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S Gay

Publications and source records attributed to S Gay.

At least 91 records · Page 5Linked to original sources
PubMed2000

Gene transfer: from concept to therapy.

In the last few years, several novel strategies have been proposed for the treatment of rheumatoid arthritis (RA). Among them, gene therapy is considered a promising concept bearing the potential of highly specific targeting of relevant pathomechanisms. Early studies using gene transfer focused mainly on studying disease mechanisms, whereas recent research has put potential clinical applications to the forefront of attention. This has provided new answers to the question of how to deliver genes into the rheumatoid synovium as well as which pathways to target. Thus, significant progress has been made in the continued development of viral systems, including retro- and adenoviruses, as well as in the exploration of novel tools such as herpes virus-based systems or liposomes in combination with viral fusion proteins. When potential targets for gene transfer in RA are considered, two strategies have emerged: the first focuses on the delivery of secreted proteins, mainly cytokines and cytokine receptors, to inhibit inflammation in arthritic joints. Based on our growing knowledge about the pathogenesis of RA, however, there has also been substantial progress in exploring approaches that aim at interfering specifically with signaling pathways involved in the activation and apoptosis of synovial cells. The data from recent studies indicate the ability to selectively target specific disease processes by the differential expression of therapeutic genes in varying cell types and at different stages of disease, thus demonstrating the potential of gene transfer as an arthritis therapy.

Animals↗
PubMed2000

Microsatellite analysis in rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic disease characterised by irreversible destruction of the affected joints. As aggressive transformed-appearing synovial fibroblasts are commonly found at the site of invasion of the rheumatoid synovium into the adjacent cartilage and bone, the presence of microsatellite instability (MSI) and expression of mismatch repair enzymes as a possible mechanism in the alteration of these cells was examined. METHODS: DNA was extracted from the synovial fibroblasts and blood of 20 patients with long term RA undergoing joint replacement, and the presence of MSI was studied at 10 microsatellite loci. In addition, immunohistochemistry was performed to evaluate the expression of the two major mismatch repair enzymes (hMLH1 and hMSH2) in rheumatoid synovium. RESULTS: MSI could not be detected in any of the fibroblast cell populations derived from the 20 different rheumatoid synovial samples. In addition, strong expression of mismatch repair enzymes could be seen in numerous cells, including fibroblasts, throughout the synovium. CONCLUSIONS: Applying the currently used and established markers for MSI, the data show for the first time that MSI does not appear to have an important role in alteration of rheumatoid synovial fibroblasts into an aggressive phenotype. On the other hand, strong mismatch repair enzyme synthesis in rheumatoid synovium supports the hypothesis of continuing DNA repair, presumably due to long term, inflammation induced DNA damage.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Activation of synovial fibroblasts in rheumatoid arthritis: lack of Expression of the tumour suppressor PTEN at sites of invasive growth and destruction.

