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S Gay

Publications and source records attributed to S Gay.

At least 163 records · Page 9Linked to original sources
PubMed1995

Enzymatic detection systems for non-isotopic in situ hybridization using biotinylated cDNA probes.

The feasibility of various non-isotopic enzymatic detection systems was tested for in situ hybridization using biotin-labelled, nick-translated cDNA probes. For this purpose, we isolated and prepared cDNA restriction fragments encoding the proteolytic cysteine proteinase cathepsin L and analysed Kirsten murine sarcoma virus-transformed BALB/3T3 cells, which have been shown to express high amounts of cytoplasmic RNA of this ras oncogene-induced proteinase. When compared on a semiquantitative basis, colorimetric non-isotopic detection of cDNA hybrids with avidin-biotin-peroxidase conjugates visualized by silver intensification of the nickel-diaminobenzidine end-product was superior to that obtained with avidin-biotin-alkaline phosphatase using different substrates for development. When the peroxidase staining technique was applied for RNA detection, it was found that overnight incubation in methanol containing hydrogen peroxide followed by deproteination with HCl was the most effective method for inhibition of endogenous peroxidase activity. For DNA detection, non-specific nucleic staining was completely abolished when heat treatment (100 degrees C) of the cell specimens was performed prior to hybridization.

Alkaline Phosphatase↗
PubMed1995

Expression of vascular cell adhesion molecule-1 mRNA and protein in rheumatoid synovium demonstrated by in situ hybridization and immunohistochemistry.

BACKGROUND: Vascular cell adhesion molecule-1 (VCAM-1) is expressed in synovial tissue of patients with rheumatoid arthritis. VCAM-1-protein has been demonstrated in nonvascular cells beside a vascular expression of this molecule. There are conflicting results about the nonvascular cell types expressing VCAM-1. EXPERIMENTAL DESIGN: For the evaluation of VCAM-1 expression in rheumatoid synovium, this molecule has been demonstrated by alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. Furthermore, VCAM-1 mRNA has been demonstrated by in situ hybridization to evaluate de novo synthesis of this molecule in vivo. To elucidate the nature of the cell types expressing VCAM-1 mRNA, this molecule has been shown by combined in situ hybridization for VCAM-1 and immunohistochemistry in the same tissue section. Double labeling has been performed with anti-collagen type IV monoclonal antibodies to delineate endothelial cells and pericytes and with anti-CD68 antibodies to elucidate the expression of VCAM-1 mRNA in fibroblast-like (type B) or macrophage-like (type A) synoviocytes. RESULTS: Although it has been reported that VCAM-1 occurs on endothelial cells after cytokine stimulation, we show that vascular expression of VCAM-1 mRNA and protein was minimal and restricted to small vessels beneath the lining cell layer. Further expression of VCAM-1 mRNA could be demonstrated in pericytes outside the collagen type IV containing vascular basement membrane. With respect to the expression of VCAM-1 in the synovial lining layer, we could clearly demonstrate by combined in situ hybridization and immunohistochemistry that CD68 positive cells of the monocyte/macrophage lineage in the lining layer (type A cells) do not express VCAM-1 mRNA and that the expression of VCAM-1 mRNA in the lining layer was restricted to fibroblast-like synoviocytes (type B cells). Scattered stromal cells revealing VCAM-1 mRNA were also CD68 negative. CONCLUSIONS: The strong expression of VCAM-1 in the fibroblast-like cells of RA synovium and the lack of expression in the vascular endothelium suggest that the major role of VCAM-1 appears to be associated with the proliferating synovial cells prone to attach and subsequently invade articular cartilage.

Antigens, CD↗
PubMed1995

Expression of proteolytic cathepsins B, D, and L in periodontal gingival fibroblasts and tissues.

BACKGROUND: A major feature of gingivitis and periodontitis is the destruction of the collagenous matrix of the surrounding connective tissue. The hypothesis that proteolytic enzymes release from cells adjacent to the site of destruction was recently supported by the presence of increased levels of cathepsin B, D, and L in the gingival crevicular fluid of patients suffering from periodontal disease (PD). EXPERIMENTAL DESIGN: We studied the expression of mRNA of cathepsins B,D, and L in early passaged, adherent gingival fibroblasts of patients with chronic adult PD. In addition, we examined the presence of cathepsin D and L mRNA-expressing cells in periodontal tissue specimen by in situ hybridization, and we localized the respective enzymes by immunohistochemistry. RESULTS: Strong gene expression of the three cathepsin types could be detected by dot hybridization in all PD-derived gingival cells. Immunohistochemical distribution of cathepsins and mRNA for cathepsin D and L could be demonstrated mainly in PD tissue specimen, largely in the area in between the epithelium and the adjacent subepithelial connective tissue. Examination of the cell types revealed multiple macrophage- and fibroblast-shaped cells expressing positive staining for cathepsins. CONCLUSIONS: The present data support the the concept that cathepsins play a major role in tissue destruction and may represent a target for therapeutic interventions.

