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Biomedical subjects

R C Page

Publications and source records attributed to R C Page.

At least 109 records · Page 6Linked to original sources
PubMed1990

Effect of cell donor age on the synthetic properties of fibroblasts obtained from phenytoin-induced gingival hyperplasia.

Diploid fibroblasts obtained from explants of human gingiva and maintained in vitro undergo a several-fold decrease in protein and collagen synthesis as a function of increasing donor age. Using drug-induced gingival hyperplasia as a model, we performed experiments to learn whether fibroblasts derived from hyperplastic tissue behave in a similar manner. Fibroblast strains were established from explants of hyperplastic gingiva obtained from 10 patients chronically ingesting phenytoin and ranging in age from 9 to 45 years. Protein production and degradation were compared to previously reported data similarly obtained from periodontally normal donors ranging in age from 12 to 68 yr. The total quantity of protein and collagen produced by the phenytoin cells was significantly greater than previously reported for cells from normal gingiva. No donor age-related decrease in protein and collagen production nor in the proportion of cell synthetic activity committed to collagen production was observed for cultures of phenytoin cells. The gross pattern of proteins produced, as assessed by 2-dimensional gel electrophoresis, was unrelated to donor age in both normal and phenytoin cells, but three polypeptides ranging in size from about 20 kD to 40 kD that were not found in the cultures of normal cells were produced by five of seven phenytoin cells strains. The observations demonstrate that the phenytoin cells do not undergo the donor age-dependent decrease in synthesis observed for normal cells. This abnormality may account in part for the phenytoin-induced hyperplasia. The phenytoin cells appear to be a unique phenotype.

Adolescent↗
PubMed1990

Relationship between gingival crevicular fluid levels of aspartate aminotransferase and active tissue destruction in treated chronic periodontitis patients.

Data from several sources demonstrate that disease-active and disease-inactive periodontal pockets exist, and that disease progression occurs in bursts of activity. Currently used diagnostic procedures do not distinguish between disease-active and disease-inactive sites at any given point in time. We report the results of studies aimed at determining whether levels of the enzyme aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) are associated with disease activity as assessed by the level of gingival inflammation and probing attachment loss. 25 previously treated periodontitis patients participating in a quarterly recall maintenance program, who had experienced recurrent periodontal deterioration, served as experimental subjects. Patients were evaluated at 3-month intervals for 2 years. Values for plaque index, gingival index, and probing attachment level were recorded, and 30-second samples of gingival fluid harvested from the mesiobuccal aspect of the 4 first molars and the distal of the 4 lateral incisors. GCF volume was measured using a Periotron 6000, and AST activity was measured by a standard method. Sites were ranked in a hierarchy based on the degree of certainty of attachment loss as well as the severity of gingival inflammation, and the relationship of the values to AST levels was determined. Three models were used to analyze the resulting data, and all led to the same conclusion. Maximum enzyme level was significantly elevated at sites with confirmed disease activity as assessed by attachment loss, with maximum AST levels 725 units higher at these sites, on average, than at other sites (p less than 0.0001). Our data support the idea that an objective diagnostic test, based on levels of AST in GCF, that distinguishes between disease-active and disease-inactive sites may be possible.

Aspartate Aminotransferases↗
PubMed1990

Isolation of a fibroblast attachment protein from cementum.

Cementum forms the interface through which soft connective tissue of the periodontium is attached to the root surface. The interactions between cementum and connective tissue are not completely understood and whether cementum influences periodontal connective tissue formation and regeneration is not clear. We have examined the effect of cementum components on the attachment of gingival fibroblasts. Cementum was harvested from healthy human and bovine teeth and extracted sequentially in 0.5 M CH3COOH, 4 M guanidine and bacterial collagenase. Fibroblast attachment was measured using 51Cr-labelled human gingival fibroblasts on tissue culture plates previously incubated with cementum components. Results showed that all three extracts mediated fibroblast attachment and attachment was dependent on concentration and incubation time. The attachment activity was not destroyed by digestion with bacterial collagenase or by antibodies to fibronectin and laminin. However, it was inhibited by a peptide containing the amino acid sequence RGD. By gel filtration or HPLC using a DEAE-cellulose column several proteins with attachment activity were fractionated. SDS-polyacrylamide gel electrophoresis revealed that HPLC fraction eluted by 0.2-0.3 M NaCl contained a protein with molecular weight 55 kDa as a major component. This protein was isolated and shown to promote fibroblast attachment, and optimal attachment occurred at a concentration of 2 micrograms/ml. We conclude that cementum contains substances capable of mediating fibroblast attachment and that these substances play an important role in periodontal connective tissue formation and regeneration by facilitating fibroblast attachment to root surfaces.