AIMS: PTEN is a novel tumour suppressor which exhibits tyrosine phosphatase activity as well as homology to the cytoskeletal proteins tensin and auxilin. Mutations of PTEN have been described in several human cancers and associated their invasiveness and metastatic properties. Although not malignant, rheumatoid arthritis synovial fibroblasts (RA-SF) exhibit certain tumour-like features such as attachment to cartilage and invasive growth. In the present study, we analyzed whether mutant transcripts of PTEN were present in RA-SF. In addition, we used in situ hybridization to study the expression of PTEN messenger (m)RNA in tissue samples of RA and normal individuals as well as in cultured RA-SF and in the severe combined immunodeficiency (SCID) mouse model of RA. Synovial tissue specimens were obtained from seven patients with RA and from two nonarthritic individuals. Total RNA was isolated from synovial fibroblasts and after first strand complementary (c)DNA synthesis, polymerase chain reaction (PCR) was performed to amplify a 1063 base pair PTEN fragment that encompassed the coding sequence of PTEN including the phosphatase domain and all mutation sites described so far. The PCR products were subcloned in Escherichia coli, and up to four clones were picked from each plate for automated sequencing. For in situ hybridization, digoxigenin-labelled PTEN-specific RNA probes were generated by in vitro transcription. For control in situ hybridization, a matrix metalloproteinase (MMP)-2-specific probe was prepared. To investigate the expression of PTEN in the absence of human macrophage or lymphocyte derived factors, we implanted RA-SF from three patients together with normal human cartilage under the renal capsule of SCID mice. After 60 days, mice were sacrificed, the implants removed and embedded into paraffin. RESULTS: PCR revealed the presence of the expected 1063 base pair PTEN fragment in all (9/9) cell cultures (Fig.1). No additional bands that could account for mutant PTEN variants were detected. Sequence analysis revealed 100% homology of all RA-derived PTEN fragments to those from normal SF as well as to the published GenBank sequence (accession number U93051). However, in situ hybridization demonstrated considerable differences in the expression of PTEN mRNA within the lining and the sublining layers of RA synovial membranes. As shown in Figure 2a, no staining was observed within the lining layer which has been demonstrated to mediate degradation of cartilage and bone in RA. In contrast, abundant expression of PTEN mRNA was found in the sublining of all RA synovial tissues (Figs 2a and b). Normal synovial specimens showed homogeneous staining fo PTEN within the thin synovial membrane (Fig. 2c). In situ hybridization using the sense probe gave no specific staining (Fig. 2d). We also performed in situ hybridization on four of the seven cultured RA-SF and followed one cell line from the first to the sixth passage. Interestingly, only 40% of cultured RA-SF expressed PTEN mRNA (Fig. 3A), and the proportion of PTEN expressing cells did not change throughout the passages. In contrast, control experiments using a specific RNA probe fo MMP-2 revealed mRNA expression by nearly all cultured cells (Fig. 3B). As seen before, implantation of RA-SF into the SCID mice showed considerable cartilage degradation. Interestingly, only negligible PTEN expression was found in those RA-SF aggressively invading the cartilage (Fig. 3c). In situ hybridization for MMP-2 showed abundant staining in these cells (Fig. 3d). DISCUSSION: Although this study found no evidence for mutations of PTEN in RA synovium, the observation that PTEN expression is lacking in the lining layer of RA synovium as well as in more that half of cultured RA-SF is of interest. It suggests that loss of PTEN function may not exclusively be caused by genetic alterations, yet at the same time links the low expression of PTEN to a phenotype of cells that have been shown to invade cartilage aggressively. It has been proposed that the tyrosine phosphatase activity of counteracting th actions o protein tyrosine kinases. As some studies have demonstrated an upregulation of tyrosine kinase activity in RA synovial cells, it might be speculated that the lack of PTEN expression in aggressive RA-SF contributes to the imbalance of tyrosine kinases and phosphatases in this disease. However, the extensive amino-terminal homology of the predicted protein to the cytoskeletal proteins tensin and auxilin suggests a complex regulatory function involving cellular adhesion molecules and phosphatase-mediated signalling. The tyrosine phosphatase TEP1 has been shown to be identical to the protein encoded by PTEN, and gene transcription of TEP1 has been demonstrated to be downregulated by transforming growth factor (TGF)-beta. Therefore, it could be hypothesized that TGF-beta might be responsible for the downregulation of PTEN. Low expression of PTEN may belong to the features that distinguish between the activated phenotype of RA-SF and the sublining, proliferating but nondestructive cells.

Animals↗
PubMed2000

Detection of activated complement complex C5b-9 and complement receptor C5a in skin biopsies of patients with systemic sclerosis (scleroderma).

OBJECTIVE: Upregulated matrix synthesis is a hallmark of systemic sclerosis (SSc). There are indications that growth factors such as platelet derived growth factor (PDGF) are involved in proliferative pathways in SSc lesions. As activated complement releases PDGF from endothelial cells, we searched for activated complement and the complement receptor for C5a (C5aR) in skin biopsies of patients with SSc. METHODS: Snap frozen sections of 8 patients with early SSc and 5 patients with longterm SSc were examined. Using monoclonal antibodies against activated complement complex C5b-9 and the C5aR, skin biopsies derived from both clinically involved and non-involved skin were examined by APAAP immunohistochemistry. RESULTS: A pattern of activated complement C5b-9 and the CSaR could be detected in SSc microvasculature. Eleven of the 13 patients (7/8 patients with early SSc) showed positive staining for C5b-9. The CSaR was detected in 6 of the 8 patients with early SSc. In 3 patients with longterm disease, C5aR expression could also be detected in non-involved skin. CONCLUSION: Activated complement and complement receptors could be detected in early and late stages of SSc skin lesions. The presence of complement receptors in non-involved skin may indicate preclinical activation of pathways resulting in growth factor dependent matrix synthesis.

