Biomedical subjects

J S Perlish

Publications and source records attributed to J S Perlish.

At least 19 recordsLinked to original sources

PubMed1997

There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model.

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Antibodies, Monoclonal↗

PubMed1995

Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro.

The purpose of this study was to determine whether nidogen, the linkage protein of the basal lamina, is of epidermal or dermal origin. The development of the basal lamina was studied in an in vitro skin model. Preputial fibroblasts seeded onto a nylon mesh attached, proliferated, and developed a rich extracellular matrix (dermal model). Preputial keratinocytes were added to the dermal model to form a keratinocyte dermal model that ultrastructurally resembled in many respects human skin. Ultrastructural analysis revealed early stages of dermal development, including an incomplete basal lamina, aggregates of dermal filamentous material connecting to the lamina densa, bundles of 10-nm microfibrils, formation of premature hemidesmosomes, anchoring filaments, and anchoring fibrils. The cell origin of nidogen was determined in the dermal model and in the epidermal and dermal components of the keratinocyte dermal model. Specific antibodies and a cDNA probe for nidogen were used for immunofluorescence microscopy, Western and Northern blots, and for in situ hybridization studies. Our data show that fibroblasts are the only source of nidogen during early basal lamina formation. Although fibroblasts can synthesize nidogen and deposit it in the dermal matrix, no basal lamina will form unless they are recombined with keratinocytes. This suggests that the epidermis plays a major regulatory role in the production and assembly of nidogen into the basal lamina.

Basement Membrane↗

PubMed1993

Collagen fibrillogenesis in a three-dimensional fibroblast cell culture system.

The purpose of this study was to follow collagen fibril formation in a newly developed three dimensional cell culture system. Human neonatal foreskin fibroblasts were grown on a nylon mesh in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. Fibrillogenesis was initiated by the addition of 50 micrograms/ml ascorbate to confluent cultures. Sample meshes were processed for electron microscopy or immuno-electron microscopy. Fibrils approximately 20-30 nm in diameter, with 67 nm periodicity, were first detected five days after the addition of ascorbate. As cultures progressed, cells organized into parallel layers between which collagen fibers continued to form and increase in diameter. By day 50, fiber diameter ranged from 30 to 80 nm and large bundles were seen. No collagen fibril formation occurred in control cultures to which no ascorbate was added. However, large amounts of microfibrils were observed. Antibodies against the aminopropeptide of type I procollagen were found to bind to fibrils with diameters less than 34 nm while antibodies against the aminopropeptide of type III collagen bound primarily to fibers which ranged from 35-54 nm in diameter. We believe that this system, which morphologically resembles a normal dermis, will serve as an excellent model for the study of collagen fibrillogenesis.

Cells, Cultured↗

PubMed1993

Culturing keratinocytes and fibroblasts in a three-dimensional mesh results in epidermal differentiation and formation of a basal lamina-anchoring zone.

The purpose of this study was to characterize an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 3 and 5 weeks and then processed for electron microscopy. Keratinocytes showed reconstruction of an epidermis consisting of a basal layer with hemidesmosomes, a stratified epithelium with tonofilaments and desmosomes, a granular layer with keratinosomes and keratohyaline granules, and a transitional stratum corneum. Anchoring filaments, lamina densa, anchoring fibrils, bundles of elastin-associated microfibrils (diameters 10 nm) and fine collagen fibrils were formed. Collagen fibrils near the epidermis were much thinner than those in the lower levels. The present study shows that the dermal model containing metabolically active fibroblasts in their natural environment will support epidermal morphogenesis and differentiation including the formation of a basal lamina and anchoring zone.

Basement Membrane↗

PubMed1993

Collagen alterations in chronically sun-damaged human skin.

