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M Cogné

Publications and source records attributed to M Cogné.

At least 37 records · Page 2Linked to original sources
PubMed1999

Optimisation of HLA-B27 testing by association of flow cytometry and DNA typing.

HLA-B27 typing contributes to the diagnosis of ankylosing spondylitis. The classical technique of microlymphocytotoxicity is costly and can give false-negative results. We have compared 304 samples using two relatively new methods - flow cytometry and PCR-SSP - and evaluated their respective uses in routine analysis. Flow cytometric HLA-B27 testing was performed using three monoclonal anti-B27 antibodies (HLA-ABC-m3, GS145.2 and FD705 clones). Cut-off values were established to differentiate HLA-B27-positive from HLA-B27-negative samples with ROC curves. Although flow cytometric analysis with a reliable monoclonal antibody (mAb) is valuable for HLA screening, none of the HLA-B27 flow cytometry protocols was sufficient on its own to ascertain the HLA phenotype in 100% of samples. Two false negatives were observed with the FD705 mAb and the use of two different monoclonal antibodies did not increase the accuracy of HLA-B27 typing. HLA-B27 typing using molecular biology is a reliable but costly technique. Therefore we suggest that DNA typing could be used as a complementary technique and applied to samples whose HLA-B27 phenotype cannot be determined by flow cytometry. The association of flow cytometry and DNA typing is, in our experience, an economical and reliable approach.

Antibodies, Monoclonal↗
PubMed1999

HLA-G and classical HLA class I transcripts in various components of the adult human eye.

HLA-G transcripts have previously been detected in the ocular cell line HS738, in foetal eyes and in global ocular extract. Using RT-PCR and hybridization, we searched for HLA-G and classical HLA class I transcripts in the corneas of eye donors (n = 6) and in various components of eyes collected at time of surgery: lens (n = 3), iris (n = 2), vitreous body (n = 2) and retina (n = 2). Whereas HLA class I mRNA was abundant in the ocular tissues of all donors, we could not find HLA-G transcript in any ocular tissues obtained from subjects in vivo. The absence of HLA-G in adult eye cornea limits its interest as a possible target in corneal transplantation. Classical HLA class I transcripts were abundant in the cornea and other components of the eye. These results highlight the role of classical HLA class I antigen in eye immunity and corneal transplantation.

Cornea↗
PubMed1999

Age-related alterations of somatic hypermutation and CDR3 lengths in human Vkappa4-expressing B lymphocytes.

The lower avidity and/or affinity of antibodies generated by an aged immune system could be attributed to two major changes in the antibody repertoire: a shift in germline gene usage and a decrease in the rate of immunoglobulin hypermutation. In an attempt to identify the mechanisms involved in the observed humoral immune deficiency in the elderly, we studied whether differences in the somatic diversity of a particular Vkappa region occurred with ageing. By using the polymerase chain reaction and sequencing, we analysed and compared Vkappa4-Jkappa rearrangements isolated from young (mean age 21 years) and aged (mean age 83 years) healthy adults. Mutations in the Vkappa4 gene compared with the germline sequence were determined as well as the length and structure of the CDR3 sequence. We analysed in detail various mechanisms contributing to CDR3 and Vkappa variability in rearrangements involving the Vkappa4 gene. Our data revealed that, despite strong individual variations, significantly lower levels of somatic mutation were found in the aged group, both for complementarity-determining regions (CDRs) and framework regions (FRs) encoding Vkappa4 sequences. This decrease mostly affected mutations responsible for replacements and thus resulted in a lowered somatic diversification of the encoded Vkappa4 proteins in aged individuals. Moreover, comparison of the CDR3 regions of the Vkappa4-Ckappa cDNA revealed changes in light-chain junctional diversity that correlated with age. Altogether these data suggest an impaired light-chain somatic diversity in connection with human senescence.

Adult↗
PubMed1998

Insertion of the IgH locus 3' regulatory palindrome in expression vectors warrants sure and efficient expression in stable B cell transfectants.