Antigens, CD↗
PubMed2000

Altered diurnal rhythm of prolactin in systemic sclerosis.

OBJECTIVE: Mild hyperprolactinemia has been reported in systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). We investigated whether the elevated serum level of prolactin (Prl) detected in SSc is due to a sustained increase over 24 h and/or a shift in the diurnal rhythm, and whether Prl autoantibodies--originally described in SLE--may interfere in the assay. METHODS: The serum level of Prl was measured by ELISA and compared between 73 patients with SSc and 73 age and sex matched controls (78% women, age 56 +/- 11 years). The diurnal rhythms of Prl and thyrotropin (thyroid stimulating hormone, TSH) were compared between 3 patients with SSc and 10 healthy controls. Blood was taken at 2-3, 6-7, 10-11 a.m., and 2-3, 6-7, 10-11 p.m. The serum level of Prl autoantibodies was measured by ELISA and compared between matched patients with SSc and SLE and controls (n = 42 each). Standard curves of the Prl ELISA were spiked with 10% sera containing high levels of Prl autoantibodies to test interference. RESULTS: Serum levels of Prl measured in the morning (8-10 a.m.) were significantly higher in patients with SSc (17.9 +/- 7.7 ng/ml), compared with controls (9.3 +/- 4.2 ng/ml; p < 0.05). In SSc, 40% of patients had Prl levels > 20 ng/ml, but no correlation was found with Scl-70 or Prl autoantibodies. Younger patients (< 50 years, n = 23/73) showed higher serum levels of Prl than older patients (21.3 +/- 10.3 vs 16.3 +/- 6.2 ng/ml; p < 0.05). The diurnal rhythm of Prl revealed that both a sustained increase over 24 h and some shift occurred in SSc. Peaks of secretion were detected between 6 and 11 a.m., instead of 2-6 a.m. The median levels of TSH over 24 h in patients with SSc ranged within the normal limits. Nevertheless, in SSc, a significant correlation (r = 0.59, p < 0.01) was found between diurnal rhythms of Prl and TSH. The prevalence of Prl autoantibodies in serum was 8% in SSc, 27% in SLE, and < 5% in controls. However, the presence of Prl autoantibodies did not interfere with our assay. CONCLUSION: Our data confirm that mild hyperprolactinemia occurs in a subgroup of patients with SSc, and showed that the elevated serum level of Prl is due to both a sustained increase over 24 h and a shift in the diurnal rhythm. The correlation between diurnal rhythms of Prl and TSH suggests common regulatory mechanisms.

Adult↗
PubMed2000

Expression of the precursor of secretoneurin, secretogranin II, in the synovium of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: Secretoneurin (SN) is a neuropeptide that is chemotactic for mononuclear cells and it has been suggested to be involved in the mediation of pain; there is also evidence that SN is involved in the inflammation process. As secretogranin II (SGII) is the precursor of SN, we investigated expression of SGII mRNA and SN protein in the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Snap frozen synovial tissue specimens from 12 patients with RA and 11 patients with OA were examined. RNA was isolated and cDNA copied by reverse transcription. The expression of SGII was determined by polymerase chain reaction and in situ hybridization (ISH). SGII expressing cells were compared with CD68 positive cells stained by immunohistochemistry. SN protein was also detected by immunohistochemistry. RESULTS: A 524 bp SGII-specific fragment could be amplified by PCR from the cDNA of all specimens. ISH showed scattered expression of SGII in both RA and OA synovial tissue; its expression pattern was characterized by positive staining for SGII in both the lining and the sublining layer. Immunohistochemical double labeling with anti-CD68 antibodies revealed expression of SGII in CD68 negative, fibroblast-like cells, whereas CD68 positive macrophages did not. In RA and OA, the SGII staining by ISH was positive with a diffuse staining throughout the entire synovial tissue. SN protein expression was scattered in RA but more intense in OA synovium. CONCLUSION: The expression of SGII mRNA in RA and OA synovial fibroblasts clearly supports the hypothesis that SN is involved in the synovial tissue inflammation in both diseases. The significant lower SN expression in RA could be due to an inhibitory mechanism with respect to the SN levels in synovial fluid. SN might be involved in the modulation of afferent nerve transmission and therefore might play a role in the sensation of pain, especially in patients with OA.