Amino Acid Sequence↗
PubMed1990

Assessment of the efficacy of a nonsteroidal anti-inflammatory drug, Naprosyn, in the treatment of gingivitis.

A double-blind, placebo-controlled trial was conducted to determine the effects of the nonsteroidal anti-inflammatory drug Naprosyn (naproxen) on gingival inflammation. The enrollment of 114 patients provided 102 patients valid for efficacy evaluation, each having a mean gingival index (GI) score of 1.5 or greater at test-teeth sites. Patients were given oral Naprosyn 500 mg b.i.d. or placebo for 30 days. At 28 d, full-mouth prophylaxis was performed. Gingival index, modified sulcular bleeding index (SBI), and plaque index (PII) scores were taken at baseline, at 28 d, and at 30 d. When the 28-d index measurements were compared to baseline, the drug had no significant effect on plaque index scores or gingival inflammation. Statistically, Naprosyn enhanced the resolution of gingival inflammation following removal of microbial plaque. Thus, although this drug does not suppress the inflammation-inducing properties of plaque, Naprosyn may enhance recovery following plaque removal.

Adult↗
PubMed1990

Effect of sampling time and repetition on gingival crevicular fluid and aspartate aminotransferase activity.

Tests based on the composition of gingival crevicular fluid (GCF) for detection of active periodontitis require a better understanding of sampling variables than currently exists. We have studied the effects of sample time and repetition on the presence and activity of aspartate aminotransferase (AST). Two 30-second samples of GCF were harvested within 10-minute intervals from 192 teeth with periodontitis. GCF sample size and AST activity were measured. GCF volume and AST activity of first samples were each approximately 10% greater than for second samples. The differences were significant. AST activity correlated positively with gingival index scores and probing pocket depth. Samples were also harvested from groups of 4 teeth during 5-, 10-, 20- and 30-s periods with 1-min intervals and varying sample order. For these samples, first samples contained the greatest amount of enzyme activity, regardless of sample time. When only first samples were considered, the 5- and 10-s samples showed more total activity than the 20- and 30-s samples, and differences were statistically significant. Flow rate for the 5-s sample was always higher than for all other samples, regardless of its position in the sampling sequence. Our observations are consistent with the existence of a reservoir of AST activity that is, in major part, depleted during the first 5 to 10 s of sampling, and that requires a time period of more than 10 min to reequilibrate. Five- to 10-s samples may distinguish active disease better than 20- or 30-s samples, since most of the activity is taken onto the strip in the first few seconds, and the activity is subsequently partly inactivated or diluted by the uptake of fluid less rich in enzyme activity.

Aspartate Aminotransferases↗
PubMed1990

Measurement of interleukin-1 alpha and -1 beta in gingival crevicular fluid: implications for the pathogenesis of periodontal disease.

Samples of gingival crevicular fluid (GCF) were harvested from sites manifesting features characteristic of active disease including inflammation, periodontal attachment loss, and radiographic signs of alveolar bone destruction in untreated patients with advanced periodontitis. The presence and concentrations of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) were measured using ELISAs specific for these cytokine molecules. IL-1 alpha and/or IL-1 beta were identified in the GCF of 15 of 15 patients having untreated periodontitis. Ninety percent (71 of 79) of the sites tested contained measureable amounts of IL-1, with IL-1 beta as the more frequently occurring form. IL-1 alpha levels ranged from 0.23 nM to 13.9 nM in the GCFs. IL-1 beta levels were between 0.04 nM and 5.28 nM. Marked reductions of total IL-1 levels were observed following effective treatment. Both forms of IL-1 messenger RNA (mRNA) were detected in 17 of 17 gingival tissue samples from 6 patients. These results demonstrate that IL-1 is produced and released locally in periodontal disease at concentrations sufficient to mediate tissue inflammation and bone resorption. IL-1 may serve as a marker of periodontal tissue destruction.

