Biomedical subjects

B Pau

Publications and source records attributed to B Pau.

At least 37 records · Page 2Linked to original sources

PubMed1999

T cell response pattern to glutamic acid decarboxylase 65 (GAD65) peptides of newly diagnosed type 1 diabetic patients sharing susceptible HLA haplotypes.

Autoantibodies and autoreactive T lymphocytes directed against several pancreatic beta cell proteins such as GAD65 have been identified in the circulation before and at the onset of clinical type 1 (insulin-dependent) diabetes. Using GAD65 synthetic peptides, we studied the proliferative response of peripheral blood mononuclear cells (PBMC) either from recently diagnosed type 1 diabetic patients, of whom the majority share the disease-associated HLA class II haplotype (DR4-DQB1*0201 or DR3-DQB1*0302), or from HLA-matched control subjects. We found that 67% (14/21) of the type 1 diabetic patients and 39% (9/23) of the control subjects exhibited a positive proliferative response. Compared with control subjects, however, PBMC from diabetic patients proliferated more frequently (P < 0.05) in the presence of peptide pools from the C-terminal region of GAD65 (amino acids 379-585). Diabetic patients with the same HLA-DQ or HLA-DR alleles showed partially identical T cell reactivity, but no clear correlation could be made between MHC class II specificity and T cell epitopes because of multiple combinations of class II alleles. In addition, by flow cytometry, we studied the direct binding of GAD65 peptides to MHC class II molecules of Epstein-Barr virus (EBV)-transformed B (EBV-B) cells obtained from a diabetic patient. We found that 11 GAD peptides were able to bind to the highly susceptible haplotype DRB1*0301/0401-DQA1*0301/0501-DQB1*0302/0201 on the surface of EBV-B cells in partial correlation with the results obtained in the proliferation assays.

Adolescent↗

PubMed1999

GABA(A) and GABA(C) receptors have contrasting effects on excitability in superior colliculus.

We have recently found that GABA(C) receptor subunit transcripts are expressed in the superficial layers of rat superior colliculus (SC). In the present study we used immunocytochemistry to demonstrate the presence of GABA(C) receptors in rat SC at protein level. We also investigated in acute rat brain slices the effect of GABA(A) and GABA(C) receptor agonists and antagonists on stimulus-evoked extracellular field potentials in SC. Electrical stimulation of the SC optic layer induced a biphasic, early and late, potential in the adjacent superficial layer. The late component was completely inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione or CoCl(2), indicating that it was generated by postsynaptic activation. Muscimol, a potent GABA(A) and GABA(C) receptor agonist, strongly attenuated this postsynaptic potential at concentrations >10 microM. In contrast, the GABA(C) receptor agonist cis-aminocrotonic acid, as well as muscimol at lower concentrations (0.1-1 microM) increased the postsynaptic potential. This increase was blocked by (1,2,5, 6-tetrahydropyridine-4-yl)methylphosphinic acid, a novel competitive antagonist of GABA(C) receptors. Our findings demonstrate the presence of functional GABA(C) receptors in SC and suggest a disinhibitory role of these receptors in SC neuronal circuitry.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1999

Human single-chain Fv fragments from a combinatorial library using the loxP-Cre recombination system.

A human scFv display library has been constructed from peripheral blood lymphocytes of a patient suffering from Hashimoto's thyroiditis. Upon induction of Cre recombinase, the amplified VH and VL genes were recombined via two loxP sites inserted in amplification primers to construct in vitro scFv genes. Either soluble scFvs or scFvs displayed on phage were screened for binding to human thyroglobulin after two pannings with this antigen. Three scFvs were obtained which showed very similar nucleotidic sequences. The VH genes expressed display 96.4% nucleotide sequence homology with the germline VH251 gene, one of the two functional members of the small VH5 family and are mutated in sites already described as "selectively neutral" mutations and the VL genes are close to the germline DPL8 gene. These scFvs bind not only to human thyroglobulin but also to other self and exogenous antigens.

Amino Acid Sequence↗

PubMed1999

A new approach for clinical biological assay comparison and standardization: application of principal component analysis to a multicenter study of twenty-one carcinoembryonic antigen immunoassay kits.

