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B Pau

Publications and source records attributed to B Pau.

At least 55 records · Page 3Linked to original sources
PubMed1997

In-cell assembly of scFv from human thyroid-infiltrating B cells.

The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Although useful for the production of recombinant antibodies, this method is not adapted to the study of the autoantibody repertoire formed in vivo during autoimmune diseases. To attain this objective, we describe the use of the in-cell PCR together with Cre-recombination applied, to our knowledge, for the first time to human B cells to obtain in situ pairing of the variable (V) region genes of the immunoglobulin heavy (H) and light (L) chains. Our method is based on amplification and recombination of the VH and VL genes within CD19+ B cells isolated from human thyroid tissue. Nested primers were designed to amplify the known major human VH and VL gene families. After reverse transcription PCR and three rounds of PCR including recombination between VH and VL using the Cre-loxP system, we obtained a unique 800-bp band corresponding in size to scFv fragments. We provide evidence that recombination between VH and VL genes occurred inside the same cell. This in-cell amplification and association procedure is a potentially useful tool for the study of autoantibody gene families and the VH/VL pairing that occurs during the autoimmune process.

B-Lymphocytes↗
PubMed1996

Characterization of the type-1 repeat from thyroglobulin, a cysteine-rich module found in proteins from different families.

The amino acid sequence of human thyroglobulin is known to enclose cysteine-rich repetitive regions. In this study, we report the existence of an eleventh type-1 repeat within the human thyroglobulin sequence, and we characterize the thyroglobulin type-1 repeat as a protein module. The 11 thyroglobulin type-1 repeats possessed the same number of cysteine residues (six in type A, four in the two type B repeats), a fairly constant number of residues between cysteines and a conserved sequence pattern. By scanning protein sequence databases, 29 proteins belonging to six different families were found to enclose at least one, and up to three, thyroglobulin type-1 repeats in their sequence. Although the repeat was present in numerous proteins possessing binding properties, an examination of the information available in the literature showed that a direct role of the repeat in protein-protein interaction has rarely been assessed. A distance analysis of the sequences indicated that all repeats segregate into four clusters of phylogenically close sequences. A consensus sequence of type-1 repeats was derived from sequence similarity analysis; it comprised a central core of conserved residues including two highly conserved motifs, QC and CWCV. The type-1 repeat from thyroglobulin was found to differ from several previously described cysteine-rich modules, in particular from the epidermal-growth-factor-like module with which it has sometimes been confused. Therefore, our results provide a complete characterization of the repeats which will help in the detection of these repeats in newly characterized proteins, a necessary step for understanding the structural/biological role of this module.

Amino Acid Sequence↗
PubMed1996

The type-1 repeats of thyroglobulin regulate thyroglobulin degradation and T3, T4 release in thyrocytes.

Thyroglobulin (Tg) proteolytic steps are central phenomena in Tg processing and thyroid hormone release in thyrocytes. Based on recent literature data, we propose that the type-1 repetitive units present in the Tg sequence could act as binders and reversible inhibitors of the proteases implicated in Tg processing. The pH-dependent interactions of proteases with the repeats could permit (i) protection from degradation of low iodinated Tg to be recycled; (ii) restriction of early proteolytic attacks to N- and C-terminal hormone formation sites; (iii) increase of the half-time of acidic proteases necessary for the final, extensive degradation steps of Tg.

Animals↗
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

The natural mutation Y248C of human angiotensinogen leads to abnormal glycosylation and altered immunological recognition of the protein.

Common molecular variants of the angiotensinogen gene have been associated with human hypertension. The rare Tyr to Cys change at residue 248 of mature angiotensinogen was identified in one pedigree. Heterozygous individuals (Y248C) had a 40% decrease in plasma angiotensinogen concentration and a 35% reduction of the angiotensin I production rate. Recombinant wild-type (Tyr-248) and mutant (Cys-248) proteins were stably expressed in Chinese hamster ovary cells. Angiotensinogen monoclonal antibodies revealed marked differences in the epitope recognition of the mutant protein and allowed the demonstration of its presence in plasma of Y248C individuals. Similar kinetic constants of angiotensin I production with human renin were observed for both proteins. Western blot analysis showed similar heterogeneities; however, a 3-kDa increase in molecular mass for the Cys-248 protein was observed after immunopurification. Metabolic labeling of the intracellular Cys-248 protein showed a 61-kDa band in addition to the 55.5- and 58-kDa bands observed for the Tyr-248 protein, with all bands being sensitive to endoglycosidase H. In addition, pulse-chase studies revealed a slower intracellular processing for the Cys-248 protein. In conclusion, the Cys-248 mutation alters the structure, glycosylation, and secretion of angiotensinogen in Chinese hamster ovary cells and is accompanied by a decrease in plasma angiotensinogen concentration in Y248C individuals.

Amino Acid Sequence↗
PubMed1996

Cardiac troponin I in the diagnosis of myocardial injury and infarction.