Antigens, CD↗
PubMed2000

Functional MR imaging and traumatic paraplegia: preliminary report.

PURPOSE: To evaluate residual activity in the sensorimotor cortex of the lower limbs in paraplegia. METHODS: 5 patients suffering from a complete paralysis after traumatic medullar lesion (ASIA=A). Clinical evaluation of motility and sensitivity. 1. Control functional MR study of the sensorimotor cortex during simultaneous movements of hands, imaginary motor task and passive hands stimulation. 2. Concerning the lower limbs, 3 fMRI conditions: 1-patient attempts to move his toes with flexion-extension, 2-mental imagery task of the same movement, 3-peripheral passive proprio-somesthesic stimulation (squeezing) of the big toes. RESULTS: Activations were observed in the primary sensorimotor cortex (M1), premotor regions and in the supplementary motor area (SMA) during movement and mental imaginary tasks in the control study and during attempt to move and mental imaginary tasks in the study concerning the lower limbs. Passive somesthesic stimulation generated activation posterior to the central sulcus for 2 patients. CONCLUSION: Activations in the sensorimotor cortex of the lower limbs can be generated either by attempting to move or mental evocation. In spite of a clinical evaluation of complete paraplegia, fMRI can show a persistence of sensitive anatomic conduction, confirmed by Somesthesic Evoked Potentials.

Adult↗
PubMed1999

[Rheumatoid arthritis: new molecular and cellular aspects].

BACKGROUND: Rheumatoid arthritis is a chronic systemic disorder of unknown etiology, that is characterized by inflammation, synovial hyperplasia and destruction of the affected joints. Novel molecular biology techniques have identified important cellular and molecular pathways in the pathogenesis of rheumatoid arthritis during the last years. RESULTS: The cellular activation of aggressively growing, matrix-degrading synovial fibroblasts is a key event in the pathogenesis of rheumatoid arthritis. The cellular activation results in an altered expression of apoptosis regulating molecules (for example CD 95 and Sentrin) as well as of protooncogenes (for example RAS and MYC). Important extracellular stimuli such as the pro-inflammatory cytokines interleukin-1 and TNF-alpha are overexpressed in the rheumatoid arthritis synovium. First clinical trials with cytokine inhibiting molecules (interleukin-1 receptor antagonist, recombinant soluble TNF-alpha receptor/Etanercept and monoclonal TNF-alpha antibodies/Remicade) revealed promising results. Etanercept is now available for the treatment of rheumatoid arthritis in the USA. In addition, gene transfer methods could help to overcome the problem of a continuous expression of therapeutic molecules in the affected joints; gene delivery of the interleukin-1 receptor antagonist is currently tested in a human trial. Finally, the inhibition of matrix degrading enzymes such as matrix metalloproteinases, that mediate the joint destructive features of the activated synovial fibroblasts, could be another therapeutic approach. CONCLUSIONS: The elucidation of important molecular and cellular pathways in the pathogenesis resulted in novel concepts in the therapy of rheumatoid arthritis. Gene transfer methods are of importance in studying the pathogenesis of the disease, however, their clinical safety and usefulness have to be proven in additional studies.

Antibodies, Monoclonal↗
PubMed1999

A molecular scanner to automate proteomic research and to display proteome images.

Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today's commonly used high-throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification and annotation in 2-DE databases: the sequential excision process of pieces of gel containing protein; the enzymatic digestion step; the interpretation of mass spectra (reliability of identifications); and the manual updating of 2-DE databases. We present a highly automated method that generates a fully annoated 2-DE map. Using a parallel process, all proteins of a 2-DE are first simultaneously digested proteolytically and electro-transferred onto a poly(vinylidene difluoride) membrane. The membrane is then directly scanned by MALDI-TOF MS. After automated protein identification from the obtained peptide mass fingerprints using PeptIdent software (http://www.expasy.ch/tools/peptident.html + ++), a fully annotated 2-D map is created on-line. It is a multidimensional representation of a proteome that contains interpreted PMF data in addition to protein identification results. This "MS-imaging" method represents a major step toward the development of a clinical molecular scanner.

Automation↗
PubMed1999

Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts.

UNLABELLED: Articular destruction by invading synovial fibroblasts is a typical feature in rheumatoid arthritis (RA),. Recent data support the hypothesis that key players in this scenario are transformed-appearing synovial fibroblasts at the site of invasion into articular cartilage and bone. They maintain their aggressive phenotype toward cartilage, even when first cultured and thereafter coimplanted together with normal human cartilage into severe combined immunodeficient mice for and extended period of time. However, little is known about the upregulation of genes that leads to this aggressive fibroblast phenotype. To inhibit this progressive growth without interfering with pathways of physiological matrix remodelling, identification of pathways that operate specifically in RA synovial fibroblasts is required. In order to achieve this goal, identification of genes showing upregulation restricted to RA synovial fibroblasts is essential. AIMS: To identify specifically expressed genes using RNA arbitrarily primed (RAP)-polymerase chain reaction (PCR) for differential display in patients with RA. METHODS: RNA was extracted from cultured synovial fibroblasts from 10 patients with RA, four patients with osteoarthritis (OA), and one patient with psoriatic arthritis. RAP-PCR was performed using different arbitrary primers for first-strand and second-strand synthesis. First-strand and second-strand synthesis were performed using arbitrary primers: US6 (5'-GTGGTGACAG-3') for first strand, and Nuclear 1+ (5'ACGAAGAAGAG-3'), OPN28 (5'GCACCAGGGGG-3'), Kinase A2+(5'-GGTGCCTTTGG-3') and OPN24 (5'AGGGGCACCA-3') for second strand synthesis. PCR reactions were loaded onto 8 mol/l urea/6% polyacrylamide-sequencing gels and electrophoressed. Gel slices carrying the target fragment were then excised with a razor blade, eluated and reamplified. After verifying their correct size and purity on 4% agarose gels, the reamplified products derived from the single-strand confirmation polymorphism gel were cloned, and five clones per transcript were sequenced. Thereafter, a genbank analysis was performed. Quantitative reverse transcription PCRj of the segments was performed using the PCR MIMIC technique. In-situ expression of centromere kinesin-like protein-E (CENP-E) messenger (m)RNA in RA synovium was assessed using digoxigenin-labelled riboprobes, and CENP-E protein expression in fibroblasts and synovium was performed by immunogold-silver immunohistochemistry and cytochemistry. Functional analysis of CENP-E was done using different approaches (eg glucocorticoid stimulation, serum starvation and growth rate analysis of synovial fibroblasts that expressed CENP-E). RESULTS: In RA, amplification of a distinct PCR product suitable for sequencing could be observed. The indicated complementary DNA fragment of 434 base pairs from RA mRNA corresponded to nucleotides 6615-7048 in the human centromere kinesin-like protein CENP-E mRNA (GenBank accession No. emb/Z15005). The isolated sequence shared greater than 99% nucleic acid (P=2.9e(-169)) identify with the human centromere kinesin-like protein CENP-E. Two base changes at positions 6624 (A to C) and 6739 (A to G) did not result in alteration in the amino acid sequence, and therefore 100% amino acid identity could be confirmed. The amplification of 10 clones of the cloned RAP product revealed the presence of CENP-E mRNA in every fibroblast culture examined, showing from 50% (271.000 +/- 54.000 phosphor imager arbitrary units) up to fivefold (961.000 +/- 145.000 phosphor image arbitrary units) upregulation when compared with OA fibroblasts. Neither therapy with disease-modifying antirheumatic drugs such as methotrexate, gold, resochine or cyclosporine A, nor therapy with oral steroids influenced CENP-E expression in the RA fibroblasts. Of the eight RA fibroblast populations from RA patients who were receiving disease-modifying antirheumatic drugs, five showed CENP-E upregulation; and of the eight fibroblast populations from RA patients receiving steroids, four showed CENP-E upregulation. Numerous synovial cells of the patients with RA showed a positive in situ signal for the isolated CENP-E gene segment confirming CENP-E mRNA production in rheumatoid synovium, whereas in OA synovial tissue CENP-E mRNA could not be detected. In addition, CENP-E expression was independent from medication. This was further confirmed by analysis of the effect of prednisolone on CENP-E expression, which revealed no alteration in CENP-E mRNA after exposure to different (physiological) concentrations of prednisolone. Serum starvation also could not suppress CENP-E mRNA completely. DISCUSSION: Since its introduction in 1992, numerous variants of the differential display method and continuous improvements including RAP-PCR have proved to have both efficiency and reliability in examination of differentially regulated genes. The results of the present study reveal that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts. The mRNA, which has been found to be upregulated in rheumatoid synovial fibroblasts, codes for a kinesin-like motor protein named CENP-E, which was first characterized in 1991. It is a member of a family of centromere-associated proteins, of which six (CENP-A to CENP-F) are currently known. CENP-E itself is a kinetochore motor, which accumulates transiently at kinetochores in the G2 phase of the cell cycle before mitosis takes place, appears to modulate chromosome movement and spindle elongation,and is degraded at the end of mitosis. The presence or upregulation of CENP-E has never been associated with RA.The three-dimensional structure of CENP-E includes a coiled-coil domain. This has important functions and shows links to known pathways in RA pathophysiology. Coiled-coil domains can also be found in jun and fos oncogene products, which are frequently upregulated in RA synovial fibroblasts. They are also involved in DNA binding and transactivation processes resembling the situation in AP-1 (Jun/Fos)-dependent DNA-binding in rheumatoid synovium. Most interestingly, these coiled-coil motifs are crucial for the assembly of viral proteins, and the upregulation of CENP-E might reflect the influence of infectious agents in RA synovium. We also performed experiments showing that serum starvation decreased, but did not completely inhibit CENP-E mRNA expression. This shows that CENP-E is related to, but does not completely depend on proliferation of these cells. In addition, we determined the growth rate of CENP-E high and low expressors, showing that it was independent from the amount of CENP-E expression. supporting the statement that upregulation of CENP-E reflects an activated RA fibroblast phenotype. In summary, the results of the present study support the hypothesis that CENP-E, presumably independently from medication, may not only be upregulated, but may also be involved in RA pathophysiology.