Adult↗
PubMed1995

Detection of insulin-like growth factor I and II in synovial tissue specimens of patients with rheumatoid arthritis and osteoarthritis by in situ hybridization.

OBJECTIVE: To study the expression of insulin-like growth factor I and II (IGF I and II) in synovial tissue specimen of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Synovial tissue sections were examined for the expression of IGF I and II by in situ hybridization using digoxigenin labeled antisense and sense RNA probes. RESULTS: The antisense probe of IGF I reacted with all specimens. IGF II mRNA was expressed in 7/7 RA and 4/5 OA tissues. Cells of the synovial lining and subsynovial layer bound both antisense probes, whereas inflammatory infiltrates of RA tissues were labeled rarely. CONCLUSION: The significant number of cells in the synovium that express IGF I and II mRNA suggests a role of IGF in repair mechanisms of articular cartilage in response to injury and effects on fibroblast growth within the synovium.

Arthritis, Rheumatoid↗
PubMed1995

Oncogenes in rheumatoid arthritis.

The evolving knowledge of the actions and interactions of (proto)oncogenes in cancer has deeply influenced the understanding of other nonmalignant diseases. In RA, the longstanding pathohistologic evidence of transformed-appearing synovial cells at the site of bone and cartilage attachment and joint destruction can now be explained in terms of alterations of cell regulation, cell cycle, and apoptotically triggered cell death. The detection of upregulated oncogenes and their gene products at these sites supported the hypothesis of an aberrant synovial cell type invading the joint. Interestingly, there are hints that this transformation of synovial cells may require more than one activated oncogene. A model was introduced by Carson and Ribero in 1993. In this model, a primary stimulus affects the cell and leads to the enhanced transcription of an oncogene (i.e., c-myc). A second stimulus activates other oncogenes and determines if this cell (i.e., a synovial fibroblast) proliferates (marked by the presence of bcl-2 mRNA) or undergoes apoptosis (marked by fas mRNA and Fas expression at the cell surface). This co-upregulation might explain why some investigators could not detect a significant upregulation of oncogenes in cultured synovial fibroblasts devoid of their normal milieu. Based on the results of the specific activity of Fas and perforin and recent data from our laboratory, we have modified Carson's model to include these data. As there exists an established retroviral model in which the tax sequence of the HTLV retrovirus initiates central oncogene transcription similar to those activated in RA, the retroviral particles, which do not resemble any other known retrovirus but are detectable in the synovial fluid, might well be an important stimulus in the pathogenesis of RA. To simplify the puzzling events of oncogene interactions in RA, we have summarized the data and propose that an oncogene network acts as a pathogenic mechanism in the synoviocytes of the rheumatoid joint. Similar to the "cytokine network" regulating the T-cell-dependent pathway, the "oncogene network" is presumably the major T-cell-independent pathway in RA (Fig. 4).

Apoptosis↗
PubMed1994

A new model for rheumatoid arthritis generated by engraftment of rheumatoid synovial tissue and normal human cartilage into SCID mice.

OBJECTIVE: A new animal model was used to study the interaction between rheumatoid synovial cells and cartilage and to explore the cellular basis of rheumatoid joint destruction. METHODS: Fresh synovial tissue derived from patients with rheumatoid arthritis was implanted with normal human cartilage into SCID mice, either subcutaneously or under the renal capsule, for up to 304 days. The implants were analyzed by light and electron microscopy, as well as by immunohistochemistry and in situ hybridization. RESULTS: Human synovial tissue and cartilage implanted in SCID mice are maintained by the animals for up to 304 days. After 35 days, focal erosions occur at the site of attachment of synovial lining cells to the cartilage. After 105 days, a pannus-like formation, consisting of proliferating synovial fibroblast-like cells invading the cartilage, is observed. The fibroblast nature of these cells was supported by observation of only focal expression of the macrophage markers CD14 and CD68. Cells at the immediate site of cartilage destruction express messenger RNA for cathepsin L, whereas cathepsin D messenger RNA was detected in subsynovial regions away from the site of destruction. The human origin of the tissue involved in cartilage destruction was demonstrated using monoclonal antibodies to HLA-ABC and human type IV collagen. CONCLUSION: The present approach introduces a novel in vivo model of rheumatoid arthritis for the study of the molecular and cellular mechanisms of rheumatoid joint destruction at sites of synovial attachment to cartilage. In this model, the SCID mouse acts as a useful host for studying the properties of rheumatoid synovium in the absence of circulating human blood components.

