Search PubMed⌕ Search

Biomedical subjects

R E Gay

Publications and source records attributed to R E Gay.

At least 91 records · Page 5Linked to original sources
PubMed1995

Demonstration of granzyme A and perforin messenger RNA in the synovium of patients with rheumatoid arthritis.

OBJECTIVE: To examine the gene expression of 2 highly specific markers of cytotoxic T lymphocyte (CTL) activation, the serine protease granzyme A and the pore-forming protein perforin, in synovial tissue of patients with rheumatoid arthritis (RA), and to compare the findings with those in osteoarthritis (OA) synovial tissue. METHODS: Snap-frozen synovial tissue specimens from 9 patients with RA and 5 patients with OA were examined. The number of CTL that expressed granzyme A or perforin messenger RNA was determined by in situ hybridization using nonradioactive riboprobes for granzyme A and perforin, and by a novel in situ reverse transcriptase technique. The signals were visualized by an immunogold-silver immunohistochemistry technique and compared with immunohistochemical labeling of T and B cells. Additional double-labeling was achieved using anti-type IV collagen, anti-macrophage (anti-CD68), anti-T lymphocyte (anti-CD45RO), anti-B lymphocyte (anti-CD20), and anti-natural killer cell (anti-CD56) antibodies in an alkaline phosphatase-anti-alkaline phosphatase assay. RESULTS: Granzyme A and perforin messenger RNA (mRNA) was observed in CTL in synovial specimens from all of the RA patients, whereas in specimens from OA patients only a few, single cells with a positive mRNA signal for these molecules could be detected. In the RA specimens, the number of lymphocytes showing a positive mRNA signal for granzyme A or perforin varied from 10% to 50%, reflecting the recent findings of other investigators studying synovial fluid. CONCLUSION: Our results demonstrate that gene expression of at least 2 CTL products, granzyme A and perforin, is up-regulated in the synovium of patients with RA compared with that in the synovium of patients with OA. These molecules presumably play an important role not only in lymphocyte-mediated cytotoxicity, but also in facilitating the migration of blood-borne mononuclear cells through the vascular basement membrane into the rheumatoid synovium.

Adult↗
PubMed1995

Expression of E-selectin messenger RNA and protein in rheumatoid arthritis.

OBJECTIVE: To examine the de novo synthesis and cellular distribution of the E-selectin adhesion molecule in synovial tissues obtained from patients with rheumatoid arthritis (RA). METHODS: Immunohistochemistry techniques combined with in situ hybridization were used to examine RA synovium. RESULTS: There were numerous endothelial cells positive for E-selectin and E-selectin messenger RNA in the RA synovial membranes. Moreover, E-selectin expression appeared to correlate with inflammatory activity. CONCLUSION: The strong vascular expression of E-selectin indicates an activation of endothelial cells in the recruitment of cells associated with the chronic inflammation of RA.

Adult↗
PubMed1995

Comparative analysis of cathepsin L, cathepsin D, and collagenase messenger RNA expression in synovial tissues of patients with rheumatoid arthritis and osteoarthritis, by in situ hybridization.

OBJECTIVE: To compare the expression of cathepsin L, cathepsin D, and collagenase messenger RNA (mRNA) in synovial specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: The expression of cathepsins L and D as well as collagenase mRNA in synovial tissues from 8 patients with RA, 6 patients with OA, and 2 patients with noninflamed joints was evaluated using in situ hybridization with digoxigenin-labeled RNA probes. RESULTS: Both RA and OA synovial tissue expressed cathepsins L and D as well as collagenase mRNA. The expression of the cathepsins was markedly higher in interstitial regions and, to some extent, in perivascular infiltrates of RA synovial tissue compared with OA specimens. CONCLUSION: Cathepsins L and D mRNA are expressed differently in RA and OA synovial tissues, supporting the concept that these enzymes may contribute to the influx of mononuclear cells into RA synovium. Moreover, the data reveal that the expression of collagenase and cathepsins in RA and OA synovial lining is otherwise largely similar, and suggest that the adhesion of synovial cells to cartilage mediates the invasive destructive process in RA.

Arthritis, Rheumatoid↗
PubMed1995

Progressive joint destruction in a human immunodeficiency virus-infected patient with rheumatoid arthritis.

This article reports the case of a 63-year-old patient with rheumatoid arthritis (RA) whose symptoms of RA improved after the occurrence of a secondary human immunodeficiency virus (HIV) infection; however, the HIV infection did not affect the histologic parameters of joint destruction to the same extent as it did the clinical symptoms. Histologic and immunohistologic joint examinations of this patient revealed an ongoing production of cartilage- and bone-degrading enzymes by macrophages and fibroblasts, without the presence of T cells. These findings demonstrate that progressive joint destruction in RA can occur in the absence of T cells. Moreover, our results support the hypothesis that both T cell-dependent and T cell-independent pathways play a significant role in the pathogenesis of RA.

Arthritis, Rheumatoid↗
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

Overexpression of zinc-finger transcription factor Z-225/Egr-1 in synoviocytes from rheumatoid arthritis patients.

In rheumatoid arthritis (RA) the proliferation of synovial lining cells appears to be one of the initial pathologic changes that contributes to the destruction of articular joints. To understand the pathomechanisms involved in these functional changes, we analyzed the transcriptional regulation of the zinc-finger gene 225 (Z-225/Egr-1), a transcription factor expressed in the immediate early events of cellular activation. We found that Z-225 transcripts were significantly up-regulated in RA synoviocytes. In primary and long term culture Z-225 was spontaneously transcribed at elevated levels. In situ hybridization of zinc-finger probe showed characteristic Z-225 transcripts in RA synovial tissues. Identity of these signals to the Z-225 gene product were confirmed in freshly isolated synovial tissue by enzymatic amplification of cDNA by the PCR technique. Z-225 transcripts were also detected and characterized in a cDNA library established from a RA synovial explant. We therefore conclude that RA synoviocytes spontaneously produce Z-225 gene products at elevated levels. Because early growth response gene Z-225 is involved in the regulation of expression of other genes such as proto-oncogenes c-ras and c-sis, which are also up-regulated in the RA synovium, activation of Z-225 transcription in RA may represent a key event in articular joint destruction.

Arthritis, Rheumatoid↗
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

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↗
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↗