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Biomedical subjects

H A Ménard

Publications and source records attributed to H A Ménard.

At least 19 recordsLinked to original sources
PubMed1997

Expression of recombinant human cyclooxygenase isoenzymes in transfected COS-7 cells in vitro and inhibition by tenoxicam, indomethacin and aspirin.

The recent discovery of cyclooxygenase-2 (COX-2), an isoenzyme associated mainly with inflammation created the need to reevaluate cyclooxygenase inhibitors with reliable screening methods. In the present study we standardized a technique to determine the IC50S of cyclooxygenase inhibitors on recombinant human COX-1 and COX-2 expressed in mammalian cells and used it to study the compounds tenoxicam, aspirin and indomethacin. The IC50S of aspirin, indomethacin and tenoxicam for human COX-1 were 0.41 +/- 0.07 microgram/ml, 0.008 +/- 0.003 microgram/ml, and 7.94 +/- 3.28 micrograms/ml, respectively, and for human COX-20.64 +/- 0.16 microgram/ml, 0.09 +/- 0.05 microgram/ml, and 10.61 +/- 1.50 micrograms/ml, for aspirin, indomethacin, and tenoxicam. Tenoxicam had the lowest IC50hCOX-2/IC50hCOX-1 ratio (1.34), followed by aspirin (1.53) and indomethacin (10.82). The system described in the present study provides a simple and efficient way to determine the specificity of NSAID inhibition for each of the human cyclooxygenase isoenzymes separately.

Animals↗
PubMed1996

Autoantigenic epitopes on hY5 Ro RNA are distinct from regions bound by the 60-kDa Ro and La proteins.

We recently reported the identification in human anti-Ro serum of Abs specifically immunoprecipitating deproteinized hY5 RNA. In the present report, we characterized the epitopes recognized by anti-hY5 RNA Abs. Using deletion and site-directed mutagenesis of hY5 cDNA and in vitro transcribed RNAs with intact and 3'-shortened ends, we have defined two conformational antigenic determinants distinct from the regions known to bind Ro and La proteins. One of these epitopes (epitope A) is present in the middle portion of hY5 RNA and is dependent on the presence of a four-nucleotide sequence (AACC at position 58-61) that may form a single-stranded loop. Deleting these four nucleotides or modifying the stem structures proximal or distal to this loop abolishes recognition of the mutated RNAs by Abs. The second epitope (epitope B) requires the presence of another four-nucleotide sequence (CUUG at position 74-77) in between the Ro and La binding sites. Deleting this CUUG sequence or modifying nucleotides on the 5' side of the stem structure below the Ro60 binding site severely compromises the interaction with Abs. Since Abs to deproteinized hY RNAs are restricted to anti-hY5 RNA and target determinants not involved in interactions with known hY5 RNA-binding proteins, human RohY5 particles may play a direct role in the immunization process, leading to the production of anti-hY5 RNA autoantibodies.

Antibody Specificity↗
PubMed1996

RNA-labelled Ro and La ribonucleoprotein complexes reassembled in vitro; characterization by gel shift analysis.

Ro and La RNP complexes were reassembled from in vitro labelled hY5 RNA and HeLa cell extracts. These complexes were then visualized through retardation of migration of labelled hY5 RNA in non-denaturing polyacrylamide gels. Three major complexes (named A, B, and C) were formed when crude cellular extracts (S100 fraction) were used. Using monospecific anti-60-kD Ro (Ro60) and anti-La antibodies to retard RNPs containing these antigens during migration in the gels, the three major complexes were shown to contain Ro60 (C), La (B), or both proteins (A). The specificity of RNA-protein interactions in the reassembled complexes was further demonstrated using two 3'-shortened hY5 RNA transcripts lacking the La-binding site (hY5-Alu I RNA) and both the Ro60 and La-binding sites (hY5-Hha I RNA). hY5-Hha I RNA still formed a single, minor complex when incubated with S100 extract, suggesting interaction with a yet undefined protein. In addition, we used the capacity of specific antibodies to retard the migration of the reassembled complexes to design a detection assay for anti-Ro and anti-La autoantibodies. Using 84 human sera, our assay was shown to approximate the specificity and sensitivity of an immunoprecipitation assay where 32P-labelled cell extracts are used as source of antigens. Our assay may be used to detect low levels of antibodies to conformational determinants on Ro60 and La proteins in human sera and antibody preparations.