We used a cardiospecific enzymoimmunometric assay to measure cardiac troponin I (cTnI) in samples serially drawn from 78 patients with acute myocardial infarction (AMI), 7 patients with unstable angina (Braunwald class III), 22 multi-traumatized patients, and in 30 athletes after eccentric exercise, as well as in 101 non-traumatic chest pain patients on admission to the emergency department. cTnI assay crossreactivity with crude human skeletal muscle homogenates was < 0.1%. cTnI could not be detected in athletes or multi-traumatized patients except for 2 trauma patients with myocardial damage. Increased cTnI concentrations were found in 6 of 7 patients with unstable angina at rest and in all AMI patients. After AMI, cTnI increased about 3.5 h (median) after the onset of chest pain, reached peak values parallel to CKMB, and stayed increased for at least 4 days. Cardiac troponin T (cTnT) increased and mostly peaked parallel to cTnI. cTnT sensitivity on the 7th day after AMI was significantly higher than that of cTnI. In contrast to cTnI, cTnT mostly showed a second, usually smaller, peak about day 4 after AMI. During the first 4 h after the onset of chest pain and before thrombolytic therapy the sensitivities of myoglobin (0.43) and CKMB mass (0.56) were significantly higher than those of both troponins (cTnI, 0.29; cTnT, 0.25). Areas under receiver operator characteristic curves indicated only moderate diagnostic accuracies of bio-chemical markers for early AMI diagnosis in non-traumatic chest pain patients that cTnI is a highly sensitive and specific marker for myocardial damage which is suitable for early and late diagnosis.

Adult↗
PubMed1996

AD2, a phosphorylation-dependent monoclonal antibody directed against tau proteins found in Alzheimer's disease.

Alzheimer's disease is characterized by an intraneuronal aggregation of hyperphosphorylated tau proteins into paired helical filaments. The hyperphosphorylation of tau proteins induces a decrease in their electrophoretic mobility, resulting in a pathological tau triplet referred to as tau 55, 64 and 69 or tau-PHF. We have developed monoclonal antibodies directed against this pathological tau triplet. In the present article, we report the properties of antibody AD2, which detects the hyperphosphorylated tau proteins forming paired helical filaments during Alzheimer's disease. Using immunoblotting, AD2 exclusively labeled the tau triplet, while normal tau proteins from control cases were not immunodetected. Furthermore, AD2 is highly specific in that it was able to detect the triplet not only in tau preparations but also in total brain homogenates from Alzheimer's disease patients. The binding of this monoclonal antibody to tau proteins is phosphorylation dependent. Characterization of this antibody allowed us to identify its epitope as containing phosphorylated Ser-396 with the participation of phosphorylated Ser-404. AD2 was also shown to label normal tau proteins from rapidly processed brain tissues, but its epitope is rapidly dephosphorylated during postmortem intervals. However, in autopsic brains, AD2 still represents a valuable tool to investigate neurofibrillary degeneration at the biochemical and immunocytochemical levels.

Adolescent↗
PubMed1996

Release kinetics of serum cardiac troponin I in ischemic myocardial injury.

OBJECTIVES: The study was undertaken to evaluate the release kinetics of cardiac troponin I (cTn-I) in ischemic myocardial injury. DESIGN AND METHODS: The reference range for cTn-I was established by determination of cTn-I in sera and plasma obtained from 622 healthy volunteers (Group 1). cTn-I was compared to: (a) Creatine kinase (CK) MB mass and myoglobin in 12 patients with severe skeletal muscle damage (Group 2); (b) CK-MB activity in 48 patients with myocardial infarction (MI) receiving intravenous thrombolysis (Group 3) (in this group, an additional 43 patients with MI were analyzed separately to characterize cTn-I patterns in thrombolyzed and nonthrombolyzed populations): and in 44 patients with unstable angina (Group 4). RESULTS: In Groups 1 and 2, no positive results (> or = 0.1 microgram/L) were obtained. In Group 3, the time-courses of cTn-I were mostly monophasic in form. A pathologic increase occurred earlier in cTn-I than in CK-MB activity (p = 0.0002); the period with increased cTn-I was longer (p = 0.001), the overall sensitivity of cTn-I (93.9%) was higher than that of CK-MB activity (p = 0.00001). cTn-I was more sensitive at admission (p = 0.0004). In additional patients, the cTn-I peak occurred and cTn-I disappeared significantly later in nonthrombolyzed than in the thrombolyzed group. In Group 4, positive tests results were detected in 45% of patients for cTn-I, 16% for CK-MB activity, and 32% for CK-MB mass. CONCLUSIONS: The cTn-I assay appears to be ideally suited for the detection of ischemic myocardial injury in complex clinical situations because of its high specificity; cTn-I indicates myocardial tissue damage in patients with unstable angina and is superior to CK-MB activity and mass in this respect.

Adult↗
PubMed1996

CD4 masking during human immunodeficiency virus type 1 infection, quantified on peripheral blood lymphocytes, is a potential marker of disease progression.

