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J C Mani

Publications and source records attributed to J C Mani.

At least 55 records · Page 3Linked to original sources
PubMed1996

Analysis of the individual contributions of immunoglobulin heavy and light chains to the binding of antigen using cell transfection and plasmon resonance analysis.

We have cloned the Tg10 murine monoclonal antibody, which is specific for a human thyroglobulin (hTg) epitope targeted by autoantibodies in several thyroid pathologies. Transfection of COS-7 cells with plasmids expressing Tg10H and -kappa chains combined with surface plasmon resonance analysis (BIAcore) of culture supernatants showed that the entire cloned Tg10 antibody displays an affinity comparable to that of the parental antibody. This approach also permitted determination of the probable role of each chain to the recognition of the cognate epitope due to the ability of COS-7 cells to secrete independently each of the two constituting immunoglobulin chains. Tg10 heavy chain recognizes hTg in the absence of the light chain, but with a ten-fold lower affinity mainly due to an increase in kappaoff. In contrast, the light chain is unable to bind hTg on its own. This suggests that the latter is probably involved in stabilization rather than in initiating the formation of the antibody/antigen complex and that the specificity of Tg10 is mostly, if not exclusively, carried by the heavy chain. The potential applications of combined cell transfection and surface plasmon resonance to our understanding of antigen/antibody interactions are discussed.

Amino Acid Sequence↗
PubMed1996

Characterization of the interaction between annexin I and profilin.

Annexin I belongs to a family of calcium-dependent phospholipid-binding and membrane-binding proteins. Although many of the biochemical properties and the three-dimensional structure of this protein are known, its true physiological roles have yet to be thoroughly defined. Its putative functions include participation in the regulation of actin microfilaments dynamics, proposed after the discovery of an interaction with actin. In accordance with this hypothesis, we found that annexin I can also interact with profilin. We used different methods, overlay and surface plasmon resonance (BIAcore), to measure the parameters of the association equilibrium, i.e. k(on), k(off) and k(d). The affinity of annexin I for profilin was between 10(7) M and 10(8) M. High concentrations of KCl did not prevent the interaction, although a slight decrease in affinity was observed. Calcium, a modulator of annexin I functions interfered only marginally with the association, in a manner comparable to magnesium. Proteins or compounds known to interact with annexin I or profilin were found to inhibit the annexin-I--profilin interaction when added in the reaction medium. Recombinant profilin exhibited a slightly lower affinity than natural platelet protein when measured with BIAcore. Due to the submembrane localisation of annexin I and the regulatory activity of profilin on the cytoskeleton, an interaction between annexin I and profilin may therefore be implicated in the regulation of some cellular functions, particularly those governing membrane-cytoskeleton dynamic organization.

Actins↗
PubMed1996

Human lymphocytes shed a soluble form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that binds iC3b and CD23.

We report on a soluble (s) form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that is spontaneously released by B and T lymphocytes. Immunoprecipitation with anti-CD21 mAb of culture supernatants of surface and biosynthetically labeled B and T cell lines revealed a single band with an apparent molecular mass of 135 kDa. The molecule exhibited a molecular mass 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) was time dependent and correlated with a parallel decrease in the expression of the membrane-associated molecule. The protein was also found in culture supernatants of tonsillar B cells and normal human thymocytes. Epitopic analysis using combinations of anti-CD21 monoclonal antibodies (mAb) indicated that sCD21 and membrane CD21 were similarly recognized by mAb directed against short consensus repeats (SCR) 1-2, SCR 4-5 and SCR 9-11. Affinity-purified sCD21 was capable of binding to purified human iC3b and to human recombinant CD23, as assessed by enzyme-linked immunosorbent assay and by using the BIAcore technology. In addition, normal human serum was found to contain a soluble form of CD21 that exhibited a similar molecular mass to that of the molecule shed by B and T cells in culture. The serum form of CD21 was recognized by all anti-CD21 mAb that we tested and showed a high reactivity with mAb directed against SCR 1-2. Our observations suggest that B and T cells shed the extracellular portion of CD21 and release a soluble molecule that retains the ligand-binding properties of CD21, thus having a potential role in immunoregulation.

B-Lymphocytes↗
PubMed1996

Identification of a novel antigenic structure of the human receptor for interleukin-6 involved in the interaction with the glycoprotein 130 chain.