Autoantigens↗
PubMed1995

Autoantibodies in human anti-Ro sera specifically recognize deproteinized hY5 Ro RNA.

We report the existence of a novel autoantibody specificity linked to anti-Ro antibodies. Sera from two patients with anti-Ro ribonucleoprotein (RNP) antibodies also contained antibodies that immunoprecipitated specifically either the deproteinized RNA component of the RohY5 RNP particle, or intact in vitro transcribed hY5 RNA. No serum recognized specifically the other hY RNAs. A mutant hY5 RNA with additional nucleotides (nt) at both extremities was not immunoprecipitated, possibly because of altered secondary structure. Following digestion of hY5 RNA with ribonuclease T1, the smallest immunoprecipitable RNA fragments were 27 and 31 nt long, and respectively mapped to the 5' and 3' ends of hY5 RNA, excluding the La-binding region. Base pairing between the 27 and 31 nt long fragments was required for recognition by antibodies. Our data indicate that the epitope bound by anti-hY5 RNA antibodies is conformational. We have previously reported that most anti-Ro sera contain a population of antibodies specific for the RohY5 RNP. Since antibodies to the deproteinized hY RNAs within anti-Ro sera are also restricted to anti-hY5 RNA, a direct role for the human-specific RohY5 particles in the immunization process leading to the production of anti-Ro antibodies is suggested.

Antibodies, Antinuclear↗
PubMed1995

Purification of antigenically intact Ro ribonucleoproteins; biochemical and immunological evidence that the 52-kD protein is not a Ro protein.

Anti-Ro sera immunoprecipitate Ro ribonucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whose functions are unknown and whose exact composition remains controversial. In addition to 60-kD Ro and to La proteins, a 52-kD polypeptide (p52) has been proposed to be a stable component of the Ro RNPs. To confirm the immunological studies supporting this hypothesis, we have biochemically purified Ro RNPs from HeLa cells using non-denaturing conditions. Ro RNPs segregated into three distinct populations, one of which only contained hY5 RNA (RohY5 RNPs). No p52 co-purified with Ro RNPs. Despite the absence of p52, purified Ro RNPs had biochemical and immunological properties identical to those of unfractionated Ro RNPs. Many anti-Ro sera only recognize p52 in immunoblots, and are said to be monospecific anti-p52. Preincubation with purified RohY5 RNPs (free of p52) of all human anti-Ro (including so-called monospecific anti-p52) sera abolished their capacity to immunoprecipitate Ro RNPs from unfractionated HeLa cell extracts. Conversely, preincubation of anti-Ro sera with purified p52 protein specifically inhibited recognition of p52 in immunoblots, but did not interfere with immunoprecipitation of Ro RNPs. Our data demonstrate that anti-p52 antibodies do not target intact Ro RNPs, nor do they target the native 60-kD Ro protein. Contrary to previous reports, p52 protein is not a stable component of antigenically intact Ro RNPs.

Animals↗
PubMed1995

Detection and expression of a cDNA clone that encodes a polypeptide containing two inhibitory domains of human calpastatin and its recognition by rheumatoid arthritis sera.

RA is the most frequent and most destructive inflammatory arthropathy. Rheumatoid factors, in spite of their lack of disease specificity, are important serological markers for RA and appear important in its immunopathogenesis as well. In search of more disease-specific autoimmune systems, we have screened a human placenta lambda gt11 cDNA expression library using selected sera from patients with classical erosive RA. We have identified one clone (RA-1) that is recognized by three of five screening sera. The 950-bp insert shows a complete nucleotide sequence homology to the cDNA encoding the two COOH-terminal domains of calpastatin. The deduced open reading frame of the RA-1 cDNA predicts a 284-amino acid protein, with a calculated mol wt of 35.9 kD. Calpastatin is the natural inhibitor of calpains, which are members of the cysteine proteinases recently implicated in joint destruction in rheumatic diseases. The two domains encoded by the RA-1 clone each contain the structural features associated with the inhibitory activity of human calpastatin. By Western blotting, 45.5% or 21/44 RA sera specifically recognized both the fusion and the cleaved recombinant protein. This is in contrast to 4.7% (2/43) in nonrheumatoid sera and 0/10 in normal sera. Anticalpastatin autoantibodies could represent a disease-associated marker in chronic erosive arthritis of the rheumatoid type and could hypothetically play a dual pathogenic role, directly via an immune interference and indirectly through an immune complex mechanism.