Adult↗
PubMed1990

Relationship between levels of aspartate aminotransferase in gingival crevicular fluid and gingival inflammation.

Data from several sources demonstrate that disease-active and disease-inactive periodontal pockets exist, but currently available diagnostic procedures do not permit identification of disease-active sites at any given point in time. Using the experimental gingivitis model, we have performed studies aimed at determining whether levels of the enzyme aspartate aminotransferase (AST) in gingival crevicular fluid correlate with the presence and extent of periodontal inflammation. Gingival inflammation was assessed using the Gingival Index and the Sulcular Bleeding Index, and enzyme activity was measured using a standard procedure. Our data reveal a statistically significant association between AST values and Gingival Index scores for spontaneously occurring lesions (p less than 0.02-0.04) and experimentally induced lesions (p less than 0.0001), as well as the extent of change in these values during developing experimental gingivitis (p less than 0.0001) and resolving experimental gingivitis (p less than 0.0001). The data demonstrate that AST levels can be used to assess the presence and extent of periodontal inflammation.

Adolescent↗
PubMed1990

Serum antibody response to antigens of oral gram-negative bacteria by cats with plasma cell gingivitis-pharyngitis.

The etiology of a form of periodontal disease in domestic cats known as plasma cell gingivitis-pharyngitis is not understood. Actinobacillus actinomycetemcomitans and Bacteroides species have been strongly implicated as the cause of periodontitis in humans and other mammalian species, and most affected patients manifest serum antibodies reactive with the infecting bacteria. We and others have isolated Bacteroides species from the oral flora of cats. Using enzyme-linked immunosorbent assay (ELISA) and immunoblot procedures, we measured serum antibodies in affected and control cats reactive with human isolates of A. actinomycetemcomitans, B. gingivalis, and B. intermedius, and purified lipopolysaccharide (LPS) from these and other species, and Bacteroides of cat origin. Affected cats had serum antibody titers reactive with these Gram-negative anaerobic bacteria that were significantly elevated relative to those of normal control cats. The quantitatively major antigens recognized by cat serum antibodies are proteins; this contrasts sharply with serum antibodies from humans with juvenile periodontitis, where LPS is the quantitatively major antigen fraction. Our data support the idea that plasma cell gingivitis-pharyngitis in cats may have a bacterial etiology, and that Gram-negative anaerobes similar to those that cause periodontitis in humans and other mammals may be involved.

Animals↗
PubMed1989

Collagen synthesis by human fibroblasts. Regulation by transforming growth factor-beta in the presence of other inflammatory mediators.

We have examined the combined effects of transforming growth factor-beta (TGF-beta), serum and gamma-interferon (gamma-IFN) on collagen synthesis by fibroblasts and compared the response of fibroblast subpopulations to TGF-beta. Human diploid fibroblasts were treated with TGF-beta alone and with serum of gamma-IFN. Cells were labelled with radioactive amino acids, and collagen production was measured as collagenase-digestible radioactivity. Collagen mRNA was determined by a solution-hybridization assay using procollagen-alpha 1[I] cDNA clone HF 677. The results showed that either serum or TGF-beta increased incorporation, collagen production and mRNA by fibroblasts approx. 2-fold; however, collagen synthesis relative to total protein synthesis and collagen mRNA relative to total polyadenylated [poly(A)+] RNA were not affected. Only serum activated cell growth. Collagen production increased approx. 4-fold in cells exposed to both TGF-beta and serum, and this increase was equal to that expected for an additive effect by both components. Treatment with gamma-IFN decreased collagen production and collagen mRNA to 44 and 40% respectively, whereas total incorporation and poly(A)+ RNA were affected only marginally. Cells exposed simultaneously to both gamma-IFN and TGF-beta produced less collagen and contained less mRNA than did those treated with TGF-beta alone. The gamma-IFN decreased collagen synthesis in control and TGF-beta-treated cultures to a similar extent, and TGF-beta increased collagen synthesis 2-fold in cells pre-treated with gamma-IFN. Fibroblast strains obtained in medium containing plasma-derived serum synthesized approximately half as much collagen as did cells derived from the same explant in the presence of fresh human serum, and TGF-beta stimulated collagen production and mRNA in both cell strains. We conclude that TGF-beta, serum and gamma-IFN regulate collagen synthesis by independent mechanisms, and that the combined action of these components plays a significant role in regulating collagen synthesis during wound healing and tissue repair.