The receptor for interleukin-6 (IL-6) is characterized by a ligand-binding glycoprotein 80 (gp80) transmembrane chain (IL-6R) which associates with a signal-transducer gp130 chain. We previously raised a series of monoclonal antibodies (mAb) recognizing different epitopes of the human IL-6R and interfering with the function of the receptor. One of them, M182, was able to diminish the proliferation of IL-6-dependent plasmacytoma cell lines although it was found unable to inhibit the binding of IL-6 to its receptor. Using an enzyme-linked immunosorbent assay for measuring the binding of IL-6 IL-6R to the gp130 chain, we showed that M182 was directed against a structure directly involved in the IL-6R gp130 interaction. M182 was able to potentiate the inhibitor effect of anti-IL-6R mAB which interfere with the binding of IL-6, leading to complete inhibition of the proliferation of IL-6-dependent cell lines. M182 was also found to synergize with inhibitory anti-IL-6 mAb. Therefore this structure appears to be an important regulatory domain of the IL-6R and a valuable target for inhibiting IL-6 signalling.

Antibodies, Monoclonal↗
PubMed1996

Selective recognition of M235T angiotensinogen variants and their determination in human plasma by monoclonal antibody-based immunoanalysis.

The common M235T mutation of human angiotensinogen has been shown to be associated with a 10-20% increase in plasma angiotensinOgen level and increased frequency of essential and pregnancy-induced hypertension. The detection of such a common factor in the plasma of individuals at risk could be a useful tool for modern molecular-based medicine. The recognition of M235T variants was investigated using four monoclonal antibodies (mAbs) directed against human angiotensinogen; two immunometric assays were developed. The first assay (using mAbS 7B2 and 4G3) allowed the direct determination of angiotensinogen concentrations and did not show a significant difference with the enzymatic measurement of angiotensinogen. The second assay (using mAbs 1H8 and 1C11) showed a fine distinction between the T235 mutant and M235 wild-type forms of angiotensinogen, with a greater affinity for the latter, as confirmed by biosensor BIAcore experiments. This assay was extremely sensitive in measuring the proportions of the M235 and T235 forms present in the test samples, the first time such a distinction has been achieved in the serpin family. The simple immunoanalysis of the plasma allowed the direct determination of the M235T genotype of the individual tested. Furthermore, it was shown that the T174M mutation, described as being in complete linkage disequilibrium with the M235T mutation, had no influence on these results. Moreover, this assay suggested the presence of the M235 and T235 angiotensinogens in approximately equal amounts in heterozygous plasmas. In conclusion, the immunometric assay described in this study should provide original tools for investigating the relationship between M235T genotype, plasma angiotensinogen levels, and regulation of blood pressure.

Angiotensinogen↗
PubMed1995

New monoclonal antibodies directed against the propart segment of human prorenin as a tool for the exploration of prorenin conformation.

Six monoclonal antibodies (MAbs) directed against human prorenin were produced by immunizing BALB/c mice with a peptide corresponding to the sequence (-17 to +9) of prorenin. The new MAbs were screened for their ability to first bind to the immobilized peptide and then to prorenin previously captured by an anti-total renin MAb. The specificity of the MAbs was confirmed by the total lack of binding to active renin. Using BIAcore technology, equilibrium affinity constants of the MAbs were determined and ranged from 3.2 x 10(8) to 5.7 x 10(9) l/mol. Immunoradiometric assays (IRMA) for prorenin were performed using the anti-total renin MAb and the anti-prorenin MAbs. The best results were obtained when an anti-prorenin MAb was immobilized and the anti-total renin MAb was used as tracer in a one-step procedure. Moreover, the signal was significantly increased by the presence of the renin inhibitor SR 43845 suggesting that the inhibitor-induced conformational change of prorenin could be detected by the MAbs.

Amino Acid Sequence↗
PubMed1995

Epitope mapping and binding analysis of insulin-specific monoclonal antibodies using a biosensor approach.

