Vincent Thomas

A role for tissue transglutaminase in α-gliadin peptide cytotoxicity

In coeliac disease, gliadin peptides p56–88, p57–68 and p31–49 have been demonstrated to be involved in the pathogenic damage of the small intestine via their immunogenicity or toxicity to epithelial cells. To try to understand the mechanism of their toxicity, we investigated the effect of synthetic peptides (p31–49, p56–88, p57–68, p69–82) and of their deamidated analogues on Caco2 and FHs 74 Int cell toxicity and tissue tranglutaminase activity. Apoptosis, necrosis and cell viability were assessed by flow cytometry, and peptide deamidation was determined indirectly by measuring its capacity to inhibit tTG activity. The results showed that p56–88 and p57–68 reduced cell growth and concomitantly inhibited tTG activity in both cell types. This effect was abolished when Caco2 cells were treated with antibodies to tTG. Deamidated peptide p57–68 (E65) lost practically all of its inhibitory effect on cell growth and on tTG activity. Cellular toxicity was also observed with p31–49, which was not a substrate for tTG. p69–82 was not cytotoxic but became so when glutamine 72 was substituted by glutamic acid. These findings provide evidence for the existence of three types of toxicity among gliadin peptides: (i) peptides that are intrinsically toxic and are not substrates of tTG; (ii) peptides that are non‐toxic but become so when they act as substrates of tTG; and (iii) peptides that are non‐toxic and are not substrates of tTG but become so when deamidated. A mechanism other than that involving tTG could be responsible for the deamidation of glutamine residues of gliadin in the intestinal tract.

A novel covalent enzyme-linked immunoassay (CELIA) for simultaneously measuring free and immune complex bound antibodies of defined specificity I. Application to naturally occurring antipolyamine antibodies in human sera

A simple covalent enzyme-linked immunoassay procedure (CELIA) is described for the routine determination of free and immune complex-bound antibodies in sera. Assays for the latter could not have been performed by adsorption ELISA due to the high ionic strength of the reassociating buffer. For the measurement in human sera of free naturally occurring IgG and IgM antibody directed against the hapten spermine, polycarboxystyrene microtiter plates with covalently coupled spermine were used. For the determination of immune complex-bound antipolyamine IgG and IgM antibody titers, serum was first dissociated at pH 2.3 in tubes and then reassociated at pH 8.1 in the wells of a microtiter plate containing covalently bound spermine. The reactivity of anti-spermine antibodies was increased from 2- to 13-fold after dissociation and reassociation compared to that of non-dissociated area. The apparent reaction constant (Rapp.) of free IgG antibodies to spermine in the sera of 19 bronchopulmonary patients with cancer differed significantly from Rapp. values of IgG antibodies having this specificity in ten other patients with non-malignant disease.

Hydrophilized and functionalized microtiter plates for the site-specific coupling of antigens and antibodies: Application to the diagnosis of viral cardiac and autoimmune diseases

Abstract Extensive adsorption of macromolecules (antigens and antibodies) took place when they were covalently bound to non-hydrophilized polystyrene microtiter plates. Precoating polystyrene surfaces with gelatin reduced this non-specific adsorption only partially, whereas precoating with unchanged polymers such as poly(ethylene glycol) (PEG) and polysaccharides eliminated the problem totally. On such hydrophilized plates functionalized with epoxide groups, antigens and antibodies were randomly bound. On those functionalized with acid hydrazide, antibodies were site-specifically bound by their carbohydrate residues. These site-specifically bound antibodies retained greater reactivity and more of their native antigenic structure than randomly coupled antibodies. They therefore permitted the measurement of a minor analyte (D-dimer) in the presence of an excess of major components such as fibrinogen. Enzyme immunoassays which were uninterpretable on non-hydrophilized plates because of the adsorption of immunoglobulin gave meaningful results on hydrophilized plates. This held true for immunoglobulin aggregates formed artificially either by successive cycles of freezing and thawing or by the acid treatment of serum to dissociate immune complexes. The latter approach permitted us to obtain unequivocal evidence for the presence of antibodies specific for human T-cell lymphotropic virus type 1 (HTLV1) in immune complexes in sera with no detectable free antibody. For naturally occurring aggregates which are often found in the sera of patients with autoimmune diseases, the use of hydrophilized plates also permitted antibody levels to be measured in instances where background noise was greater than the signal on non-hydrophilized plates. This combination of hydrophilization and site-specific coupling of monoclonal antibodies of a defined specificity should provide a distinct advantage when developing routine covalent enzyme linked immunoassays (CELIAs) for minor analytes, in a mixture with major constituents, as is often encountered, in both human and veterinary medicine and in the food industry.