The major histological characteristic of sun-damaged skin is the accumulation of an elastotic material that appears to replace collagen. This elastotic material consists primarily of elastin and histological studies suggest a large loss of collagen in the dermis of chronically sun-damaged skin. In this study, we examine the content and distribution of collagen and procollagen in sun-damaged human skin. The total collagen content of sun-damaged skin was 20% less than nonsolar-exposed skin (524 micrograms collagen per mg total protein in sun-damaged skin and 667 micrograms collagen per mg total protein in nonsolar-exposed skin). In addition, there was a 40% decrease in the content of intact amino propeptide moiety of type III procollagen in sun-damaged skin (0.68 U per 50 mg wet weight) as compared to nonsolar-exposed skin (1.12 U per 50 mg wet weight). The data suggest that this change in collagen content is due to increased degradation. The distribution of collagen in sun-damaged skin was examined by indirect immunofluorescence. Mild digestion of sun-damaged skin with elastase removed the elastin and revealed the presence of collagen in the elastotic material. Therefore, the elastin appears to mask the presence of collagen fibers in the dermis of sun-damaged skin.

Aged↗

PubMed1993

Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.

Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.

Cell Adhesion Molecules, Neuronal↗

PubMed1993

[Physiopathology of scleroderma].

Scleroderma (systemic sclerosis) is a connective tissue disease characterized by an excessive accumulation of collage in the main body organs. The subsequent progressive fibrosis may result in severe functional impairment of the tissue(s) involved. In this paper, recent advances in the understanding of the disease are reviewed. Particular emphasis is placed on the role played by vascular lesions, inflammatory cell infiltrates, autoimmunity and possibly abnormal secretion of cytokines in the disturbance of connective tissue production. A better knowledge of the pathophysiological process involved in scleroma might lead to the development of new therapeutic approaches.

Antibody Formation↗

PubMed1992

Immunochemical analysis of human kidney reticulin.

This study characterized the nature of reticulin fibrils from human kidney cortex by immunochemical analysis. Controls consisted of type I collagen fibrils derived from the kidney parietal capsule. Most of the fibrils in the capsule ranged in diameter from 60 to 80 nm whereas reticulin fibrils from the cortex ranged from 30-45 nm. Immunochemistry by light and electron microscopic examinations was carried out with antibodies directed against type I and type III collagens, their corresponding aminopropeptides, and decorin (PG-II). The ratio of type I to type III collagen was determined by cyanogen bromide peptide digests. This study showed that reticulin fibrils are hybrids of type I and type III collagens. Double immunoelectron microscopic examination showed that fibrils 20-25 nm consisted mainly of type I collagen some of which retained their aminopropeptide. Larger fibrils 30-35 nm labeled simultaneously for type I and type III collagens. However, most fibrils with diameters between 40-55 nm labeled for type III collagen and its corresponding aminopropeptide. No decorin was detected at the surface of reticulin fibrils. Purified reticulin consisted of 82% type III and 18% type I collagen whereas collagen derived from the capsule revealed 76% type I and 24% type III. The presence of the aminopropeptide of type III procollagen in reticulin fibrils is a striking feature and may play a role in regulating their diameter.

Collagen↗

PubMed1991

Decorin interacts with fibrillar collagen of embryonic and adult human skin.

Biglycan (PG-I, BGN) and decorin (PG-II, DCN) are small proteoglycans that have been isolated in cartilage, skin, and bone. Although the function of biglycan is unknown, there is biochemical evidence that decorin interacts with fibrillar collagens (type I, type II). The purpose of this study was to perform immunofluorescence and immunoelectron microscopy and immunoblotting of human embryonic and adult skin with antibodies directed against biglycan and decorin. These antibodies were developed against synthetic peptides of the core proteins of biglycan (amino acid sequence 11-24) and decorin (amino acid sequence 5-17). Immunofluorescence microscopy showed that decorin stained embryonic and adult collagen fibrils. Biglycan did not stain collagen, but it appeared to stain the pericellular matrix of embryonic mesenchymal cells. Immunoelectron microscopy revealed labeling of all collagen fibrils with decorin antibodies regardless of their diameter, often at 60-nm periodicity. Positive stains suggest that most of the labeling was in the gap of the D-period (d and e bands) and also in one of the steps (c band). Decorin was identified by immunoblotting in fetal and adult skin. Also, significant amounts of core protein was identified lacking the dermatan sulfate chain. This study suggests that the core protein of decorin interacts with collagen fibrils although its specific function remains unknown.