Amino Acid Sequence↗
PubMed1995

Detection of anti-neutrophil cytoplasmic antibodies by immunoprecipitation.

Immunoprecipitation (IP) of radiolabeled PMN extracts was used as the gold standard for anti-proteinase 3 (PR-3) autoantibody detection to validate immunofluorescence (IF) and alpha granule (alpha) ELISA. A myeloperoxidase (MPO) ELISA was also used in parallel. We studied 48 patients with strictly defined vasculitic syndromes in the initial active phase of their disease. The 3 methods confirmed the high (> 90%) sensitivity and specificity of anti-PR-3 for patients with Wegener's granulomatosis (WG). Similarly, a high (86%) sensitivity of MPO-ELISA was found in microscopic polyarteritis as defined. Using alpha-ELISA, we could not improve the detection rate of anti-PR-3 obtained by IF-cANCA-pattern reading. Moreover, a small proportion (< 15%) of biopsy-proven WG patients had anti-MPO antibodies detected by IF, usually as pANCA but also, even if rarely, as bona fide cANCA (< 5%). Thus, IF would seem to be the most reliable screening method and alpha-ELISA should be used for confirmation. On the other hand, because MPO-ELISA detected twice as many anti-MPO positive sera as did pANCA pattern reading by IF, we suggest that in the clinical context of a vasculitis, MPO-ELISA should also be used as a screening test. Although IP is not designed for routine clinical use, it should be required when reporting the presence of anti-PR-3 in vasculitis-like diseases that are fertile grounds for false positive reactions.

Adult↗
PubMed1995

Clinical and serological identification of 2 forms of complete heart block in children.

OBJECTIVE: To study the association of maternal antibodies to Ro(SSA) and/or La(SSB) with isolated complete congenital heart block (CCHB) in children according to the child's age at detection. METHODS: Sera from 17 mothers of 18 children with CCHB of unidentified cause were studied. Autoantibodies were measured by double immunodiffusion, enzyme linked immunosorbent assay (ELISA), Western blot, and immunoprecipitation from cell extracts. Statistical analysis used the chi 2 test with Yates' correction. RESULTS: CCHB was diagnosed in 12 children of 11 mothers before the age of 3 mo (Group A) and in 6 children of 6 mothers after the age of 17 mo (Group B). Seven Group A mothers and no Group B mother had connective tissue disorders; autoantibodies were found in 9/11 Group A and in 1/6 Group B mothers (p < 0.01). Eight Group A children needed a pacemaker and one other died of cardiac insufficiency, whereas only one of the 6 Group B children needed a pacemaker. Interestingly, this latter child was the only one from Group B whose mother's serum contained autoantibodies. Irrespective of their age at diagnosis, the children with CCHB who needed a pacemaker and the one who died were born to mothers with autoantibodies (p < 0.001). CONCLUSION: CCHB detected before the age of 3 mo is highly associated with the presence of anti-Ro(SSA)/La(SSB) in the mothers, while CCHB diagnosed later is generally not. For epidemiological studies, the former type should be considered early onset as opposed to late onset CCHB in the latter type. Establishing this clinicoserological distinction is also important for the children, since it alerts the clinician to a more severe prognosis (necessity of a pacemaker), even in the rare occurrence of late diagnosed CCHB.

Adolescent↗
PubMed1994

Expression of prostaglandin endoperoxide synthase-1 and prostaglandin endoperoxide synthase-2 in human osteoblasts.

A Prostaglandin endoperoxide synthase isoenzyme was recently identified in several cell lines. Osteoblasts possess Prostaglandin endoperoxide synthase activity, but it is not known which isoenzymes are present in these cells. Our objective was to identify these isoenzymes in human osteoblasts. Resting cells in culture did not produce measurable amounts of PGE2 and did not express Prostaglandin endoperoxide synthase-1 or Prostaglandin endoperoxide synthase-2 mRNAs detectable by Northern blot. Treatment with rhIL-1 alpha or rhTNF alpha induced both the expression of Prostaglandin endoperoxide synthase-2 mRNA and the synthesis of PGE2, rhIL-1 alpha being more potent on an equimolar basis than rhTNF alpha. Dexamethasone inhibited the increase in Prostaglandin endoperoxide synthase-2 mRNA and the production of PGE2 induced by both cytokines. These results suggest that Prostaglandin endoperoxide synthase-2 may be the relevant isoenzyme for prostanoid production in human osteoblasts in culture.