We have explored the effect of inserting 3' immunoglobulin heavy chain (IgH) locus transcriptional regulatory elements in stable expression vectors driven by a heavy chain variable gene promoter (pVH). A cassette was constructed, associating three enhancer elements from the palindromic part of the 3' IgH regulatory region, namely Calpha3'/hs3 reverse, alpha3'E/hs1-2, and hs3. As regard to stable expression, this cassette carried some features of a locus control region (LCR) and conferred expression to an associated cat reporter gene in the majority of B cells having integrated the transgene. The palindromic cassette was inserted in an expression vector carrying Ig light chain coding sequences. In this construct, transcription driven by a pVH promoter/Emu cassette upstream of the transcription initiation site was boosted by the palindromic cassette located downstream of the coding sequence. This potent expression plasmid mimicking the architecture of endogenous Ig loci, definitely manifested a potent stimulatory activity for stable transcription, outscoring conventional ubiquitous or B-cell specific expression vectors.

Animals↗
PubMed1998

Modification of HLA expression on peripheral lymphocytes and monocytes during aging.

Immunosenescence involves modifications of humoral and cellular immunity. Here we report the analysis of human leukocyte antigen (HLA) expression on T lymphocytes, B lymphocytes and monocytes of 58 healthy subjects aged 23-95 years old. Using a double staining immunofluorescence and flow cytometry analysis, we have determined the percentages of cells expressing HLA class-I and HLA-DR antigens. The number of antigenic sites expressed per cell were evaluated for HLA-ABCw, HLA-A, HLA-B, HLA-DR locus with a flow cytometry quantification technique. With advancing age, we observed: (i) a significant decrease of the percentage of T cells and B cells expressing HLA-A products; (ii) a decrease of the number of HLA class-I antigenic sites expressed per cell on the three populations tested, predominantly on B cells and in a locus-dependent fashion; (iii) a decrease of the number of HLA-DR molecules expressed per T cell, although the percentage of T cells expressing DR products was increased; (iv) a significant diminution of the percentage of B cells expressing HLA-DR molecules, without changes of the number of HLA-DR antigenic sites per cells. These changes in HLA expression with increasing age could contribute to the decreased level of immunologic responsiveness observed with ageing and contribute to the modification of antigen recognition.

Adult↗
PubMed1998

Complete primary sequences of two lambda immunoglobulin light chains in myelomas with nonamyloid (Randall-type) light chain deposition disease.

We herein report on the first two primary sequences (BOU and RAC) of monoclonal light chains of the lambda type responsible for nonamyloid lambda light chain deposition disease. Both patients were affected with severe forms of myeloma complicated with renal failure. The pathological presentation typically featured Congo red-negative deposits along tubular basement membranes but differed somewhat from the typical "Randall-type" kappa light chain deposition disease: they lacked the prominent glomerulosclerosis pattern often featuring nonamyloid kappa deposits and were associated with cylinders or myeloma casts. Both protein sequences were deduced from those of the corresponding complementary DNAs in the bone marrow plasma cells. For each chain, products of three independent amplifications by polymerase chain reaction were sequenced and found to be identical. BOU and RAC lambda mRNAs had a normal overall structure consisting of Vlambda2 segments rearranged to Jlambda2Clambda2 but displayed a number of unusual features within their primary sequences. These substitutions are likely responsible for changes in light chain conformation that promote their aggregation and deposition along renal tubule basement membranes.

Amino Acid Sequence↗
PubMed1997

Identification of a homolog of the C alpha 3'/hs3 enhancer and of an allelic variant of the 3'IgH/hs1,2 enhancer downstream of the human immunoglobulin alpha 1 gene.