Adult↗

PubMed1991

Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis.

Collagen monomers, oligomers, and fibrillar structures were isolated from chick tendons at various stages of development and studied by rotary shadowing. Monomers of Type I collagen, solubilized in 0.15 M NaCl solutions, were mostly present as collagen, pN-collagen, and pC-collagen with few procollagen molecules. They did not form polymers, nor were they associated with a carrier. Dimers of fibrillar collagen molecules were arranged in a 4-D stagger, suggesting that this was the preferred molecular interaction for the initiation of collagen fibrillogenesis. Type XII collagen molecules were mostly free, but some were attached by their central globular domain to one end of free fibrillar collagen molecules. Tenascin and Type VI collagen were also identified. The fibril populations consisted of collagen and beaded structures. These fibrils consisted of beads (globular domains) about 23 nm in diameter, separated by a period about 27 nm in length. Beads were linked by filamentous structures. These beaded fibrils probably represent the microfibrils of elastin.

Animals↗

PubMed1991

Self reactive repertoire of tight skin mouse: immunochemical and molecular characterization of anti-topoisomerase I autoantibodies.

Tight skin (TSK) mice develop cutaneous hyperplasia accompanied by histopathological alterations of skin and collagen metabolism similar to those described in human scleroderma. Diffuse scleroderma, the most severe form of progressive systemic sclerosis, is associated with the production of autoantibodies specific for Scleroderma 70 antigen (topoisomerase I). Our studies show that there is an increase in the level of serum anti-topoisomerase I (topo I) autoantibodies in aged TSK mice. The monoclonal antibodies isolated from TSK mice bind to epitopes which interact with autoantibodies from scleroderma patients. A significant number of TSK monoclonal anti-topo I antibodies and serum immunoglobulin (Ig) from aged TSK mice bear a cross reactive idiotype (Id) recognized by a syngeneic monoclonal anti-Id antibody obtained from a 2 month-old TSK mouse. Analysis of V gene usage by monoclonal anti-topo I antibodies showed that the majority of these antibodies are encoded by VH genes derived from VHJ558 family pairing with VK genes from various families in a stochastic manner.

Animals↗

PubMed1990

Type I and type III collagen interactions during fibrillogenesis.

There is some evidence that type I and type III collagens may be present in the same fibril. In order to demonstrate this, double labeling immunofluorescence microscopy and immunoelectron microscopy were performed with antibodies directed against the collagen molecule and the aminopropeptide domains of type I and type III procollagens using embryonic (postabortion) and adult human skin. Double indirect and protein A immunoelectron microscopy were carried out with 5- and 15-nm gold particles. Skin extracts were also studied by immunoblotting. Double immunofluorescence microscopy with antibodies against type I and type III collagen molecules revealed patterns of fluorescence that were identical in both fetal and adult skins. Immunofluorescence microscopy using an antibody directed against the aminopropeptide of type III procollagen labeled the entire dermis in both embryonic and adult skins. In contrast, although the aminopropeptide of type I procollagen was present throughout embryonic dermis, it was markedly reduced in adult dermis, except for the epidermo-dermal junction. Double immunoelectron microscopy of fetal skin revealed labeling of the aminopropeptide of type I and type III procollagens on the same thin (20-30 nm) fibrils. Large type I fibrils (90-100 nm) were coated with type III collagen molecules and their corresponding aminopropeptide but not with the aminopropeptide of type I procollagen. The aminopropeptide of type I procollagen was present on thin fibrils only at the epidermo-dermal junction in adult skin. Immunoblotting of skin extracts revealed the presence of both pN-type III procollagen (collagen plus the aminopropeptide) and pN-type I procollagen in fetal skin, but only pN-type III in adult skin. This study demonstrates that type I and type III collagens coexist within the same fibril and that the aminopropeptide of type III procollagen is present at the surface of type I collagen fibrils that apparently have reached full growth.