Molecular interactions between the human insulin molecule and a panel of seven monoclonal antibodies (mAbs) against insulin were investigated by surface plasmon resonance. Using real-time analysis (BIAcore technology), the antigenic sites recognized on the human insulin molecule by these mAbs were mapped. Three overlapping regions could be defined on the insulin molecule coupled to the dextran matrix on the basis of its recognition by three mAbs groups: group I (mAbs 19, 10), group II (mAbs 13, 5, 25, 15) and group III (mAb P10). The antigenic determinants of the molecule in solution were mapped using a two-site assay in which insulin was presented to each mAb, bound to a rabbit anti-mouse IgG Fc gamma 1 coupled to the dextran. Four antigenic regions were defined: regions I and III were defined by the same antibodies as above whereas region II was recognized differently by mAbs 13, 5 (region IIa) and mAbs 25,15 (region IIb). The Ka value of the mAbs determined using the BIAcore were in the same range (from 3 x 10(7) M-1 to 6.6 x 10(9) M-1) whether insulin was coupled to the dextran matrix or in solution. Furthermore, mAbs 19 and P10, which recognize two distinct regions on the insulin molecule, were selected to develop a highly sensitive immunoassay for measurement of insulin in serum.

Allergy and Immunology↗
PubMed1995

Monoclonal digoxin-specific antibodies induce dose- and affinity-dependent plasma digoxin redistribution in rats.

The effect of three monoclonal digoxin-specific antibodies on total and free digoxin plasma disposition was studied in rats in order to determine the role of affinity constant (Ka) and dose. Thirty minutes after digoxin infusion, administration of a stoichiometrical dose of the ICIO, 6C9 and 9F5 IgG (Ka = 6 10(9), 3.1 10(8) and 2.5 10(7) M-1, respectively) resulted in a plasma digoxin increase linearly related to Ka. The mean free plasma digoxin was 0.6 +/- 0.4, 7.8 +/- 3.3 and 43 +/- 22% respectively after 1C10, 6C9, and 9F5 IgG infusion in comparison to 70 +/- 9% in the control group. When the IgG:digoxin ratio increased from 1 to 5, plasma digoxin Cmax and AUCT also increased as a function of both affinity (Ka) and dose (N), but not linearly. The product of NKa defined an immunoreactivity factor that was well fitted to the digoxin redistribution parameters (Cmax and AUCT) by a Hill equation.

Animals↗
PubMed1994

Role of N- and C-terminal amino acids in antithrombin binding to pentasaccharide.

We have used a monoclonal antibody-based binding procedure to determine the dissociation constants of the interactions between the essential antithrombin-binding pentasaccharide and a series of 13 distinct N- and C-terminal antithrombin substitution mutation variants with defective binding interaction with heparin. The reduction in binding affinity of the pentasaccharide with the N-terminal variants (with substitution mutations Pro-41-->Leu, Arg-47-->Cys and His, Leu-99-->Val and Phe, Gln-118-->Pro, Arg-129-->Gln) compared to normal antithrombin, Kd 200 nM, ranged from 15-984-fold and was generally less than 150-fold. Reduced binding affinity is assumed to arise mostly by perturbation, direct or indirect, of the initial contact of pentasaccharide with basic residues of antithrombin. Surprisingly, the binding interaction of the pentasaccharide with the C-terminal variants (with substitution mutations in or near strand 1C/4B, Phe-402-->Leu, Cys, and Ser, Ala-404-->Thr, Pro-407-->Thr, Pro-429-->Leu) was more uniformly and yet substantially (135-482-fold) decreased, despite the spatial separation between the site of mutation and the proposed primary contact site of the pentasaccharide. These results demonstrate that strand 1C/4B region integrity is required for optimum interaction with the pentasaccharide, suggesting its involvement in transmission of the induced conformation change required for high affinity binding.

Amino Acids↗
PubMed1994

Characterization of a monoclonal antibody produced in an attempt to mimic the active site of HIV aspartyl protease using haptens based on inhibitor models.

The high binding affinity and specificity of antibodies for a great variety of ligands has been widely exploited in structure-activity relationship studies of biomolecules and more recently in the development of new catalysts for several chemical reactions. It is assumed that antibodies generated against haptenic protease inhibitors would recognize both these haptens and the substrate of the model proteolytic reaction. We have produced antibodies against HIV PRp12 aspartyl protease substrate analogues, chemically modified at the scissile bond, Phe-Pro. Identical chemical modifications have been reported for related HIV protease inhibitors. We finally selected an anti-hapten monoclonal antibody that specifically recognized the substrate and those haptens with both the phenylalanyl side chain and the prolyl pyrrolidine ring. This selectivity of recognition suggests that such an antibody might mimic the catalytic site of the model protease.

