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Biomedical subjects

J Ripoche

Publications and source records attributed to J Ripoche.

At least 37 records · Page 2Linked to original sources
PubMed1995

Inhibition of chronic myelogenous leukemia cells harboring a BCR-ABL B3A2 junction by antisense oligonucleotides targeted at the B2A2 junction.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL hybrid gene. Two types of hybrid BCR-ABL mRNA have been found, B2A2 and B3A2. As the BCR-ABL rearrangement is specific to leukemic cells, selective inhibition of leukemic cell growth by BCR-ABL antisense oligonucleotides (ASO) has been reported in vitro for CML patients and cell lines. However, controversial results have been obtained from preclinical studies using anti-BCR-ABL ASO, as nonspecific inhibition of leukemic cell growth was evidenced in some cases. B3 exon secondary structure was deduced from its sequence and found to be a loop. According to this predictive structure of exon B3, a 56-mer antisense oligonucleotide targeting the polypurine bases from the B2A2 junction was devised which would inhibit proliferation (MTT assay) of B3A2 junction cell lines (K562 and a murine cell line Ba/F3 transfected with the B3A2 junctional sequence). This ASO had a hairpin-like secondary structure and was found to be much more resistant to the action of nucleases than control 18-mer standard oligonucleotides. Hybridization to its target mRNA occurs via formation of a triplex structure. A concentration of 5 microM of specific 56-mer B2A2 ASO was necessary to demonstrate 50% optical density (OD) reduction for K562 cell line and Ba/F3 transformed by B3A2 cDNA. Sense and non-sense 56-mer sequence or 18-mer linear ASO showed no effect for these concentrations. Western blot showed a partial inhibition of P210 protein; expression of P145abl remains unchanged. The 56-mer ASO also inhibited the proliferation of B2A2 junction cell line BV173 at the same concentration and showed no effect on the HL60 cell line used as control.

Base Sequence↗
PubMed1994

Location of Golgi membranes with reference to dividing nuclei in syncytial Drosophila embryos.

The role of cytoskeletal elements in the cellularization of syncytial Drosophila embryos is becoming evident; however, the distribution and role of organelles such as the Golgi complex, essential for membrane biogenesis, remain unknown. We have cloned a Golgi-membrane-associated polypeptide, beta-COP, from Drosophila. Immunocytochemical studies of syncytial Drosophila embryos with anti-Drosophila beta-COP antibody reveal that Golgi membranes are spatially segregated from the rapidly dividing nuclei. In early embryos, the Golgi membranes are located in the embryonic cortex and nuclei are confined to the core. This distribution of Golgi membranes may serve in preparation of the embryonic cortex for the accommodation of nuclei upon their eventual migration to the cortex and in biogenesis of the excessive plasma membrane needed for cellularization of syncytial embryos.

Amino Acid Sequence↗
PubMed1993

Local and systemic activation of the whole complement cascade in human leukocytoclastic cutaneous vasculitis; C3d,g and terminal complement complex as sensitive markers.

We have studied complement activation both in plasma samples and in lesional skin from patients with leukocytoclastic cutaneous vasculitis (LCV). Enzyme immunoassay (EIA) quantification of the complement activation markers, C3d,g and the terminal complement complex (TCC) in plasma, showed that their levels were significantly increased in 66% and 55% of the patients, respectively (n = 29) compared with healthy controls, whereas the standard measurements of C3, factor B, C1q, C4 and C2 were generally within normal range. Elevations of C3d,g and TCC levels in plasma were significantly correlated. Importantly, a significant correlation was found between the severity of the vasculitis and both C3d,g and TCC plasma levels. Immunofluorescence studies of skin biopsy specimens demonstrated simultaneous presence of perivascular dermal deposits of C3d,g and TCC in lesional skin from 96% and 80% respectively of the patients (n = 25). There was a significant correlation between the intensity of the deposits of both markers. Clusterin, a TCC inhibitory protein, was always found at the same sites of perivascular TCC deposits. Immunofluorescence studies at the epidermal basement membrane zone (BMZ) revealed in each case deposits of C3d,g which were accompanied by TCC deposits in 52% of the biopsy specimens. These data demonstrate that there is a local and systemic activation of the whole complement cascade in human LCV. The presence of both C3d,g and clusterin-associated TCC perivascular deposits suggests an intervention of a regulatory mechanism of local complement activation in LCV. Finally, measurement of plasma C3d,g and TCC appears to be a sensitive indicator of systemic complement activation and disease severity in LCV.

Aged↗
PubMed1992

Expression of complement components of the alternative pathway by glioma cell lines.