Adult↗

PubMed1989

Alterations in dermal collagen in ultraviolet irradiated hairless mice.

Chronic exposure of the skin to sunlight results in severe dermal connective tissue damage that is characterized by the basophilic degeneration of collagen and the accumulation of an elastotic material. The aim of this study was to identify changes in collagen (the major structural protein of the skin) in ultraviolet irradiated mouse skin using immunochemical and biochemical techniques. Specific antibodies directed against the aminopropeptide of type III procollagen were used in immunofluorescence and immunoblotting studies. Immunofluorescent staining of irradiated and nonirradiated mice skin showed that the aminopropeptide of type III procollagen was distributed throughout the dermis in a pattern similar to that observed for type I collagen. Extracts of irradiated (5 and 10 weeks) and nonirradiated skins were then subjected to immunoblotting techniques. Levels of pN alpha 1, type III procollagen (measured by radioimmunoassay) were reduced in the extracts prepared from skins of mice that were irradiated for 5 and 10 weeks. Immunoelectron microscopy verified the loss of pN alpha 1 type III procollagen in irradiated skin. Collagen fibers of nonirradiated skin demonstrated normal labeling with antibody directed against the aminopropeptide of type III procollagen. In contrast, collagen fibers of 10 week irradiated skin failed to label with this antibody. The pN alpha 1 type III collagen is known to coat type I collagen fibers of normal skin. Therefore, its absence from the surface of type I collagen fibers of irradiated skin may play a role in the development of the elastotic material.

Animals↗

PubMed1989

Immunological modulation of dermal fibroblasts in scleroderma.

Scleroderma can be considered as a model to study the mechanism of fibrosis. Lymphocytes and monocytes/macrophages are recruited during the inflammatory process associated with this disorder. These activated blood cells release a series of factors--lymphokines and monokines--which in turn recruit more fibroblasts, possibly stimulate their proliferation, and regulate their synthesis of collagen and glycosaminoglycans. These factors represent both positive and negative modulators for these processes. It may be the absence of an inhibitory factor(s) and/or presence of other stimulator(s) which allow the fibroblast to synthesize collagen in an uncontrolled manner, leading to the increased accumulation of this protein in the dermis. Although there is an increase in collagen accumulation in vitro, the ratio of type I/type III collagen remains unaltered. It has been suggested that an increase in T-helper cells could explain the production of collagen stimulatory lymphokines. Collagen production may also be regulated by a feedback mechanism involving the amino and carboxyl propeptides of procollagen. In scleroderma this mechanism may be altered by the lymphokine/monokine factor(s) which affect collagen synthesis. Fibrosis probably is not the primary event in scleroderma but represents the final or terminal stage of the disease. Future research on the interactions which take place between the cells of the immune system and fibroblasts may clarify the mechanism of fibrosis in scleroderma and other disorders.

Cell Division↗

PubMed1988

Identification of collagen fibrils in scleroderma skin.