In human immunodeficiency virus type 1 (HIV-1)-infected adults, the proportion of gp120-free CD4 molecules on the surface of T lymphocytes was measured by double-epitope EIA and expressed as a CD epitope concentration ratio. In 51% of these patients (n=81), CD4 T cells showed a significant decrease (up to 100%) in the accessibility of the CD4 epitope in the D1 domain remained accessible. Of interest, a significant increase in the CD4 gp120 binding site concentration, without a change in T cell counts, was observed within 10 days after initiation of zidovudine treatment. Furthermore, CD4 masking by gp120 was associated with a poor clinical patient status. The assessment of the CD4 epitope concentration ratio is proposed as a surrogate marker of disease progression in HIV-1-infected patients.

Adult↗
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↗
PubMed1996

Highly sensitive immunoradiometric assay for serum thyroglobulin with minimal interference from autoantibodies.

Five monoclonal antibodies (MAbs) directed against antigenic domains on thyroglobulin (Tg) not recognized by most anti-Tg human autoantibodies (aAbs) have been used to develop an improved IRMA for serum Tg with a limit of detection of 0.2 micrograms/L. Samples are incubated for 3 h in tubes coated with four anti-Tg MAbs. After washing, the tubes are incubated with the tracer MAb for 20 h at room temperature. Dilution and reproducibility tests demonstrated assay reliability. Tests performed on samples with (n = 361) or without (n = 283) aAbs showed that the TG IRMA Pasteur is largely independent of the marked interference generally caused by aAbs. These results were confirmed with an extended population of 2759 samples. For a cutoff of 1 micrograms/L, sensitivity and specificity were 0.97 and 1, respectively, in a follow-up of differentiated thyroid carcinoma in patients treated by total thyroidectomy.

Animals↗
PubMed1996

[Value of human cardiac troponin I determination in the diagnosis of acute myocardial infarction].

Immunoenzymatic assay (IEMA) of human cardiac Troponin I (TnI c) was used in patients admitted to the coronary care unit with acute myocardial infarction (AMI). TnI c was detected in all patients with AMI. The detection of TnI c was earlier after the onset of pain (4.5 +/- 2.3 hours) than that of CKMB activity (6.3 +/- 3.6 hours), p = 0.003. The kinetics of TnI c are usually monophasic and parallel to that of CKMB activity. The peak value occurs 12.2 +/- 4.6 hours and 15.8 +/- 9.0 hours after the onset of pain in patients treated by thrombolysis. The TnI c disappears from the plasma between 5 and 9 days after the onset of pain, later than CKMB activity (p = 0.0001). In 49 patients admitted for AMI treated by thrombolysis, the comparative sensitivities of TnI c (threshold: 0.1 ng/ml) and of CKMB activity (threshold: 15 IU/l; CK > or = 100 Ul/l) were, at the first sampling on admission, 61% and 22% respectively (p = 0.0002) (average interval from onset of pain to first blood sampling: 3.4 +/- 1.3 hours). TnI c was not detected in the plasma of 145 normal subjects nor in any of the 6 patients with severe muscular trauma or rhabdomyolosis (specificity: 100%). This IEMA is a specific and a sensitive method of diagnosing acute and subacute myocardial infarction. It is ideal for the detection of myocardial necrosis in complex clinical situations when the usual enzymatic markers may be ineffective.

Adult↗
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

New immunoassay for the mapping of neurofibrillary degeneration in Alzheimer's disease using two monoclonal antibodies against human paired helical filament tau proteins.

Monoclonal antibodies against human paired helical filament tau (PHF-tau) proteins were produced. Two of these antibodies, AD1 and AD2, were shown by immunoblot to be directed against distinct hyperphosphorylated epitopes of the PHF-tau proteins. Using AD1 and AD2, an antigen-capture ELISA specific for PHF-tau proteins was developed and used to map the neurofibrillary degeneration of several Broadmann areas from an Alzheimer's disease patient. The results confirm that the neurofibrillary degeneration predominates in parietal and temporal lobes.

Aged↗
PubMed1995

Immunoanalysis of human insulin using monoclonal antibodies reveals antigenicity of evolutionarily conserved residues.

Seven anti-human insulin monoclonal antibodies (mAb) were produced according to an efficient immunization protocol elaborated in our laboratory. Their affinity constants for the binding to the insulin molecule ranged from 5.0 x 10(8) M-1 to 1.0 x 10(10) M-1 when insulin was in solution and from 6.0 x 10(6) M-1 to 2.5 x 10(8) M-1 when insulin was adsorbed onto the microtiter plate. The antigenic sites on the insulin molecule recognized by these mAbs were mapped using two approaches. MAb pairs capable of binding simultaneously to human insulin in solution (using a two-site ELISA) or adsorbed onto a microtiter plate (using a competitive ELISA) were first sought. Three antigenic regions were defined on the surface of adsorbed human insulin and four on soluble insulin. Two distinct antigenic regions common to both the adsorbed and the soluble forms of insulin were defined by our mAbs. In a second approach, the immunological cross-reactivities of these mAbs with species variants of insulin, chemically modified insulin of known structure and a panel of 78 overlapping nonapeptides covering the entire sequence of human proinsulin were assessed. Evidence was obtained that the epitopes recognized by the mAbs included residues conserved during evolution in the insulin molecule. The epitopic specificity of one group of mAbs (group I) was precisely defined. This group recognized a highly conserved region of the insulin molecule including residues 10-17 of the A chain.

Amino Acid Sequence↗