Glioma cell lines express proteins of the complement alternative pathway, namely C3, factor B, factor H, and factor I. Secretion of these proteins was shown by a sensitive and specific ELISA. C3 and factor H were rapidly secreted by glioma cell line CB193 and reached a concentration of 140 ng/ml/10(6) cells after 72 h of culture. Factor B and factor I were secreted at a lower rate and reached concentrations of 25 and 15 ng/ml/10(6) cells, respectively. Western blot and immunoprecipitation experiments showed that secreted proteins were identical to the corresponding plasma proteins. For factor H, besides the well known 150-kDa species, an additional polypeptide of 45 kDa with factor H immunoreactivity was observed. This species corresponded to the N-terminal truncated form found in plasma. In preliminary experiments, we observed control of these syntheses by cytokines. IL-1 beta significantly increased C3 secretion, with no effect on factor H. Secretion of factor H was enhanced by IFN-gamma. These results show that a glioma cell line could be a useful tool to study complement biosynthesis by glial cells in humans.

Complement C3↗
PubMed1992

In vitro biosynthesis of complement factor I by human endothelial cells.

We have studied the secretion of the complement regulatory protein factor I by human umbilical vein endothelial cells (HUVEC). Northern and Western blot analysis and biosynthetic labeling experiments indicate that HUVEC secrete factor I at very low levels in basal conditions and that this secretion is significantly enhanced by interferon-gamma. Analysis of the proteolytic inactivation of C3b by HUVEC supernatants show that factor I is secreted in a functional form and can promote the specific proteolytic inactivation of C3b to iC3b. Together with previous studies establishing the secretion of complement factor H by HUVEC, this work demonstrates that the endothelial cell is able to secrete in its environment two complement regulatory proteins, factor I and factor H, which can mediate the degradation of C3b to iC3b. The secretion of factor I by HUVEC provides a useful in vitro model to analyze the modulation of this secretion and may be relevant to the local deposition of iC3b at the surface of the endothelium during the inflammatory reaction.

Cells, Cultured↗
PubMed1992

Differential modulation by glucocorticoids of alternative complement protein secretion in cells of the monocyte/macrophage lineage.

The effect of the synthetic glucocorticoid dexamethasone (DXM) on the secretion by human monocytes of alternative complement proteins C3, factor B and factor H was investigated. Results indicated that DXM modulates this secretion in a direction which would be consistent with its anti-inflammatory properties. DXM, at therapeutic concentrations, had a suppressive effect on C3 and factor B secretion and a stimulatory effect on factor H secretion by monocytes. This differential modulation on C3, factor B and factor H secretion was similar in mature macrophages. Together with previous studies showing that DXM had a suppressive effect on C3 and factor B secretion and a stimulatory effect on factor H secretion by human endothelial cells, our results indicate that DXM appears to have the general property of regulating local production of complement components so as to control complement activation.

Cell Differentiation↗
PubMed1990

Expression of complement alternative pathway proteins by endothelial cells. Differential regulation by interleukin 1 and glucocorticoids.

We have studied the secretion of proteins of the alternative pathway of complement C3, factor B and factor H by human umbilical vein endothelial cells (HUVEC). Results showed that factor H and factor B are quantitatively secreted in abundance whereas C3 could only be detected when the cells are maintained in culture during long periods of time. Interferon-gamma stimulated factor H, factor B and, to a lesser extent, C3 secretions. Interleukin (IL) 1 had a differential effect on spontaneous C3, factor B and factor H secretions. In the presence of IL 1, there was a significant secretion of C3 occurring within a short period of culture. IL 1 also stimulated factor B secretion. There was a synergistic stimulating effect between IL 1 and interferon-gamma to bring C3 and factor B productions by HUVEC to very high levels. In contrast, factor H secretion was consistently inhibited by IL 1. Local increase in C3 and factor B secretions by endothelial cells in the presence of IL 1 may have important implications in the inflammatory reaction. In striking contrast, the glucocorticoid dexamethasone (DXM) had modulatory effects which are consistent with its anti-inflammatory properties. DXM, at therapeutic concentrations, decreased C3 and factor B secretions and increased factor H secretion. Local modulation of complement protein secretion by DXM appears to be a new mechanism by which this glucocorticoid may control inflammation.

Complement C3b Inactivator Proteins↗
PubMed1990

[Study of the complement system. General principles and perspectives].

Measurement of complement in clinical medicine is traditionally based on the determination of CH50 and immunochemical and/or functional measurement of complement proteins C1q, factor B, C3 and C4. The interpretation of these measurements, as far as complement activation is concerned, can however be difficult as these tests do not allow to discriminate between consumption due to activation, hereditary deficiency, increased rate of synthesis or even hyposynthesis. This explains why their use as markers of evolutivity in diseases where complement activation is occurring has given variable results. New tests for complement activation have been more recently introduced. These are mainly the measurements of the anaphytotoxins, the degradation products of C3 and the membrane attack complex. As these tests reflect more directly complement activation, they may be more reliable markers. The immunochemical and functional measurements of C1-inhibitor are of special interest as they are the tests which allow definitive diagnosis of the hereditary angio-oedema. General principles for the interpretation of the different tests used to evaluate the complement system are presented and discussed.