Alkaline Phosphatase↗
PubMed1994

The Sa system: a novel antigen-antibody system specific for rheumatoid arthritis.

OBJECTIVE: To describe a novel autoimmune system (Sa/anti-Sa) specific for rheumatoid arthritis (RA). METHODS: Antibodies were detected in immunoblots using human spleen and placenta extracts as antigens. Sera from 482 patients with various rheumatic diseases as well as from healthy controls were evaluated to define the disease associations of anti-Sa antibodies. RESULTS: Sera from 88 of 206 (42.7%) unselected patients with RA recognized specific protein bands (the Sa antigen) in immunoblots of spleen or placenta extracts, including 9 of 31 (29%) patients seen in the first few months after disease onset. Anti-Sa antibodies were found both in rheumatoid factor (RF) negative (17/63 or 27%) and in RF positive patients with RA (71/143 or 50%). They were nevertheless absent in RF positive patients with other connective tissue diseases (0/39). Antibodies to Sa were essentially found in sera from patients with RA (specificity 98.9%) being found only in 3 patients whose arthritides did not fulfill the ACR criteria. The positive predictive value of anti-Sa antibodies for RA was 96.7%, while its negative predictive value was 69.8%. Anti-Sa antibodies were predominantly of the IgG isotype, with titers varying from 1/50 to > 1/1000. The Sa antigen was characterized as a poorly soluble protein that is present in normal human tissues and that is distinct from all previously described RA associated autoimmune systems. CONCLUSION: Anti-Sa antibodies are a novel serological marker highly specific for RA. Since anti-Sa antibodies occur independently of RF, they can be used as an additional diagnostic tool. The molecular nature of the Sa antigen as well as its potential pathogenic role in a significant proportion of patients with chronic articular inflammation of the rheumatoid variety merit further definition.

Antibody Specificity↗
PubMed1994

Chronic modulation of the autoimmune response following parvovirus B19 infection.

We describe 2 patients with prolonged autoimmune alterations following parvovirus B19 infection. B19 induced aplastic crises were the revealing manifestations of asymptomatic hemolytic conditions in the 2 patients: a Coombs' positive hemolytic anemia induced by D-penicillamine in the first and congenital spherocytosis in the second. Both patients had transient clinical and serological manifestations highly suggestive of systemic lupus erythematosus. In addition, prolonged clinical and serological remission of rheumatoid arthritis was observed in the first patient, while arthralgias, FANA, and anti-Ro antibodies persisted in the second, previously healthy patient. Our data suggest that parvovirus B19 infection may lead to chronic modulation of the autoimmune response in predisposed individuals.

Adult↗
PubMed1993

Heterogeneity of ANCA sera showing atypical, peripheral and classical cytoplasmic immunofluorescence patterns.

Atypical (A) ANCA immunofluorescence (IF) patterns have been described in several disease groups. We have previously reported a distinct cytoplasmic A-ANCA in 7-10% of patients with SLE and/or RA. Here, we show that these rheumatic disease associated A-ANCAs are best identified using U937 cells as substrate and that they do not target either a serine proteinase or a peroxidase. Furthermore, these sera immunoprecipitate a 40 kDa or a 42 kDa band using in vivo 35S-amino acid labelled HL60 or U937 cell extracts, respectively. Although these bands are the only one seen with pure A-ANCA sera, they can also be found in addition to the expected bands of PR3 or MPO in up to 30% of bona fide C- or P-ANCA sera. These data confirm and extend our previous observations. They also suggest that target heterogeneity of ANCA antibodies is frequent. Care should thus be taken in interpreting in a cause and effect relationship, an IF pattern or a biological effect produced by a serum with ill documented monospecificity. The exact nature and significance of this (these) new antigen(s) have yet to be clarified.

Antibodies, Antineutrophil Cytoplasmic↗