Skin from early and late stages of scleroderma has been shown to contain large amounts of thin (30-40 nm diameter) collagen fibrils that may be present in bundles or intermingled with large diameter fibrils (90-120 nm). The nature of these fibrils is unknown. Skin biopsies were obtained from involved areas of nine patients with progressive systemic sclerosis (PSS), one case of generalized morphea, one case of morphea, and six normal controls. Intact skin was analyzed by immunoelectron microscopy (IEM), while extracts were subjected to sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE), Western immunoblotting, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Fine fibrils 20-40 nm in diameter in the mid to lower dermis of scleroderma skin were labeled with antibodies directed against the aminopropeptide (AP) of type III procollagen. Antibodies directed against the AP of type I procollagen labelled fine fibrils in the lower dermis. Larger fibrils (80-120 nm) did not label. pN alpha 1 (III) was found to be present in both normal and scleroderma skin. Extracts of scleroderma skin contained 2.5 times the amount of pN (III) collagen and 3.0 times the amount of fibronectin as did extracts of normal skin. The data indicate that the increase in thin fibrils in scleroderma skin is most likely due to an increase in type III collagen, which retains the AP at its surface.

Adult↗

PubMed1988

Procollagen intermediates during tendon fibrillogenesis.

The purpose of this study was to correlate ultrastructural features of tendon collagen fibrils at various stages of development with the presence of procollagen, pN-collagen, pC-collagen, and the free amino propeptides and carboxyl propeptide of type I procollagen. Tendons from 10-, 14-, and 18-day chicken embryos reveal small, well-defined intercellular compartments containing collagen fibrils with diameters showing a unimodal distribution. At 21 days (hatching) and 9 days (post hatching) and at 5 weeks (post hatching), the compartments are larger, less well-defined, and there is multimodal distribution of tendon fibril diameters. Procollagen and the intermediates pN-collagen and pC-collagen are present in tendons up to 18 days. Thereafter there is a marked reduction in procollagen, whereas the intermediates persist throughout all stages of development. Similarly, free amino propeptides and carboxyl propeptides of type I procollagen were found at all stages. The amino propeptide of type III procollagen was restricted to the peritendineum until 7 weeks post hatching. At that time, a network of fibrils containing the amino propeptide of type III procollagen was seen delineating well-circumscribed compartments of collagen fibrils throughout the entire tendon. This study supports the notion that pN- and pC-collagen have an extracellular role and participate in collagen fibrillogenesis.

Animals↗

PubMed1987

Amino and carboxyl propeptides in bone collagen fibrils during embryogenesis.

Collagen fibrillogenesis was studied in tibiae of chick embryos, 9, 11, and 14 days old. Specimens were incubated with antibodies against the amino and the carboxyl propeptides of type-I collagen and subjected to ferritin-labelling immuno-electron microscopy. The amino propeptide was found in thin fibrils, 20-40 nm in diameter, distributed at 60-nm periodicity. The carboxyl propeptide antibody labelled a wide spectrum of fibrils, although the majority were in the range of 40-100 nm, distinctly larger than those labelled with the amino propeptide antibody. The presence of pN (amino propeptide plus collagen) and pC (carboxyl propeptide plus collagen) collagen was also demonstrated by Western blotting in all specimens. This study suggests that the sequence of propeptide removal may regulate collagen fibril diameter.

Animals↗

PubMed1987

The carboxylpropeptide of type I procollagen in skin fibrillogenesis.

Previous studies suggested that the aminopropeptide of type I procollagen may initiate fibril formation. The purpose of this investigation was to study the location of the carboxylpropeptide of type I procollagen during collagen fibrillogenesis. Chick embryonic and posthatching skin specimens were studied by immunofluorescence and immunoelectron microscopy and by immunoblotting with antibodies against the amino and carboxylpropeptide of type I procollagen. The carboxylpropeptide was demonstrated at the surface of collagen fibrils, 20-40 nm in diameter (10-day embryos) and in fibrils, 40-65 nm (21-day embryos). In addition, the carboxylpropeptide was found at the cell surface and free in the ground substance. The aminopropeptide was only seen in fibrils, 20-30 nm in diameter, as previously reported. Ratios of pN-collagen/pC-collagen increased from 16 days embryonic to 3 and 9 days postembryonic skins. This study suggests that both pN-collagen (aminopropeptide plus collagen) and pC-collagen (carboxylpropeptide plus collagen) participate in fibrillogenesis.

Animals↗