BACKGROUND: Principal component analysis (PCA) is a powerful mathematical method able to analyze data sets containing a large number of variables. To our knowledge, this method is applied here for the first time in the field of medical laboratory analysis. METHODS: PCA was used to evaluate the results of a blind comparative study of 21 carcinoembryonic antigen (CEA) reagent kits used to determine CEA concentration in a panel of sera from 80 patients. RESULTS: The mathematical technique first eliminated the variations attributable to the use of different calibrators. The PCA representation then gave a global view of the dispersion of the kits and allowed the identification of a main homogeneous group and of some discrepant kits. CONCLUSIONS: PCA applied to the in vitro diagnostic reagent field could contribute to the standardization process and improve the quality of medical laboratory analyses. A standardization method using a panel of patient sera is proposed.

Biomarkers, Tumor↗

PubMed1999

Effects of bone fracture and surgery on plasma myosin heavy chain fragments of skeletal muscle.

OBJECTIVE: Myosin heavy chain (MHC) fragment is part of a structural or force-bearing protein expressed in the thick filament of muscle fibres. Since MHC fragment is a contractile protein, an increase in plasma MHC concentrations after muscle injury indicates degradation of the contractile apparatus. This study was conducted to determine whether MHC concentrations could be a tool in the assessment of tissue damage in patients with myoskeletal injuries. DESIGN: Prospective, controlled study. SETTING: A UK University National Health Service Centre. PATIENTS: Thirty-eight orthopedic patients, of whom 14 received surgical treatments within the 2-day study period. Patients were compared with 16 nonorthopedic control subjects. OUTCOME MEASURES: Serum levels of MHC, creatine kinase, cardiac troponin I (cTnI), and myoglobin were measured at the time of admission and 24 hours later. Data from patients undergoing surgical repairs were obtained 24 hours after surgery. A competitive radio-immunoassay for beta-type MHC was used. RESULTS: Plasma MHC concentration was higher in the patients than in the controls. The peak levels were observed 24 hours after injury or surgery (p < 0.05). cTnI concentrations were consistently below the assay detection limit of 0.3 microgram/L, thus excluding protein release from the heart muscle (cardiac beta-type MHC). Creatine kinase and myoglobin concentrations were significantly higher on admission in the non-surgical patients than in the surgically treated cases. CONCLUSIONS: Serum MHC levels could be a useful supplementary retrospective, prognostic or diagnostic tool in the study of myoskeletal disturbances involving muscle injury or bone fractures that result in membrane leakage of myoskeletal cells.

Adult↗

PubMed1999

Analysis of VH and VL genes of a monospecific human anti-myosin antibody produced by a B cell from the primary repertoire.

Epstein-Barr virus (EBV) transformation of B lymphocytes from a Glanzmann's thrombasthenia patient with a serum antibody to the integrin alpha IIb beta 3, led to the immortalization of a B cell secreting a monospecific IgM monochonal antibody (MAb), B7, reactive with platelet myosin. Analysis of B7 V genes revealed minimally mutated sequences: the immortalized B cell issued from the primary repertoire, with no evidence of an in vivo selection by myosin. The V genes were here compared with sequences of human MAbs available on databases to more clearly understand the monospecificity of the B7 MAb. B7 V genes were closely identical to rearranged V genes in clones with self-specificities, often secreting polyreactive antibodies. In contrast, B7 is an unmutated monoreactive human MAb able to recognize myosin with a high avidity. Comparison of the CDR3H sequence with that of MAbs in databases supports a central role for the CDR3H subdomain in determining monospecificity. Our results suggest the existence of a monospecific autoreactive B cell compartment, besides the well-known polyspecific one, susceptible to be the template of pathogenic autoreactivity, characterized by antibodies of high affinity and specificity.

Amino Acid Sequence↗

PubMed1999

Whole blood capcellia CD4/CD8 immunoassay for enumeration of CD4+ and CD8+ peripheral T lymphocytes.