Although four regulatory elements are known downstream of the mouse IgH alpha gene, a single enhancer homologous to hs1,2 has been thus far described downstream of each human alpha gene (Chen, C. and Birshtein, B. K., J. Immunol. 1997. 159: 1310). We characterized a 10-kb region downstream of the human alpha 1 gene. Two B cell-specific regulatory elements homologous to the murine C alpha 3'/hs3 and hs1,2,3' enhancers were found, which are duplicated downstream of alpha 2. The hs1,2 element is in inverted orientation by comparison with a recently reported alpha 1 hs1,2 element: it appears as a common allelic variant carrying an internal tandem repeat insertion and its prevalence in the human population is 60%. As in the mouse, the human hs1,2 enhancer is flanked with long inverted repeats which may have promoted inversion events through homologous recombination. Although the palindromic organization of the region is maintained in human, sequence identity with rodents focuses on core enhancer elements rather than on flanking repeats. Concerted divergence of both sides of the dyad symmetry suggests that inverted repeats are not just evolutionary remnants but rather play an architectural role in the LCR function.

Alleles↗
PubMed1997

Reversal of the relative expression of cardiac and skeletal alpha1 subunit isoforms of L-type calcium channel during in vitro myogenesis.

Cardiac and skeletal type of excitation-contraction coupling (ECC) are quite different. Those differences could be explained by structural ones in the molecular entities involved in ECC, ie dihydropyridines (DHP) receptors (alpha1 subunit of L-type calcium channels) of the sarcolemma or ryanodine receptors of the sarcoplasmic reticulum membrane. As previously demonstrated by means of electrophysiology, the two types of ECC coexist during the first stages of in vitro development of skeletal muscle, whereas the skeletal type predominates at the later ones. In order to see whether evolution of ECC could be correlated with the one of alpha1 subunit expression, we determined by Northern Blotting which isoforms of alpha1 subunit are expressed during the in vitro myogenesis. mRNA corresponding to the cardiac isoform are present in myoblasts (before fusion), but patch-clamp experiments showed that they are not functional. After fusion, skeletal and cardiac mRNA are coexpressed in myotubes, with different intensities: whereas expression of skeletal mRNA (which are the more intensive) stabilized at the later stages tested, cardiac mRNA decreased. We conclude that evolution in mRNA alpha1 subunit isoforms expression could partly explained evolution of ECC features during in vitro myogenesis.

Animals↗
PubMed1997

Membrane isoforms of human immunoglobulins of the A1 and A2 isotypes: structural and functional study.

As for IgM, human IgA occurs either as soluble molecules in plasma and various other body fluids, or as membrane-bound molecules on differentiated B cells, where they are part of the B-cell receptor for antigen (BCR). We studied the structure of transcripts encoding the membrane-anchored alpha-chain of the human BCR alpha, which may be present in two different forms resulting from alternate splicing of the alpha-chain mRNA (type I or type II). The ratio of type I versus type II did not vary upon stimulation of a B-cell line with various cytokines. Rather, it differed strikingly in cells expressing either the IgA1 or IgA2 isotype of the BCR alpha, with virtually no type II alpha-chain in the latter. Co-modulation experiments also yielded different results for both isotypes, since they demonstrated a physical association of both membrane (m)IgA1 and mIgA2 with CD79b, the beta component of the BCR Ig alpha/Ig beta heterodimer, but only of mIgA1 with CD19. Whatever the isotype, the BCR of the IgA class was able to carry out signal transduction upon cross-linking by specific monoclonal antibodies but, in contrast to mIgM, it relied mainly on the entry of extracellular Ca2+ rather than on the release of intracellular stocks.

Alternative Splicing↗
PubMed1996

Structural peculiarities of a truncated V kappa III immunoglobulin light chain in myeloma with light chain deposition disease.

We report on the primary sequence of the monoclonal immunoglobulin light chain (LC) REV involved in myeloma-associated light chain deposition disease (LCDD). This sequence was deduced from that of the corresponding complementary (c)DNA in bone marrow plasma cells. Products of three independent amplifications by polymerase chain reaction (PCR) were sequenced and found to be identical. The kappa mRNA encoding this N-glycosylated LC showed an overall normal structure consisting of a V kappa III segment rearranged to J kappa II. Direct N-terminal amino acid sequencing of the circulating monoclonal IgA2, kappa showed identity with the bone marrow-derived sequence. The kappa-chain presented several unusual features affecting both the leader sequence and the variable (V) region. Four unique amino acid substitutions were found at positions -8, -3, -2 and -1 in the leader sequence and probably resulted in an unusual cleavage by signal peptidase, thus making the LC truncated by one residue and accounting for its unique hydrophobic N-terminus: Ile-Ile-Leu. Additional peculiarities were observed in the V region, including a Thr74-->Asn substitution creating a N-glycosylation site, and Thr53-->Ile, which was only reported once among human kappa III chains, in another LCDD case, and may be of special significance at a position usually harbouring a polar amino acid.