Cells, Cultured↗
PubMed1989

Effects of interleukin-1 on fibroblast extracellular matrix, using a 3-dimensional culture system.

This study describes the alterations induced by Interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta) on fibroblast-synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen-coated Terasaki wells for 3 or 6-9 weeks to create 3-dimensional cell-containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL-1 alpha or IL-1 beta (10(-9) M) at 37 degrees C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL-1 alpha and IL-1 beta induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross-sectional area and an increased density of the matrices. Three-week matrices were reduced 26% and 45% by using IL-1 alpha and IL-1 beta, respectively. Comparable values obtained by using 6-week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL-beta than with IL-1 alpha. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3-week matrix following IL-1 beta incubation. There was also a decrease in collagen type 1 staining of 9-week matrices treated with IL-1 alpha or IL-1 beta. These studies show that IL-1 has an effect on fibroblast-synthesized extracellular matrix and indicate that the effects of IL-1 alpha and IL-1 beta may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.

Actin Cytoskeleton↗
PubMed1989

Detection of a high-affinity binding site for the globular head regions of the C1q complement protein on a human diploid fibroblast subtype.

A cultured fibroblast subtype with growth and synthetic properties expected of cells residing in healing wounds and in inflammatory lesions binds the Clq component of complement with a functional affinity much higher than that of the remaining cell population. In this study we examined the optimal conditions that favor the interaction between purified 125I-labeled Clq and this cell subtype, following its isolation from the parent culture using a cell sorter, and assessed the biologic consequences of binding. Binding of 125I-Clq to the cell surface is specific, saturable and reversible. It is maximal between pH 5.5 and 8.5 at an ionic strength of mu = 0.10 and decreases as a function of increasing salt concn, with half saturation near physiologic ionic strength. Scatchard analysis of binding data indicates a single class of sites with an average association constant in the order of 1.5 x 10(9)/M and an average number of 2.5 x 10(6) binding sites per cell. Unlabeled globular fragments of Clq inhibit intact 125I-Clq binding by 64%, while unlabeled collagen-like fragments have no effect. Thus it appears that binding of Clq to this high-affinity site is mediated by a region of the globular domain of the molecule. Only the fibroblast subtype with binding sites for the globular domain of Clq appear to have the capacity to induce non-immune activation of the classical complement cascade, as assessed by the generation of C4a and C4d fragments in normal AB serum following exposure to the cells. This activation may generate products that account for a previously reported complement mitogenicity for fibroblasts.

Binding Sites↗
PubMed1989

The role of proteoglycans in collagen gel contraction by human gingival fibroblasts.

Fibroblasts generated from biopsies of normal gingiva contract collagen gels to a significantly greater extent than do fibroblasts generated from chronically inflamed gingiva. The present study demonstrates that proteoglycans play a major role in gel contraction, and that cells from the two sources differ greatly in their ultrastructural features and their capacity to synthesize proteoglycans. Fibroblasts from normal gingiva contained more rough endoplasmic reticulum and mitochondria, and they were more well-spread than cells from chronically inflamed gingiva. We observed a fine granular electron-dense lamina of material especially prominent around the cell processes and on the surface of the collagen fibrils. This material appears to be rich in proteoglycans since it stained with ruthenium red, a dye that binds to proteoglycans, and labeled in cultures incubated for 1 or 4 hr in medium containing 35S-sodium sulfate. Proteoglycans have the capacity to bind matrix components to one another and to cells, and thereby play a major role in gel contraction. Normal fibroblasts have a significantly greater capacity to synthesize proteoglycans, as manifested by label incorporation, that do cells from inflamed tissue, and this may account for their greater capacity to contract collagen gels in vitro.

Collagen↗
PubMed1988

Binding, internalization, and intracellular localization of interleukin-1 beta in human diploid fibroblasts.