We evaluated the Whole Blood Capcellia(R) CD4/CD8, an immunoenzymatic method that provides absolute counts of CD4+ and CD8+ T cells in peripheral blood. The assay is based on the separation of T cells by use of an anti-CD2 magnetic bead suspension, followed by reaction of the CD4 or CD8 molecules with the corresponding monoclonal antibody coupled to peroxidase. CD4-positive monocytes were excluded from the assay. Freeze-dried magnetic bead-T-cell complexes were used as calibrators. Capcellia counts from HIV-1-infected patients were compared with those obtained by flow cytometry as the comparison method. The results by Capcellia correlated well with those by flow cytometric analysis: r2 = 0.95; P <0.001; (y = 0.96x - 22.1); Sy|x = 64 for CD4; r2 = 0.81; P <0.001; (y = 1.26x - 76.4); Sy|x = 139 for CD8; n = 76. The correlation between CD4+ T-cell counts determined by two trained experimenters was significant (r2 = 0.96). Our results indicate that this new ELISA technique for lymphocyte immunophenotyping is an efficient alternative to flow cytometry.

Antibodies, Monoclonal↗

PubMed1998

Anti-digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties.

A gene encoding a single-chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 Fab, whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half-lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.

Amino Acid Sequence↗

PubMed1998

Affinity of recombinant antibody and antibody fragment binding to human thyroglobulin: potential applications in gene therapy.

Recombinant antibodies and antibody fragments are currently being produced. They can be used in vitro for the structural study of antigen-antibody interactions for instance, but their in vivo production may have applications for gene therapy of certain cancers and severe viral diseases and in developing new animal models of autoimmune disease. We report here these two types of applications using a recombinant antihuman thyroglobulin (hTg) antibody.

Animals↗

PubMed1998

Peptide specificity of high-titer anti-glutamic acid decarboxylase (GAD)65 autoantibodies.

To study systematically the linear epitope specificity of anti-glutamic acid decarboxylase (GAD) autoantibodies associated with insulin-dependent diabetes mellitus (IDDM), we produced 93 overlapping 12-residue synthetic peptides derived from the sequence of the human GAD65 protein and covering the entire length of the protein. These peptides were used as antigens in an enzyme immunoassay to screen the sera from 10 IDDM patients, all of which contained at high level autoantibodies directed against GAD65. Three out of ten (30%) IDDM patients had antibodies that reacted with one or more of the synthetic peptides. Two of the peptide-reactive IDDM sera, which also bound denatured recombinant GAD65 on western blots, had the highest titers of anti-GAD antibodies in ELISA assay. Moreover, the anti-GAD antibodies-GAD complexes formed with these sera were characterized by low dissociation rates, indicative of their good stability. A fine specificity analysis, using analogs of antigen peptide 1 (residues 1-12), allowed us to identify the residues at positions 5-9 (GSGFW) as critical for antibody recognition.

Antibody Specificity↗

PubMed1998

Peptide models of immunological recognition: paratope dissection by multiple peptide synthesis.

New approaches to obtain information about the residues of the antibody paratope involved in the interaction with antigen would be very useful to help perform rational mutagenesis of antibodies for improving sensitivity or selectivity in immunoassays. We have evaluated the possibility of dissecting the antibody paratope into large sets of short (12 residues) overlapping peptides to determine the contribution of each peptide to antigen binding. Our results show that the systematic analysis of the antigen-binding properties of heavy chain variable segment and light chain variable segment derived peptides of HyHEL-5, a model anti-lysozyme antibody, can be an experimental approach to the selection of paratope-derived peptides with antigen-binding activity. Detailed analysis of the contribution of each residue from each binding peptide permitted the identification of residues contributing to antigen binding. Of the 38 residues we identified, 22 corresponded to residues that had been shown by X-ray crystallography to be at the interface between HyHEL-5 and lysozyme. The peptide analysis we have developed is thus a way to map the subset of residues from the antibody paratope involved in antigen recognition. The same peptide approach was used to map the idiotope that an anti-idiotypic antibody recognized in the paratope of its cognate antibody.

Antibodies, Anti-Idiotypic↗

PubMed1998

Human cardiac troponin I: precise identification of antigenic epitopes and prediction of secondary structure.