Amino Acid Sequence↗
PubMed1995

Role of light chain variable region in myeloma with light chain deposition disease: evidence from an experimental model.

Light chain deposition disease (LCDD) results from a propensity of some human monoclonal L chains to form tissue deposits. We designed an experimental model for in vivo expression of human kappa L chain sequences in mice and compared a somatically mutated LCDD chain with a closely related control kappa chain, both encoded by the unique V kappa IV gene. Mice secreting the LCDD chain but not those producing the control chain showed deposits with a distribution similar to that observed in patients. These data show that discrete changes in V region sequences can play a major role in tissue deposition of human L chains.

Amino Acid Sequence↗
PubMed1995

Sequences of V kappa L subgroup light chains in Fanconi's syndrome. Light chain V region gene usage restriction and peculiarities in myeloma-associated Fanconi's syndrome.

Certain monoclonal Ig light chains (LC) are responsible for marked disturbances of proximal tubule cell functions (Fanconi's syndrome, FS). In patients with FS, intracellular crystal-like inclusions containing LC determinants are commonly found in plasma cells, macrophages, and renal tubular cells. In an attempt at understanding the pathogenesis of myeloma-associated FS, we recently determined the first complete primary sequence of a kappa-LC (CHEB) responsible for the disease. We now report on the primary structure of three other kappa-LC of the V kappa l variability subgroup associated with FS (TRE, TRO, and DEL). After PCR amplification, cDNA encoding these LC were sequenced. CHEB, TRE, and TRO LC genes were found to be highly homologous to the same germline gene O2/O12. These patients had numerous intracellular crystals, whereas the fourth patient, DEL, had no detectable crystals. The LC from the latter patient was homologous to another germline gene, O8/O18. Comparison of these LC sequences to previously reported LC of the V kappa l subgroup allowed identification of a number of unusual amino acid substitutions in the V region that had rarely or never been previously described at the corresponding positions. Some of these unusual substitutions affect highly conserved amino acids located either in an external loop (residue 30) or in inner (residues 48 and 55) and outer (positions 63 and 72) beta-sheets that may be important for the structure and binding properties of the kappa-chains. These and several other substitutions, some of them shared with amyloidogenic kappa-LC, could induce conformational alterations and represent a determinant pathogenic factor.

Amino Acid Sequence↗
PubMed1995

Structure of abnormal heavy chains in human heavy-chain-deposition disease.

The sequences of two immunoglobulin gamma 1 heavy chains involved in the formation of non-amyloid tissue deposits were determined in two patients (RIC and THR) affected with plasma cell monoclonal proliferation and heavy-chain-deposition disease. The proliferating plasma cells of patients RIC and THR synthesized truncated gamma 1 chains of 45 kDa and 24 kDa, respectively, carrying internal deletions of the first constant (CH1) domain (RIC) or of the CH1, hinge and CH2 domains(THR). The shortened gamma chains were associated with lambda light chains in the monoclonal IgG component present in the serum from both patients but not in tissue deposits which lacked any detectable light chains. Bone marrow plasma cells from RIC contained short gamma 1 heavy-chain transcripts in which a VDJ exon related to the VH2 subgroup was directly joined to the hinge exon; plasma cells from THR contained short gamma 1 transcripts with a VDJ exon related to the VH3 subgroup joined to the CH3 exon. In both cases, the truncated transcripts carried precise deletions of complete exons and used regular splice sites at the variable/constant junction, consistent with the hypothesis that they originated from abnormal splicing of primary transcripts from the productively rearranged heavy-chain genes. Striking abnormalities of the variable regions were found, especially in framework regions, including replacement of an invariant tryptophan residue in protein from THR, hydrophobic residues most likely exposed to the solvent and inversion of charged amino acids probably exposed on the surface of the molecule.