Animals↗
PubMed1994

Cytokines and oncogenes in cellular interactions of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic systemic disorder of unknown etiology. Most of its debilitation sequelae are derived from progressive destruction of joints. The affected joints exhibit inflammation, abnormal immune responses and synovial hyperplasia. Although growth factors and cytokines derived from macrophages and endothelial cells contribute to the perpetuation of the inflammatory process, activated transformed-appearing synovial fibroblasts mediate cartilage and bone destruction. Based on the observation that synovial hyperplasia is associated with a transformed-appearing phenotype and an upregulated expression of protooncogenes and matrix degrading enzymes, the present studies are designed to explore the role of a heretofore unknown (retro) virus-like particle in the pathogenesis of RA.

Arthritis, Rheumatoid↗
PubMed1994

Interactions of syndecan-1 and heparin with human collagens.

Glycosaminoglycan (GAG)-collagen interactions play important roles in cell adhesion and extracellular matrix assembly; however, the chemical bases for these interactions are not fully understood. We have used affinity co-electrophoresis (ACE) (Lee, M.K. and Lander, A.D., Proc. Natl. Acad. Sci, USA, 88, 2768-2772, 1991) to study the binding of the heparan sulphate proteoglycan syndecan-1 and heparin to human collagens. [35S]Syndecan-1 [from normal murine mammary gland (NMuMG) epithelial cells] and low-M(r) (approximately 6 kDa) [125I]heparin were subjected to electrophoresis through agarose gel lanes containing human collagens at various concentrations, and binding affinities were measured from shifts in migration of the labelled materials. Results demonstrate that the affinities of each collagen for syndecan-1 and low-M(r) heparin were similar, and followed the order: type V >> type IV approximately type III approximately type I > type VI >> type II, and ranged in Kd from approximately 10(-8) to approximately 3 x 10(-6) M. These data suggest that syndecan-1 and heparin may contain similar collagen-binding determinants. It was also found that the same heparin subpopulation was selectively bound with high affinity by each of the collagens. The published amino acid sequences of the six collagens were examined for what are thought to be heparin-binding consensus sequences (Cardin, A.D. and Weintraub, H.J.R., Arteriosclerosis, 9, 21-32, 1989). The presence of such sequences did not correlate with affinity for heparin or syndecan-1, and collagens I, II and III lacked such sequences entirely. The data suggest that collagens may use novel types of binding sites to interact with GAGs.

Animals↗
PubMed1994

Retroviruses and autoimmune rheumatic diseases.

In autoimmune rheumatic diseases, retroviruses have been repeatedly discussed as important etiologic factors. However, despite a considerable amount of indirect evidence that retroviruses might indeed be involved in triggering or perpetuating autoimmune rheumatic diseases, clear cut direct evidence is still missing. Studies on arthropathies associated with HIV-1 or HTLV-1 infection as well as new experimental animal models like the Tax transgene mice and new data from the MLR/lpr mouse model might help to answer the questions how and by what mechanisms retroviral infection may lead to autoimmune rheumatic diseases. From data obtained in the MLR/lpr mouse it seems obvious that a potential link of retroviruses, apoptosis and autogenes to autoimmune diseases opens exciting new approaches to the study of rheumatic disease pathogenesis.

Animals↗
PubMed1994

A new double labeling technique for combined in situ hybridization and immunohistochemical analysis.