The presence of human cardiac troponin I (hcTnI) in serum is considered to be a highly specific biochemical marker of acute myocardial infarction. To better understand the antigenic properties of hcTnI, a set of 68 overlapping peptides covering the complete amino acid sequence of hcTnI was prepared and used in epitope mapping experiments. All 16 anti-hcTnI monoclonal antibodies tested were found to recognize a peptide epitope, indicating that recognition by anti-hcTnI monoclonal antibodies was not dependent on the tertiary structure of the protein. Furthermore, the peptide reactivity with anti-hcTnI polyclonal antibodies indicated that most of the sequence of the protein was antigenic; in particular, the N- and C-terminal extremities were found to be the strongest antigenic regions. By using accurate secondary structure prediction methods, hcTnI was found to be an all-alpha type protein, with five regions predicted as helices. Matching the results of the epitope analysis with the structural prediction led us to the view that hcTnI is not a globular protein but probably adopts an extended conformation, allowing a large part of the amino acid sequence of this molecule to be recognized by the immune system. This improved knowledge of the antigenic and structural properties of hcTnI may help in developing new antibodies and immunoassays for use in diagnosing myocardial infarction.

Amino Acid Sequence↗

PubMed1998

[Biological perspectives].

The tumour biology of non-small cell bronchial cancer integrates recent developments and a dynamic schema of the phenomena of tumour progression and diffusion of the metastatic disease. There is no leap of known biological disruption between Stage II and Stage III. The latter is defined by anatomical criteria and is a transition in the continuum of the natural history of these cancers. The moto for the tumour progression is the genotypic instability and phenotypic diversification. Metastatic microscopic disease constitutes the first cause of failure in the treatment of Stage III non-small cell bronchial cancer. Among prognostic factors for survival emphasis is placed on the alterations of p53 expression, different types of aneuploidy, anomalies of the expression of cellular adhesion molecules and finally, tumour diversification towards a metastatic phenotype.

Aneuploidy↗

PubMed1997

Systematic exploration of the antigen binding activity of synthetic peptides isolated from the variable regions of immunoglobulins.

Sets of short (12 residues) cellulose-bound synthetic overlapping peptides derived from the sequences of the variable regions of the heavy and light chains of three different antibodies (an anti-thyroglobulin antibody, the HyHEL-5 anti-lysozyme antibody, and an anti-angiotensin II antibody) were used to systematically assess the antigen binding capacity of peptides from the antibody paratope outside their natural molecular context. Peptides enclosing one or several of the complementarity determining region (CDR) residues had antigen binding activity, although the most active peptides were not necessarily those bearing the greatest number of CDR residues. Several residues from the framework region, preceding or following the CDR, were found to play a role in binding. Affinity constants from 4.1 x 10(-7) to 6.7 x 10(-8) M-1 for the soluble form of 9 lysozyme-binding dodecapeptides were measured by BIAcore analysis. Alanine scanning of lysozyme-binding hexapeptides from the HyHEL-5 sequence identified 38 residues important for binding, of which 22 corresponded to residues that had been shown by x-ray crystallography to be at the interface between HyHEL-5 and lysozyme. Our results could be of interest for the rational identification of biologically active peptides derived from antibody sequences and in providing an experimental basis for mutagenesis of the antibody paratope.

Amino Acid Sequence↗

PubMed1997

Circulating cardiac troponin I in severe congestive heart failure.

BACKGROUND: Spontaneous progression of severe congestive heart failure is structurally characterized by cellular degeneration and multiple foci of myocardial cell death. The cardiac muscle isoform of troponin I is uniquely expressed in the adult human myocardium, and an increase in its circulating levels is highly indicative of myocardial injury. Accordingly, we addressed the usefulness of cardiac troponin I as a sensitive and specific molecular marker of congestive heart failure in patients with severely reduced left ventricular performance. METHODS AND RESULTS: A new generation single-step immunoenzymoluminometric assay with high analytical sensitivity was used to assess cardiac troponin I in patients with severe congestive heart failure, healthy blood donors, and hospitalized control subjects without known cardiac disease. The cardiac troponin I concentration (mean+/-SEM) was 72.1+/-15.8 pg/mL in heart failure patients and 20.4+/-3.2 and 36.5+/-5.5 pg/mL in healthy and hospitalized control subjects, respectively (P<.01 versus heart failure patients). When both control groups were considered, the mean cardiac troponin I level was 25.4+/-2.9 pg/mL (P<.01 versus heart failure patients). Creatine kinase MB mass and myoglobin concentrations remained within the normal range in all groups. CONCLUSIONS: These data (1) provide the first evidence for ongoing myofibrillar degradation and increased cardiac troponin I levels in patients with advanced heart failure and (2) show potential usefulness of cardiac troponin I as a specific and sensitive new serum marker molecule in severe congestive heart failure.