Anaphylatoxins↗
PubMed1989

Truncated forms of human complement factor H.

By the use of Western-blot analyses with polyclonal anti-(Factor H) antibodies, two low-Mr protein species of Mr 41,000 and 37,000 under non-reducing conditions and 43,000 and 40,000 under reducing conditions are consistently detected together with the well-known 155,000-Mr Factor H in human plasma and serum. These two additional species are also found in plasma, urine and synovial fluids. The 41,000-Mr species but not the 37,00-Mr species is detected by a monoclonal anti-(Factor H) antibody directed at the N-terminal part of Factor H. The 37,000-Mr species but not the 41,000-Mr species is detected by a monoclonal anti-(Factor H) antibody directed at the C-terminal part of Factor H. The 41,000-Mr and 37,000-Mr species are different from the well-characterized 36,000-Mr N-terminal tryptic fragment of Factor H. They are likely to represent translational products of the short Factor H mRNA species of 1.8 kb and 1.2-1.5 kb occurring in human liver that we have recently described.

Antibodies, Monoclonal↗
PubMed1988

Interferon gamma induces synthesis of complement alternative pathway proteins by human endothelial cells in culture.

Human umbilical vein endothelial cells grown in vitro under standard conditions contain a high level of mRNA specific for the complement regulatory factors H and I. An additional 1.8-kb mRNA encoding a truncated form of factor H is also present. IFN-gamma stimulation of the cells causes a 6-7 fold increase in both factor H mRNA species, and a greater than 10-fold increase in factor I mRNA. IL-1 and LPS slightly suppressed factor H mRNA, while TNF had no effect. mRNA for factor B is also detectable in IFN-gamma-stimulated cells, but messengers for C1q, C4bp, and CR3 beta chain were not found. Secretion of factor H protein was also stimulated by IFN-gamma. The presence of mRNA for factors H, B, and I, together with C3 secretion, demonstrated by others, suggests that endothelial cells can assemble the complete alternative complement pathway. Endothelial cell complement may be involved in leukocyte-endothelium interactions mediated by leukocyte C3 receptors.

Blotting, Northern↗
PubMed1988

Two populations of complement factor H differ in their ability to bind to cell surfaces.

Using hydrophobic affinity chromatography on phenyl-Sepharose, human complement factor H can be separated into two subpopulations, phi 1 and phi 2. Although phi 1 and phi 2 are known to differ in their aggregation properties under non-physiological low ionic strength conditions, no difference in aggregation state was detected under the conditions used for cell-binding experiments. We have investigated these two subpopulations further to determine whether functional differences exist between them. The subpopulation phi 2 was found to bind specifically and saturably to the surface of Raji cells. The binding of the other subpopulation, phi 1, was low, and essentially non-specific. A monoclonal anti-factor H antibody, BGH-1, was raised which recognizes preferentially the phi 2 subpopulation and inhibits the binding of factor H to cell surfaces.

Antibodies, Monoclonal↗
PubMed1988

The complete amino acid sequence of human complement factor H.

The complete amino acid sequence of the human complement system regulatory protein, factor H, has been derived from sequencing three overlapping cDNA clones. The sequence consists of 1213 amino acids arranged in 20 homologous units, each about 60 amino acids long, and an 18-residue leader sequence. The 60-amino-acid-long repetitive units are homologous with those found in a large number of other complement and non-complement proteins. Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma. A tyrosine/histidine polymorphism was observed within the seventh homologous repeat unit of factor H. This is likely to represent a difference between the two major allelic variants of factor H. The nature of the cDNA clones indicates that there is likely to be an alternative splicing mechanism, resulting in the formation of at least two species of factor H mRNA.

Amino Acid Sequence↗
PubMed1988

Sequence polymorphism of human complement factor H.

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2:1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.

Amino Acid Sequence↗
PubMed1988

Localization of a hydrophobic domain in human C5.