This study demonstrates internalization of interleukin-1 (IL-1) via its cell surface receptor on human diploid fibroblasts and shows intracellular localization of IL-1 beta. Binding experiments at 8 degrees C using confluent fibroblast monolayers revealed 5,000-15,000 IL-1 receptors/cell that bound both IL-1 alpha and IL-1 beta. Incubation of monolayers with 125I-IL-1 beta (10(-9) M) at 8 degrees C and then at 37 degrees C for various times up to 8 h revealed a t1/2 for internalization of receptor-bound IL-1 beta of about 1.5 h. In addition, it was shown that IL-1 beta internalized via receptors was undegraded and retained binding activity. Electron microscopic autoradiography of monolayers incubated with 125I-IL-1 beta, as above, showed a progressive increase in the ratio of cytoplasmic to cell surface-associated grains. Grains at the cell surface were primarily localized at cell processes or attachment sites, frequently close to intra- and extracellular filamentous material. During incubation at 37 degrees C, most grains were free in the cytoplasm, with few present in lysosomes or vesicles. After 1 h, approximately 15% of the grains were over nuclei. Control cultures incubated at 37 degrees C with 125I-IL-1 beta and 100-fold excess unlabeled IL-1 beta showed increased uptake of label into lysosomes and little into nuclei. This study shows that IL-1 receptors are primarily located at fibroblast processes and that receptor-mediated internalization of the ligand is slow. Nuclear localization apparently requires IL-1 receptor-specific internalization of IL-1 beta, suggesting a possible role for this process in eliciting the IL-1 signal.

Cell Membrane↗
PubMed1988

Role of platelet factors and serum complement in growth of fibroblasts with high-affinity Clq complement receptors.

Cultures of human diploid fibroblasts are heterogeneous in that a subpopulation interacts via high-affinity receptors with the globular head regions of the Clq complement protein. Growth and synthetic properties of these cells are characteristic of cells residing in healing wounds and inflammatory lesions. At these sites, fibroblasts are exposed to regulatory molecules such as complement components and factors released from blood platelets. We assessed the effects of native complement proteins and platelet-derived factors on proportions and phenotypic stability of high-affinity and low-affinity receptor cells generated from explants of adult and embryonic connective tissue, using radioligand binding assays and immunofluorescence analysis by flow cytometry. Fibroblasts expressing high-affinity Clq receptors could be generated from explants only when factors from platelets were present in the medium; native complement proteins were not essential. High-affinity receptor cells could be generated only from tissue; they could not be generated by incubating cultures of the low-affinity receptor phenotype in medium containing platelet-derived factors. High-affinity receptor cells, once established from explants in the presence of platelet-derived factors, persisted through many replications in the absence of platelets. We obtained the same fibroblast phenotypes from embryonic skin as from adult gingiva, but the proportion of high-affinity receptor cells from skin was much greater. We conclude that factors derived from platelets are essential for generating cultures containing fibroblasts expressing high-affinity Clq receptors, but not for their maintenance. High-affinity receptor cells may comprise a rapidly dividing subpopulation giving rise only to like progeny or to other, more differentiated cells.

Adult↗
PubMed1988

Isolation of noncollagenous proteins from gingival connective tissue.

Noncollagenous proteins form an integral part of gingiva and other connective tissues. We have performed studies aimed at purification and partial characterization of the gingival noncollagenous proteins. Healthy gingival tissues from mongrel dogs were extracted in neutral buffers, acetic acid, and 6 mol/L urea. Immunoblots using anti-keratin antibodies and CNBr peptide patterns revealed that the majority of the proteins present in these extracts were keratins. To exclude keratins, gingival connective tissue was separated from the epithelium and then extracted. Acid extracts of the connective tissue contained very little protein, whereas urea extracts contained collagen and other noncollagenous proteins. The noncollagenous proteins present in the urea extract were partially purified by DEAE-cellulose chromatography and separated by affinity chromatography through a Sepharose 4B-type I collagen column. At least eight proteins, which ranged in molecular size from 15 to 75 kilodaltons, were obtained by this procedure. We conclude that keratins are major components of whole gingiva extracts and that epithelium must first be removed in order for connective tissue proteins to be obtained. The gingival connective tissue appears to contain several collagen-binding proteins, and these proteins may play an important role in the structure and function of the gingival matrix.

Animals↗
PubMed1988

The effects of two therapeutic communities on illicit drug users between 6 months and 1 year after treatment.

Thirty former residents of two different therapeutic communities for illicit drug users were interviewed between 6 months and 1 year after terminating treatment to determine how successfully they had been in adjusting to society. Two findings of this research concerned the amount of time the former residents spent in their therapeutic communities: Time in treatment was associated with fewer arrests once they graduated from these programs and was also associated with less drug use.

Adult↗