Blood Donors↗

PubMed1997

Accurate topological comparison of two recombinant human growth hormones by optical surface plasmon resonance.

A strategy for the comparison of two recombinant derived human growth hormones (r-hGH) has been developed using surface plasmon resonance (SPR). Statistical analysis was systematically used on the results obtained with several batches derived from two different Escherichia coli strains. Monoclonal antibodies (MAb) directed against four different domains in the tertiary structure of natural human growth hormone were used to compare the epitopic maps of the three (two recombinant and one natural) hGH by SPR analysis. Topological studies show the homogeneity of the epitopic maps of the three hGH. The kinetic parameters, association rate, and dissociation rate constants were also analyzed for the binding of each hGH batch to all MAbs. They were found to be homogeneous between the three hormones. Furthermore, the two r-hGH were compared by more classical approaches examining recognition of lactogenic or somatogenic receptors using, respectively, a bioassay of Nb2 cell proliferation and binding to rat liver microsomes. Specific bioactivities and IC50 values calculated in radioreceptor assays did not significantly differ between different r-hGH. The method was sensitive enough to show slight differences on koff value for one MAb (3C11) between (natural) hormone and two r-hGH. These differences are discussed in relation to previous observation made in the literature and the presence of isoforms in the natural product. The strategy developed here was very useful as a new tool to establish the equivalence of the two r-hGH.

Analysis of Variance↗

PubMed1997

Development and standardization of an immuno-quantified solid phase assay for HIV-1 aspartyl protease activity and its application to the evaluation of inhibitors.

The catELISA technique was modified and standardized for measuring HIV-1 aspartyl protease activity and evaluating the potency of synthetic peptide inhibitors. This immuno-quantified solid phase assay combines the use of an immobilized C-terminal biotinylated peptide as substrate, a crude enzyme preparation, and a highly specific antiserum elicited against the C-terminal product of the enzyme reaction. A standard curve of this C-terminal product was constructed to determine the enzyme activity. This assay, which requires less enzyme and substrate, is more sensitive than the conventional HPLC method. The amounts of C-terminal peptide produced in solution as determined from ELISA and HPLC standard curves were comparable. Analogues of peptidomimetics designed in our laboratory were assayed for their potency to inhibit the enzyme. One of them, H4, which is a hydroxyethylamine isostere of the Phe-Pro peptide bond, was a powerful inhibitor.

Amino Acids↗

PubMed1997

Epitope localization of monoclonal antibodies used in human troponin I immunoenzymometric assay.

Serum troponin I isoforms have proven to be potent markers of striated muscle injury. They reach the blood-stream soon after their liberation from the damaged muscular cell and can be detected by the use of selected antibodies. Among monoclonal antibodies (MAbs) originally produced against cardiac troponin I (cTnI), two MAbs able to cross react with the skeletal isoform of troponin I (sTnI) were selected and used to develop a one-step immunoenzymometric assay which allows the quantification of both cardiac and skeletal isoforms (scTnI IEMA). The present report describes the main characteristics of this assay. By using multiple peptide synthesis methods, the localization of the epitopes recognized by the two MAbs on sTnI were determined. The capture and tracer MAbs of the scTnI IEMA were shown to recognize epitopes located within positions 121-127 and 160-167 in the sTnI sequence, respectively. The results of this epitopic analysis are discussed in light of the cross reaction of these two MAbs with cTnI.

Antibodies, Monoclonal↗