Amino Acid Sequence↗
PubMed1994

The effect of intron sequences on expression levels of Ig cDNAs.

Several cDNA expression vectors were constructed and tested by stable transfection into a murine lymphoid cell line in order to compare secretion rates of a human immunoglobulin (Ig) light chain (LC). When the cDNA was under transcriptional control of the SV40 promoter and enhancer and preceded by the SV40 19S late mRNA intron, a weak LC production was detected. Secretion rate was not improved by replacing the SV40 promoter and enhancer by a combination of a murine Ig heavy chain (HC) gene promoter and enhancer even with insertion of additional Ig enhancers. In contrast, replacement of the 19S intron by a large intron derived from a human Ig HC gene and containing the intronic enhancer dramatically increased the secretion rate. High-level production was also obtained with the same enhancer-containing intron placed downstream from the LC cDNA. Stable transfectants were obtained that secreted the human LC in amounts comparable to those obtained with Ig genes. Our results suggest that the SV40 19S late mRNA intron used in several expression vectors is not appropriate when the purpose is to produce large amounts of antibody molecules. By providing transcriptional, splicing and polyadenylation signals, the presently described vectors will be useful for easy cloning and high-level expression in lymphoid cells of cDNAs or PCR products encoding antibody molecules.

Animals↗
PubMed1994

A class switch control region at the 3' end of the immunoglobulin heavy chain locus.

We replaced the IgH 3' enhancer (3'EH) region with a neomycin resistance gene in ES cells and generated chimeric mice in which all mature lymphocytes were either heterozygous (3'EH+/-) or homozygous (3'EH-/-) for the mutation. In vitro activated 3'EH-/- B cells responded similarly to 3'EH+/- B cells with respect to proliferation and secretion of IgM and IgG1 but were specifically deficient in IgG2a, IgG2b, IgG3, and IgE secretion. These isotype deficiencies correlated with a deficiency in accumulation of transcripts from and class switching to affected CH genes. In vivo, chimeric mice containing only 3'EH-/- B cells were deficient in serum IgG2a and IgG3. We propose that the 3'EH-/- mutation disrupts the activity of a regulatory region that influences heavy chain class switching to several different CH genes that lie as far as 100 kb upstream of the mutation.

Animals↗
PubMed1993

Monoclonal Ig L chain and L chain V domain fragment crystallization in myeloma-associated Fanconi's syndrome.

Monoclonal free L chains secreted in immunoproliferative disorders are frequently involved in renal complications, including a specific proximal tubule impairment, the Fanconi's syndrome, which is generally featured by intracellular crystallization of L chain-related material. In a patient with myeloma-associated Fanconi's syndrome, hexagonal crystals (most surrounded by smooth membranes) were found in kidney proximal tubular cells and bone marrow plasma cells and phagocytes. The sequence of the patient's monoclonal kappa-chain was deduced from that of identical kappa-cDNA clones from the tumoral plasma cells. Small protein-enriched gel filtration fractions from urine yielded crystals morphologically similar and with the same 60 A periodicity on electron micrographs as those found in the cells. N-terminal sequencing and mass spectrometry studies showed that the crystals contained a 107-amino acid fragment (with a C-terminal lysine) corresponding to the V domain together with a low proportion of the entire kappa-chain. In vitro trypsin and pepsin treatment of the native entire kappa-chain yielded a homogeneous V domain fragment which, contrary to other monoclonal kappa-chains, was completely resistant to further proteolytic attack. The patient's kappa-chain also displayed an unusual self-reactivity, as demonstrated by a Western blot technique. The peculiar proneness of the V domain to resist proteolysis and to form crystals might prevent the normal cell catabolism of the L chain and lead to crystallization and renal impairment.

Adult↗