Adult↗
PubMed1999

[Analysis of gene expression patterns in rheumatoid synovial fibroblasts using RAP-PCR for differential display].

OBJECTIVE: Destruction of articular cartilage and bone by invading synovial fibroblasts is a typical histopathologic feature in rheumatoid arthritis (RA). However, little is known about specific up- or downregulation of genes leading to this aggressive phenotype. Thus, our aim was to identify genes, which are differentially expressed in RA synovial fibroblasts as compared to synovial fibroblasts derived from patients with osteoarthritis (OA) using RAP-PCR for differential display. METHODS: After extraction of total RNA, the first step of RAP-PCR was performed using various different arbitrary 10-12-base primers for first-strand cDNA synthesis. Second-strand synthesis was achieved by cycling at low stringency conditions for 35 cycles using different arbitrary 10-base primers, followed by electrophoretic separation and sequence analysis of the amplified fingerprint products. RESULTS: On average, approximately 70 different RNAs were obtained per primer, of which most were expressed both by RA and OA synovial fibroblasts. Using 26 different primer combinations, in total 12 cDNAs were differentially expressed between RA and OA synovial fibroblasts. In the RA group strong amplification of distinct PCR products suitable for sequencing could be observed. Sequence analysis identified these PCR products as highly homologous to various genes involved in regulation of cell cycle and metabolism. CONCLUSION: The data indicate that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts potentially involved in the specific pathophysiology of RA.

