearing at 48 h (stage two). Stage two processing required calcium in the medium with maximum processing occurring at 5-10 mM. Furthermore, stage two processing was inhibited by nifedipine, a calcium channel blocker, suggesting that calcium influx activates this event. Second-stage processing was also inhibited by the protease inhibitor leupeptin, implicating calpain. Confluent cells had higher levels of calpain I than subconfluent cells; in confluent cells, two immunoreactive bands were detected, comigrating with inactive (80 kDa) and activated (78 kDa) calpain I. In cells processing profilaggrin, most of the calpain I was in the 78-kDa form, implying extensive activation, supporting a role for calpain in processing.

Animals↗
PubMed1993

Antisense profilaggrin RNA delays and decreases profilaggrin expression and alters in vitro differentiation of rat epidermal keratinocytes.

Ichthyosis vulgaris is an epidermal disorder in which profilaggrin expression is decreased or absent. To determine whether the ichthyosis vulgaris phenotype could be mimicked by eliminating profilaggrin expression, a rat epidermal cell line was transfected with a plasmid that directs the constitutive expression of an RNA that is antisense to normal profilaggrin mRNA. Non-transfected and neomycin-resistant cells not containing antisense plasmid that were grown in the neomycin analogue G418 served as controls. Immunoblot and immunofluorescence analysis showed that profilaggrin protein expression and processing to filaggrin were delayed by 3 to 4 d and decreased in transfected cells. Profilaggrin mRNA was detected in both control and transfected cells only after the cells reached confluence, whereas antisense RNA was detected in transfectants at all times, even prior to confluence. Ultrastructural examination revealed that keratohyalin granules were decreased in number, globular, and heterogeneous in appearance in transfected cells in-contrast to angular structures seen in control cells. Unexpectedly, stratification was impaired, intermediate filaments were noticeably reduced, and cornified cell envelope formation was delayed in transfectants. Unlike ichthyosis vulgaris keratinocytes, where keratin expression is unaffected, appearance of K1 and K10 was delayed and K1/K10 synthesis was delayed and decreased in transfected cells. The precipitous drop in 35S-methionine incorporation into cytoskeletal protein seen at confluence in control cells was delayed by 3 d in transfected cells. We conclude that, rather than producing the ichthyosis vulgaris phenotype, antisense profilaggrin RNA has more broad-reaching effects on in vitro differentiation of rat epidermal keratinocytes.

Animals↗
PubMed1992

Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.

Filaggrin is an intermediate filament-associated protein which functions to aggregate keratin intermediate filaments in the stratum corneum of mammalian epidermis. It is synthesized as a large precursor protein, profilaggrin, that consists of multiple filaggrin units and is localized in keratohyalin granules. In this report, we describe the characterization of cosmid genomic clones containing the human profilaggrin gene coding for 11 complete filaggrin repeats of 324 amino acids each. At the amino- and carboxyl-terminal ends of human profilaggrin are leader and tail peptide sequences of 293 and 157 amino acids, respectively, which differ from filaggrin. The leader peptide is composed of two distinct domains: an 81-residue segment which shows significant homology to the S-100 family of EF hand-containing calcium-binding proteins, and a hydrophilic second domain of 212 residues. The gene is divided into three exons, with one intron (approximately 9.6 kilobase pairs) in the 5' noncoding region and a second one of 570 base pairs between the EF hands. The position of intron 2 is identical to that of other members of the S-100-like family. The presence of an S-100-like domain suggests that profilaggrin binds calcium and that the calcium binding domain is functionally significant in the formation of keratohyalin and/or the subsequent processing of profilaggrin to filaggrin, both of which may be calcium-dependent events.

Amino Acid Sequence↗
PubMed1992

CHILD syndrome: lack of expression of epidermal differentiation markers in lesional ichthyotic skin.

Congenital hemidysplasia with ichthyosiform erythroderma and limb defects (CHILD) syndrome is a rare genetic disorder. The epidermal abnormalities associated with the unilateral ichthyosis have previously been examined only by morphology. In order to describe these abnormalities more completely we analyzed the expression of markers of epidermal differentiation (keratins and filaggrin), grew keratinocytes in culture, and correlated the results with ultrastructural features. Expression of all differentiation markers was significantly reduced or absent, whereas keratins K5 and K14 and keratins K6 and K16 were strongly expressed in lesional epidermis, suggesting that basal cell keratin expression was not down-regulated as in normal epidermis and that lesional keratinocytes mature via an abnormal pathway. When removed from the tissue and grown in culture, keratinocytes from lesional and non-lesional biopsies had similar phase microscopic morphology as well as keratin and profilaggrin expression, in contrast to the extreme differences in vivo. Lesional keratinocytes also had similar contents of keratin filaments and keratohyalin, but showed abnormal accumulation of intercellular vesicles and debris and altered cell-cell and cell-substratum interaction. Comparison of the results in tissue and in culture suggests that systemic or dermal factors influence the abnormal structural protein expression and ichthyosiform epidermal differentiation seen in CHILD syndrome, but that lesional keratinocytes maintain abnormalities in the secretion and accumulation of extracellular material in vitro similar to the lesional tissue in vivo.

Abnormalities, Multiple↗
PubMed1992

Neutrophil-mediated damage to human gingival epithelial cells.

Polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of inflammatory gingivitis and periodontitis. To further study the role of PMNs in mediating gingival injury, we cocultured these cells in vitro with monolayers of human gingival epithelial cells. Scanning electron microscopy revealed that the epithelial cells were homogeneous and SDS-PAGE/immunoblot analysis identified the presence of keratins K3, K13 and the K6/16 pair which authenticated the oral origin of the cells. Injury to the gingival cells was determined by scanning electron microscopy and measurement of cell detachment and cytolysis. Unstimulated PMNs produced minimal lysis or detachment, but PMNs stimulated by phorbol myristate acetate produced marked epithelial cell detachment without lysis, which was time- and PMN-dose-dependent. Supernatants of activated PMNs were similarly effective, indicating that the mediator was a stable soluble substance. Elastase and cathepsin G, two neutral proteases of PMN origin, produced time- and concentration-dependent detachment of gingival epithelial cells, suggesting that these enzymes may mediate this form of injury. In other studies, gingival epithelial cells were exposed to PMN myeloperoxidase (MPO), chloride and glucose plus glucose oxidase (GO) as a hydrogen peroxide (H2O2) generating system. The toxic oxygen species produced by this system caused lysis of the epithelial targets which was dependent on the duration of incubation and the concentrations of MPO and GO. Azide, an inhibitor of MPO, and catalase, a scavenger of H2O2, inhibited the lytic activity of this system. Scanning electron micrographs of gingival epithelial cells cocultured with activated PMNs showed lifting of the cells from the plating surface, while target cells attacked by the MPO system revealed extensive damage of cell membranes. These studies indicate that activated PMNs cause nonlytic detachment injury to gingival epithelial cells which may be mediated by digestion of their extracellular matrix by granule neutral proteases. Furthermore, PMN MPO is capable of generating toxic oxygen species which can lyse these epithelial cells. Collectively, these actions could have profound adverse effects on the function and integrity of the gingival epithelium.

Cell Death↗