Differential recognition of free and covalently bound polyamines by the monoclonal anti-spermine antibody SPM8-2

The reactivity of an anti-spermine MAb (SPM8-2) toward polyamines either free or bound to a solid surface was investigated using equilibrium dialysis and ELISA methods. When polyamines were covalently linked to hydrophilized microtiter plates using carbodiimide, the MAb SPM8-2 reacted both with spermine and spermidine, with a higher affinity for the latter, but did not show any reactivity towards bound putrescine. In contrast, the MAb SPM8-2 reacted with all three polyamines bound to the microtiter plates with glutaraldehyde, with an affinity in the order: putrescine > spermidine > spermine. Equilibrium dialysis and competitive ELISA tests showed that the MAb SPM8-2 exhibited high affinity for free spermine and 50% and 5% cross-reactivity with free spermidine and putrescine respectively. The affinity of the MAb SPM8-2 for putrescine, spermidine and spermine appears to depend on whether the polyamine is free or bound. The antigenicity of the polyamines differs according to the nature of their link to the solid phase. These observations are discussed in the light of the structural modification produced by covalent binding of the polyamines. It is also concluded that when antibodies are used, due care has to be exercised in choosing the appropriate immunoassay for determining the specificity of antibodies directed against small haptens such as the polyamines.

A novel covalent enzyme-linked immunoassay (CELIA) for simultaneously measuring free and immune complex bound antibodies with a defined specificity. II. Application to immune complexes containing viral antigens in human sera.

The coupling of viral antigens from parainfluenza virus (PIV-1), cytomegalovirus (CMV) and human immunodeficiency virus (HIV-1) to chemically functionalized polystyrene plates has permitted us to develop a covalent enzyme-linked immunoassay (CELIA) for measuring the titers of free antibody (Ab) and immune complex (IC) bound Ab directed against each of these viruses. The method was first validated for experimentally produced IC (PIV-anti-PIV) and then applied to the analysis of IC in human sera. In the case of a renal transplant patient with CMV viremia whose free Ab titers were less than 100, the method unambiguously permitted the IC bound anti-CMV titers to be determined. In the case of a survey for HIV-1 Ab, it also allowed us to identify a sub-group of seropositives with IC anti-HIV. In view of the ease and rapidity with which CELIA can be performed, this technology should enable determinations of IC bound Ab of defined specificity to be undertaken routinely in seroepidemiological surveys.

Development and evaluation of a modified colorimetric solid-phase microassay for measuring the activity of cellular and plasma (Factor XIII) transglutaminases

Plasma TG (transglutaminase) [FXIII (Factor XIII)] stabilizes fibrin and plays an essential role in haemostasis. In the present paper, we report a simple colorimetric assay for measuring FXIII activity. The advantage of this approach, compared with all the other solid‐phase assays described so far for measuring TG activity, is that the first substrate, namely the synthetic dipeptide, N‐benzyloxycarbonyl‐l‐Gln‐l‐Gly, is coupled covalently by its C‐terminus with amine‐substituted polystyrene plates. Covalent coupling eliminates the problem of desorption of proteins such as casein and N,N′‐dimethylcasein (first substrates) when ‘blocking’ buffers containing proteins, e.g. BSA, are employed, or when samples of plasma or cell homogenates are used. The principle of the assay itself is based on the incorporation of the well‐known second substrate, 5‐(biotinamido)pentylamine, into the γ‐carboxamide of glutamine in the immobilized dipeptide. The amount of biotinylated amine bound to the plate, as measured by the phosphatase activity of Extravidin® phosphatase attached to the biotin moiety, is directly proportional to the TG activity. The method shows strong correlation (r=0.96) with the radiometric assay for FXIII activity. For plasma samples, a linear response was obtained in the range 0–1.33 i.u. (international unit)/ml, versus the Behring standard. In a preliminary clinical investigation, the method was applied to normal and pathological plasma samples from patients with liver failure and disseminated intravascular coagulation. In the isolation of TG from guinea‐pig liver, it was used to measure enzyme activity after each purification step. This method is rapid, sensitive and can easily be applied to routine clinical analyses and to specific research problems.