Arthritis, Rheumatoid↗
PubMed1999

Modeling peptide mass fingerprinting data using the atomic composition of peptides.

The peptide mass fingerprinting technique is commonly used for identifying proteins analyzed by mass spectrometry (MS) after enzymatic digestion. Our goal is to build a theoretical model that predicts the mass spectra of such digestion products in order to improve the identification and characterization of proteins using this technique. We present here the first step towards a full MS model. We have modeled MS spectra using the atomic composition of peptides and evaluated the influence that this composition may have on the MS signals. Peptides deduced from the SWISS-PROT protein sequence database were used for the calculation. To validate the model, the variability of the peptide mass distribution in SWISS-PROT was compared to two theoretical, randomly generated databases. Functions have been built that describe the behavior of the isotopic distribution according to the mass of peptides. The variability of these functions was analyzed. In particular, the influence of sulfur was studied. This work, while representing only a first step in the construction of an MS model, yields immediate practical results, as the new isotopic distribution model significantly improves peak detection in MS spectra used by protein identification algorithms.

Databases, Factual↗
PubMed1999

Improving protein identification from peptide mass fingerprinting through a parameterized multi-level scoring algorithm and an optimized peak detection.

We have developed a new algorithm to identify proteins by means of peptide mass fingerprinting. Starting from the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectra and environmental data such as species, isoelectric point and molecular weight, as well as chemical modifications or number of missed cleavages of a protein, the program performs a fully automated identification of the protein. The first step is a peak detection algorithm, which allows precise and fast determination of peptide masses, even if the peaks are of low intensity or they overlap. In the second step the masses and environmental data are used by the identification algorithm to search in protein sequence databases (SWISS-PROT and/or TrEMBL) for protein entries that match the input data. Consequently, a list of candidate proteins is selected from the database, and a score calculation provides a ranking according to the quality of the match. To define the most discriminating scoring calculation we analyzed the respective role of each parameter in two directions. The first one is based on filtering and exploratory effects, while the second direction focuses on the levels where the parameters intervene in the identification process. Thus, according to our analysis, all input parameters contribute to the score, however with different weights. Since it is difficult to estimate the weights in advance, they have been computed with a generic algorithm, using a training set of 91 protein spectra with their environmental data. We tested the resulting scoring calculation on a test set of ten proteins and compared the identification results with those of other peptide mass fingerprinting programs.

Algorithms↗
PubMed1999

Gene transfer of cytokine inhibitors into human synovial fibroblasts in the SCID mouse model.

OBJECTIVE: To investigate the effects of retrovirus-based gene delivery of inhibitory cytokines and cytokine inhibitors into human synovial fibroblasts in the SCID mouse model of rheumatoid arthritis (RA). METHODS: The MFG vector was used for gene delivery of tumor necrosis factor alpha receptor (TNFalphaR) p55, viral interleukin-10 (IL-10), and murine IL-10 into RA synovial fibroblasts. The effect on invasion of these cells into human articular cartilage and on perichondrocytic cartilage degradation was examined after 60 days of coimplantation into the SCID mouse. RESULTS: TNFalphaR p55 gene transfer showed only a limited effect on inhibition of RA synovial fibroblast invasiveness and cartilage degradation. In contrast, invasion of the RA synovial fibroblasts into the coimplanted cartilage was strongly inhibited by both viral and murine IL-10. Perichondrocytic cartilage degradation was not affected by either form of IL-10. CONCLUSION: The data show that cytokines can be successfully inserted into the genome of human RA synovial fibroblasts using a retroviral vector delivery system, and that the SCID mouse model of human RA is a valuable tool for examining the effects of gene transfer. In addition, inhibition of more than one cytokine pathway may be required to inhibit both synovial- and chondrocyte-mediated cartilage destruction in RA.