Amino Acid Sequence↗
PubMed1994

Effect of HIV-1 peptide presentation on the affinity constants of two monoclonal antibodies determined by BIAcore technology.

We studied two monoclonal antibodies (MAbs 9-11 and 41-1) which are specific for dominant and conserved epitopes located on HIV-1 transmembrane Gp41. These MAbs recognize both Gp41 and a synthetic HIV-1 envelope peptide (39GC) which is a fragment of Gp41. The interactions between MAbs 9-11 and 41-1 and 39GC either coupled to a sensor chip or to alkaline phosphatase were investigated using BIAcore technology. The association and dissociation rate constants as well as the affinity constants were determined. BIAcore technology allows real-time determination of the interaction between two molecules without the need for any labeling, neither isotopic nor enzymatic. The peptide 39GC was immobilized by coupling to dextran on the BIAcore biosensor through a disulfide bond with a cysteine residue added to the N-terminus of the synthetic peptide. The two native cysteine residues located in the loop of Gp41 were protected by ethylcarbamoyl residues (CONHC2H5); this chemical modification prevented the formation of the S-S bridge and in particular the internal loop. We specifically studied the interaction between the MAbs and either the protected peptide or the peptide whose cysteine residues had been deprotected in situ by alkaline treatment. The results showed that MAb 41-1 recognized 39GC either protected (Ka = 7.6 x 10(6) M-1) or unprotected (Ka = 1.48 x 10(8) M-1), whereas MAb 9-11 recognized only the unprotected form (Ka = 2.18 x 10(8) M-1). Our results suggest that the epitope MAb 9-11 is directed against a part of the peptide sequence which includes the two native cysteines. The difference in affinity observed for MAb 41-1 between the protected and the unprotected forms of 39GC was found to be due to a lower rate of dissociation for unprotected 39GC; these results illustrate the importance of peptide conformation on antibody recognition and might be explained by a conformational change due to reconstitution of the internal loop following deprotection of the thiol groups. MAbs 9-11 and 41-1 also recognized 39GC conjugated to alkaline phosphatase and deprotected. We observed a difference between the rate constants for MAb 41-1 binding to free peptide and its binding to the peptide-enzyme conjugate which might be due to changes in peptide flexibility. In contrast, the rate constants of MAb 9-11 were the same in both experiments, suggesting that the rigidity of the internal loop prevents changes in 9-11 epitope conformation.

Amino Acid Sequence↗
PubMed1994

Anti-CD4 activity of normal human immunoglobulin G for therapeutic use. (Intravenous immunoglobulin, IVIg).

The effects of intravenously administered normal immunoglobulin G (IVIg) in autoimmune diseases are dependent on the ability of IVIg to interact with surface molecules of lymphocytes. In the present study, we demonstrate the presence of anti-CD4 activity in IVIg by showing the ability of IVIg to bind to CD4 and to inhibit CD4-dependent cellular functions. Binding of IVIg to recombinant soluble human CD4 was assessed by ELISA, immunoblotting and real time analysis of complex formation. Anti-CD4 antibodies isolated from IVIg by affinity-chromatography bound to human CD4+ T cells. These anti-CD4 antibodies inhibited proliferative responses in MLR and infection of CD4+ human T cells with HIV. These results indicate that IVIg contains antibodies reactive with human CD4 and that these anti-CD4 antibodies exhibit biological functions. The presence of anti-CD4 antibodies in IVIg may be relevant to the immunoregulatory effects of normal polyspecific immunoglobulin G.

Adjuvants, Immunologic↗
PubMed1992

Synthesis and antibronchospastic activity of 8-alkoxy- and 8-(alkylamino)imidazo[1,2-a]pyrazines.

Theophylline still occupies a dominant place in asthma therapy. Unfortunately its adverse central nervous system (CNS) stimulant effects can dramatically limit its use, and adjustments in the dosage are often needed. We have synthesized a new series of imidazo[1,2-alpha]pyrazine derivatives which are much more potent bronchodilators than theophylline in vivo and do not exhibit the CNS stimulatory profile. In vitro studies on isolated rat uterus and guinea pig trachea confirm the high potentialities of these derivatives. 6-Bromo-8-(methylamino)imidazo[1,2-alpha]-pyrazine-3-carbonitrile (23) is identified as the most potent compound of the series. As in the case of theophylline, phosphodiesterase inhibition appears unlikely to be the unique mechanism of action of this series of heterocycles.