Naturally occurring antibodies reacting with lipoic acid: screening method, characterization and biochemical interest☆

The development of a covalent enzyme-linked immunoassay (CELIA) using lipoic acid covalently bound to modified polystyrene microplates has permitted the detection, in the sera of normal BALB/c mice, of natural antibodies reacting with lipoic acid (LA). Hybridomas producing monoclonal anti-LA antibodies were obtained from splenocytes of non-immune BALB/c mice. Two of them, of IgM isotype, recognized LA but failed to react with dihydrolipoic acid (DHLA, the reduced form of LA), suggesting that the integrity of the dithiolane ring was of importance for antibody recognition. They did not give positive reactions with other disulfide linked biological molecules such as oxidized glutathione or cystine. Anti-LA antibodies, coated on polystyrene microplates, were used for the detection of free LA in a competitive assay based on peroxidase-LA conjugate.

Protein-bound polyamines in the plasma of mice grafted with the Lewis lung carcinoma

Protein‐bound polyamines were isolated from the plasma of mice using antipolyamine antibodies covalently linked to magnetic latex spheres. Their subsequent separation by polyacrylamide gel electrophoresis (PAGE) showed that in plasma from normal mice, 3 proteins (27, 55 and 82 kDa) carrying polyamines could be visualized, whereas in mice bearing the Lewis lung carcinoma at least 8 other proteins of higher molecular mass (5 of 94, 110, 130, 145 and 160 kDa, and 3 of ⪢ 170 kDa) had bound polyamines. These protein‐bound polyamines could be detected from the first week after tumour graft; they increased during the second and third week but decreased thereafter. These proteins were not bound by immunolatex spheres preincubated with spermine bound to a protein‐carrier insulin. Moreover, the appearance of these protein‐bound polyamines was not a consequence of the inflammatory process since in mice infected with heat‐inactivated Brucella abortus, with the exception of a 65 kDa protein, polyamines were bound to the same proteins found in normal mice. In mice grafted with the Lewis lung carcinoma the concomitant decrease in transglutaminase‐mediated polyamine (e.g. putrescine) binding capacity of plasma proteins provides additional evidence for the presence in vivo of polyamines already bound to plasma proteins.

Identification of human salivary transglutaminases

Transglutaminases (TGs) expression and enzymatic activities in human saliva were investigated. Specific antibodies showed the co-existence of TG1, TG2, TG3 and TG4. TG2 and TG3 were found in native and multiple proteolytic forms. Our data indicate that TG1 and TG2 isoenzymes are highly active with the major activity attributed to TG1. These findings pave the way for future studies on the physiological role of TG in the oral cavity and the potential impact of their deregulation in TG-associated oral diseases.

Development of a sandwich ELISA assay for quantification of human tissue transglutaminase in cell lysates and tissue homogenates.

Tissue transglutaminase (tTG) belongs to the multigene transglutaminase family of Ca2+-dependent protein cross-linking enzymes. There is a strong evidence that tTG is involved in pathology, such as neurodegenerative diseases, cancer, and celiac disease. To study physiopathological implication of tTG, a sandwich immunoassay has been developed with a new monoclonal antibody for the capture and polyclonal antibody both generated in house. Using this ready to use assay, the tTG protein level can be measured in human tissue homogenates and cells extracts easily in about 4xa0h. The limit of detection is 1.7xa0ng/ml; the coefficients of intra- and inter-assay variations range from 1 to 2xa0% and from 7 to 10xa0%, respectively. The assay is specific to tTG, and no cross reactivity with TG1, TG3, TG6, TG7, or factor XIIIa was observed. Finally, in the addition to the tTG activity assay previously developed, this assay should be a valuable tool to increase our knowledge of the tTG involvement in physiologicalxa0and pathological states.