Animals↗
PubMed1999

Synovial fluid CD146 (MUC18), a marker for synovial membrane angiogenesis in rheumatoid arthritis.

OBJECTIVE: CD146 (MUC18/MCAM/S-Endo) is a marker of tumor progression and metastasis formation in human melanoma. This molecule has also been identified in smooth muscle, endothelial cells, and activated T lymphocytes. We measured the synovial fluid levels of soluble CD146 in various human joint diseases, including rheumatoid arthritis (RA). In addition, we studied the distribution of CD146 in normal and RA synovial tissues. METHODS: CD146 was isolated from MEL-OH melanoma cells and characterized by Coomassie blue staining and Western blotting. Soluble CD146 was measured by competitive enzyme-linked immunosorbent assay in synovial fluids of 3 healthy individuals and 7 cadavers (controls), as wells as in patients with traumatic joint injury (n = 10), osteoarthritis (OA; n = 10), psoriatic arthritis (PsA; n = 10), other non-RA polyarthritis (NRAP; n = 10), and RA (n = 31). Immunohistochemistry was performed on 3 normal and 3 RA synovial tissues. Flow cytometric, reverse transcription-polymerase chain reaction, and Western blot analyses were performed on enzymatically separated RA synovial tissue cells. RESULTS: Compared with controls (mean +/- SD 10 +/- 2 ng/ml), significantly elevated synovial fluid levels of soluble CD146 were detected in patients with OA, PsA, and RA (17 +/- 7, 21 +/- 11, and 39 +/- 16 ng/ml, respectively; P < 0.02-0.001), but not in patients with traumatic joint injury or NRAP. Patients with early RA (<1 year after diagnosis) revealed the highest levels (51 +/- 15 ng/ml, n = 10; P < 0.001 versus controls). In RA, soluble CD146 correlated significantly with morning stiffness (P < 0.001), the number of tender joints (P < 0.02), and the number of swollen joints (P < 0.005), but not with the erythrocyte sedimentation rate (P = 0.07) or the C-reactive protein level (P = 0.57). CONCLUSION: Since CD146 is expressed almost exclusively by vascular endothelium, high levels of soluble CD146 found in RA synovial fluid, particularly in patients with early disease, could reflect increased activity of endothelial cells and angiogenesis.

Adult↗
PubMed1999

Characterization of collagenase 3 (matrix metalloproteinase 13) messenger RNA expression in the synovial membrane and synovial fibroblasts of patients with rheumatoid arthritis.

OBJECTIVE: To study the localization and cell type-specific expression of collagenase 3 messenger RNA (mRNA) in the synovial membrane, its regulation in primary synovial fibroblasts, and the correlation with systemic markers of inflammation and radiographic damage in rheumatoid arthritis (RA). METHODS: The expression of collagenase 3 mRNA was characterized by Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization. Immunohistochemical detection of cell type-specific antigens was used in combination with in situ hybridization of collagenase 3 mRNA to characterize the cellular origin of collagenase 3 mRNA expression. RESULTS: Collagenase 3 mRNA was detected in synovial membrane specimens of 21 of 36 RA patients (58%) and correlated with an increase in erythrocyte sedimentation rate (P<0.05) and C-reactive protein levels (P<0.005). Collagenase 3 mRNA was localized in fibroblast-like cells of the lining and sublining layers, and at the synovial membrane-cartilage interface. Four of 10 primary synovial fibroblast cell cultures showed basal expression of collagenase 3 mRNA, which was stimulated 2-4-fold upon interleukin-1beta or tumor necrosis factor alpha treatment and, in contrast to interstitial collagenase mRNA, 5-10-fold by increasing the intracellular level of cAMP. The stimulation by cAMP analogs was completely abolished by protein kinase A inhibitors. CONCLUSION: Some RA patients show collagenase 3 mRNA expression in the synovial membrane, which correlates with elevated levels of systemic markers of inflammation in these patients. In synovial fibroblasts, the expression of collagenase 3 and interstitial collagenase mRNA is differentially regulated by distinct protein kinase signal transduction pathways.

1-Methyl-3-isobutylxanthine↗