BACKGROUND: A reliable, sensitive, and specific double labeling technique is required that allows the simultaneous visualization of in situ hybridization products and antigens. Currently used double labeling techniques are limited by various problems including the numerous disadvantages associated with radioactive labels, the time-dependent loss of fluorescence signals, and the high background that is associated with various peroxidase techniques. Therefore, the aim of this study was to develop an improved double labeling technique. EXPERIMENTAL DESIGN: Riboprobes were used for detection of mRNA of cathepsin D, vascular cell adhesion molecule, and endothelial leukocyte adhesion molecule in in situ hybridization and monoclonal antibodies specific for macrophages and basement membranes (collagen type IV) for immunohistochemical analysis. The in situ hybridization and immunohistochemical analysis were used to characterize cathepsin D messenger ribonucleic acid (mRNA) in macrophages expressing cells and the expression of adhesion molecule mRNA in endothelial cells delineated by the vascular basement membrane expressing collagen type IV. RESULTS: The application of in situ hybridization detection systems before immunohistochemical analysis was shown to give reliable results. In situ hybridization with digoxigenin labeled riboprobes using alkaline phosphatase linked Fab fragments visualized by 4-nitro blue tetrazolim chloride/5-bromo-4-chloro-3-indolphosphate combined with immunohistochemical detection of the antigen by the alkaline phosphatase anti-alkaline phosphatase-technique with new fuchsin as substrate is a reliable double labeling technique. Using this protocol, we could show that the reaction product is stable, there is virtually no background, and both reaction products can be easily distinguished. Vascular cell adhesion molecule-1 mRNA is expressed only in endothelial cells and certain fibroblast-like cells that do not label with antibodies against macrophages, whereas cathepsin D mRNA is coexpressed with macrophages. We also demonstrated that endothelial leukocyte adhesion molecule-1 mRNA is strongly expressed in endothelial cells that can be localized within the boundaries of the vascular basement membrane. CONCLUSIONS: A new and reliable double labeling technique for the simultaneous evaluation of in situ hybridization and immunohistochemical analysis is described that is suitable for various applications.

Arthritis, Rheumatoid↗
PubMed1994

RS3PE syndrome: no evidence for retroviruses.

OBJECTIVE: To determine whether human T cell lymphotrophic virus (HTLV) infection is associated with remitting seronegative symmetrical synovitis with pitting edema (RS3PE) syndrome. METHODS: Three patients presenting with RS3PE syndrome and 7 controls were examined for the presence of HTLV crossreactive antigens. RESULTS: Our patients were elderly men who presented with typical symptoms of abrupt onset of synovitis of the wrist, carpal joints, metacarpophalangeal, proximal interphalangeal, distal interphalangeal joints, and flexor tendons, associated with remarkable pitting edema of the hands. Transmission electron microscopy and immunohistochemical analysis for HTLV-1 P19 and P20 related antigens failed to detect retroviral presence in the sample specimens or controls. CONCLUSION: Our investigation suggests there is no evidence for HTLV synovial infection associated with RS3PE.

Aged↗
PubMed1993

Dentin sialoprotein: biosynthesis and developmental appearance in rat tooth germs in comparison with amelogenins, osteocalcin and collagen type-I.

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with 14C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.

Amelogenin↗
PubMed1993

Virus-like particles in synovial fluids from patients with rheumatoid arthritis.

Based on the elevated expression of oncogenes in proliferating transformed-appearing synoviocytes we searched for the possible involvement of a viral agent in the pathogenesis of RA. We report the detection of virus-like particles with retroviral C type morphology in SF, which lack the typical morphologic as well as immunohistochemical features of the human T-lymphotropic and immunodeficiency viruses.

Arthritis, Rheumatoid↗
PubMed1993

Pharyngeal fat in obstructive sleep apnea.

Although most patients with obstructive sleep apnea (OSA) are obese, it is not known how obesity contributes to airway collapse during sleep. The purpose of this study was to determine whether the volume of adipose tissue adjacent to the pharyngeal airway in humans is related to the degree of OSA. We studied 30 subjects, nine without OSA and 21 with OSA; two subjects were studied before and after weight loss. Adipose tissue was detected with magnetic resonance imaging using T1-weighted spin echo sequences. The volume of adipose tissue adjacent to the upper airway was determined by measuring the volume of all pixels in the intensity range of adipose tissue within the region bounded by the ramus of the mandible, the spine, the anterior border of the soft palate, and the hard palate. Polysomnography was performed with conventional techniques. All subjects had a collection of adipose tissue adjacent to the upper airway; the volume of this adipose tissue correlated with the number of apneas plus hypopneas per hour of sleep (r = 0.59, p < 0.001). Both patients who lost weight and had fewer apneas and hypopneas had a marked decrease in the pharyngeal adipose tissue volume. We conclude that adipose tissue is deposited adjacent to the pharyngeal airway in patients with OSA and that the volume of this tissue is related to the presence and degree of OSA.

Adipose Tissue↗