Animals↗
PubMed1991

Highly specific radioimmunoassay for digoxin using a monoclonal antibody selected for lack of interference by digoxin-like immunoreactive substances in cord blood sera.

Four digoxin radioimmunoassays (RIA) were evaluated using four antidigoxin monoclonal antibodies (MAb) with different binding specificities for digoxin metabolites and other glycosides. These RIA have been used to measure the apparent digoxin concentrations in the sera of patients treated with digoxin, in cord blood sera and in rat intestine aqueous extracts. Two MAb strongly recognized digoxin-like immunoreactive substances (DLIS) in cord blood sera and in rat intestine aqueous extract. In contrast, one MAb (2C2), which recognizes active digoxin metabolites and does not cross-react with inactive metabolites nor with digotoxin, showed no reactivity with DLIS in the cord blood sera tested. This MAb was used in a new digoxin RIA that was rapid, sensitive, reproducible, and specific for digoxin and its active metabolites.

Antibodies, Monoclonal↗
PubMed1990

Extracellular ATP4- permeabilizes thymocytes not only to cations but also to low-molecular-weight solutes.

Extracellular ATP increases the free cytosolic calcium concentration ([Ca2+]i) of thymocytes by changing the membrane permeability to divalent cations: (1) no [Ca2+]i elevation is observed in the absence of extracellular calcium and (2) ATP also promotes Mn2+ entry into thymocytes, as shown by a stimulated quench of the fluorescence of Quin2-loaded cells. Using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5- hexatriene, a dye which usually does not pass through membranes, we showed that ATP permeabilizes thymocyte membranes not only to cations but also to low-molecular-weight solutes. The ATP effect is dose-dependent, specific and reversible; ATP4- appears to be the active species and probably acts through specific receptor(s). Both medullary and cortical thymocytes respond to ATP whereas splenocytes do not; this shows that the ATP effect is restricted to certain populations of lymphocytes. The thymoma-derived cell line, EL-4, the permeability of which is also sensitive to ATP, could provide a useful model for studying the putative ATP4- receptor(s) of thymocytes.

Adenosine Triphosphate↗
PubMed1989

The [Ca2+]i increase induced in murine thymocytes by extracellular ATP does not involve ATP hydrolysis and is not related to phosphoinositide metabolism.

We have previously demonstrated that exogenous ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis. In the present study, using the highly sensitive calcium indicator Indo-1, we have measured the effect of exogenous nucleotides on the cytosolic-free calcium concentration [Ca2+]i of thymocytes, and determined inositol phosphate (IP) formation in the same cells, in parallel assays. The results were compared to those obtained with the mitogenic lectin concanavalin A (ConA) in similar experiments. They show that ATP does not mobilize calcium from its internal stores but stimulates its influx from the extracellular medium. Nevertheless, these data do not rule out the possibility that the nucleotide acts through specific P2 purinergic sites.

Adenosine Triphosphate↗
PubMed1989

Cyclooxygenase and lipoxygenase inhibitors act differently on oxidative product formation by immune mononuclear cells: a flow cytometric investigation.

The action of indomethacin, a cyclooxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on oxidative products formed by immune mononuclear cells was studied by flow cytometry. Jurkat T-cells and peripheral blood mononuclear cells were incubated with 2',7'-dichlorofluorescin diacetate; this substance is hydrolyzed in the cells leading to non-fluorescent 2',7'-dichlorofluorescin which is oxidized by oxygen reactive species into highly fluorescent 2,7'-dichlorofluorescein. Using this fluorescent probe, the formation of oxygen reactive species in phytohemagglutinin-stimulated mononuclear cells treated by NDGA or indomethacin was followed by flow cytometry. We observed that NDGA caused a marked decrease, both in the level of fluorescence intensity and in the number of fluorescent cells, whereas indomethacin caused a small increase in fluorescence intensity. On the other hand, NDGA inhibited and indomethacin increased the incorporation of tritiated thymidine by phytohemagglutinin-stimulated lymphocytes. These results suggest that oxygen reactive species are involved in the stimulation of immune mononuclear cells.

Cells, Cultured↗