The fifth component of human complement (C5), under physiological salt conditions, bound firmly to phenyl-Sepharose. This indicated the presence of hydrophobic sites on C5. These sites may play an important role in C5 haemolytic activity since this activity was inhibited by chaotropic ions (Mg2+, SCN-, I-) at relatively low concns (0.6-1 M). Charge shift experiments performed on purified C5 provided further evidence for the presence of hydrophobic sites within C5. Bidirectional charge shifts of C5 mobility were observed with the two detergent systems TX-100 + DOC and TX-100 + CTAB. In order to localize these sites, C5 was subjected to trypsin digestion. Three major fragments were evidenced by two-dimensional electrophoresis (agarose/SDS-PAGE and SDS-PAGE/SDS-PAGE), and were isolated by hydrophobic affinity chromatography. The first fragment, C5FI, was composed probably of a mixture of six alpha chain fragments (alpha 1-alpha 6) linked to the beta chain by disulphide bridges. The second fragment, C5FII, was composed of the beta chain linked to two alpha chain fragments: alpha 2 (Mr = 58 K) and alpha 4 (Mr = 32 K). The third fragment, C5FIII, contained two covalently linked chains (beta chain and alpha 4). The rank order for mol. wt, agarose gel electrophoretic mobility and hydrophobicity was respectively: C5 greater than or equal to C5FI greater than C5FII greater than C5FIII; C5 less than or equal to C5FI less than or equal to C5FII much less than C5FIII; and C5FII greater than or equal to C5 greater than C5FI much greater than C5FIII. From the relative hydrophobicity, the hydrophobic sites of C5 can be localized to the alpha 2 fragment (Mr = 58 K) since C5FIII lacked the alpha 2 fragment and was not hydrophobic. Cleavage and the liberation of the N-terminal part of C5 induced an increase in hydrophobicity of the remaining part of C5 (C5b and C5FII), suggesting a possible role of the hydrophobic sites in association of C5b with membranes.

Complement C5↗
PubMed1987

Marmoset red blood cell receptor for membrane-associated complement components is not related to human CR1: partial characterization of the C3-binding proteins responsible for the spontaneous rosette formation between marmoset red blood cells and human leukocytes.

Cells from all the human B-lymphoblastoid cell lines tested and most human monocytes form rosettes with marmoset red blood cells (MaRBC). Because previous reports suggested the involvement of complement components in this phenomenon, the mechanism of rosette formation and the eventual similarities between the MaRBC receptor and the CR1 receptor present on human erythrocytes have been analyzed herein. The binding of MaRBC to human leukocytes strongly differs from the immune adherence phenomenon: rabbit anti-human CR1 did not react with MaRBC and the MaRBC receptor-binding activity is Ca2+-dependent. Rosette formation required intact energy metabolism and cytoskeleton integrity of leukocytes. Our attempts to purify the receptor from MaRBC membranes revealed the absence of CR1. Nevertheless, C3-binding proteins were isolated by selective desorption by Sepharose iC3 column chromatography. A three-band pattern was observed under reduced conditions with 74,000, 70,000, and 53,000 molecular weights. It was not possible to further separate these components. This protein complex inhibited the rosette phenomenon between MaRBC and both Raji and U-937 cells, exhibited a very poor cofactor activity, and had no decay-accelerating activity toward the classical C3 convertase. This material did not cross-react with antibodies directed to human proteins. These results showed that erythrocytes from new world monkeys do not express a receptor analogous to the human CR1, but expressed C3-binding protein with low cofactor activity that could recognize membrane-associated complement components.

Animals↗
PubMed1987

Sequence analysis of a cDNA clone encoding the C-terminal end of human complement factor H.

Peptide sequencing of the complement system regulatory protein, factor H, permitted the synthesis of a mixed sequence oligonucleotide probe. Human liver cDNA libraries were screened and factor H-specific clones selected. No full-length clone was obtained, but the largest available clone, R2a, was found to encode the C-terminal 657 amino acids of factor H. The derived amino acid sequence consists of 10 contiguous internally homologous segments, each about 60 amino acids long. Sequences homologous to these are found in several other complement and non-complement proteins. Such sequences are likely to represent a particular type of tertiary structure subunit.

Amino Acid Sequence↗
PubMed1986

Loss of complement receptor type 1 (CR1) on ageing of erythrocytes. Studies of proteolytic release of the receptor.

Complement receptor type 1 (CR1) is a glycoprotein of Mr about 250 000 present on erythrocytes and other cell types. CR1 acts as a cofactor in the factor I-mediated breakdown of complement fragment C3b to form iC3b. Using an assay of cofactor activity, a wide variation in mean CR1 levels between erythrocytes from individual donors is observed. CR1 levels also decrease on ageing of erythrocytes in vivo, and again the rate of loss is widely variable between individuals. However, variable loss of CR1 during ageing of erythrocytes is likely to make only a minor contribution to the observed variation in mean CR1 levels. CR1 is very sensitive to proteolysis, and random proteolytic removal of CR1 from erythrocytes is likely to be an important factor in loss of CR1 on ageing of red cells in vivo. In vitro, mild trypsin treatment, plasmin or thrombin digestion of erythrocytes results in the loss of the factor I cofactor activity from the cell surface, and appearance of this activity in the supernatant. We conclude that an active fragment of CR1 is released from the cell surface on proteolysis. Subsequent prolonged trypsin treatment destroys most of the activity of this fragment. Proteolytic removal of CR1 from red cells may account not only for loss on ageing of cells, but also for the acquired CR1 deficiencies observed by others in systemic lupus erythematosus.

Erythrocyte Aging↗