Biomedical subjects

A Gardas

Publications and source records attributed to A Gardas.

At least 19 recordsLinked to original sources

PubMed2000

Human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface.

The enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides. Antisera to peptide sequence 599-617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15-80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope. None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four antipeptide antibodies did not give any inhibition of Fabs to TPO. The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO. The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised.

Animals↗

PubMed2000

Identification of an immunodominant region recognized by human autoantibodies in a three-dimensional model of thyroid peroxidase.

Autoimmune thyroid diseases (AITD) are characterized by the presence of autoantibodies to thyroid peroxidase (TPO). This response is dominated by autoantibodies to two conformational determinants, termed A and B, that have been defined by monoclonal antibodies but whose structures and location within TPO are unknown. We have modeled the three-dimensional structure of the extracellular region of TPO, raised antisera to prominent surface structures, and identified an epitope that we show to be a critical part of the B determinant. Antibodies to this epitope inhibit the binding to TPO of human autoantibodies in virtually all serum samples from 65 patients with AITD that were tested. This first description of a model of the three-dimensional structure and location of a major autoantigenic determinant within the TPO molecule may provide structural clues for identifying causative agents or developing novel therapeutic strategies.

Amino Acid Sequence↗

PubMed1999

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.

Animals↗

PubMed1998

Antiphospholipid antibodies and lipoprotein (a) in women with recurrent fetal loss.

OBJECTIVE: To estimate the occurrence of lupus anticoagulant, both isotypes of anticardiolipins, IgG and IgM, and lipoprotein (a) in women with recurrent fetal loss. METHOD: 43 women with two or more fetal losses were included in this open, prospective study. They were divided into a group of 32 nullipara who have had only miscarriages and a group of 11 women who besides the fetal losses were able to give birth to at least one live newborn. Two control groups were also added: 35 women who were never pregnant and 15 parous who did not have any miscarriage. The presence of lupus anticoagulant was assessed by the Staclot LA clotting test. IgG and IgM isotypes of anticardiolipins and lipoprotein (a) were determined using the immunoenzymatic methods. RESULTS: Lupus anticoagulant was discovered in only one of 23 examined patients. Abnormally high values of IgG isotype of anticardiolipins were found in two subjects of each control group and in two nullipara with miscarriages only. The increased level of IgM isotype was noted in approx. 50% of patients with recurrent fetal loss, but at least one successful delivery and in control subjects who were never pregnant. In contrast, the frequency of the increased level of this antibody was diminished by half in the remaining two groups: women who delivered a live child (children) without any miscarriage and women who had only miscarriages. Elevated levels of lipoprotein (a) were found in approx. 30% of non-pregnant control subjects and in the patients of both groups; the abnormally high concentration of this lipoprotein was encountered in only 13% of women who gave birth to live newborn(s) and did not have any miscarriage. CONCLUSION: Of all four determined variables, only abnormally high values of lipoprotein (a) may have some prognostic significance in women predisposed to the recurrent fetal loss.

Abortion, Habitual↗

PubMed1998

Prevalence of high IgM anticardiolipins in patients with ischemic stroke.

Lupus anticoagulant (LA), IgG and IgM isotypes of anticardiolipins (aCL), lipoprotein (a), and the resistance to activated protein C were determined in patients with ischemic stroke. The raised concentration of the aCL-IgM isotype was noted in 42% of patients with this type of stroke, and it was in contrast with an 8% frequency of an increased level of aCL-IgG isotype in these cases. The high level of lipoprotein (a) was found with similar frequency in stroke patients and in age-matched control subjects. It is concluded that the elevated concentration of IgM isotype of anticardiolipin antibodies can be regarded as significant in the ethiological work-up in elderly stroke patients.Copyright Lippincott-Raven Publishers

Journal Article↗

PubMed1997

Purification and crystallisation of the autoantigen thyroid peroxidase from human Graves' thyroid tissue.

Milligram quantities of the human membrane autoantigen thyroid peroxidase (TPO) have been purified to a high degree of homogeneity by a combination of detergent solubilisation, monoclonal antibody affinity, and ion exchange chromatography, from pooled Graves' disease thyroid glands. The purified TPO of greater than 90% purity was enzymatically active as judged by its ability to oxidise guaiacol. Crystals of TPO have been grown from solutions of the protein solubilised in sodium deoxycholate, in the presence of ammonium sulphate. The crystals exhibited birefringence under polarised light, indicative of molecular order. Crystallisation of this large, membrane autoantigen represents the first step in delineating the complete three-dimensional structure of a human autoantigen involved in destructive thyroiditis.

Autoantigens↗

PubMed1997

Human thyroid peroxidase (TPO) isoforms, TPO-1 and TPO-2: analysis of protein expression in Graves' thyroid tissue.

Thyroid peroxidase (TPO) is the key enzyme involved in the biosynthesis of thyroid hormones and is an important autoantigen in autoimmune thyroid disease. Different messenger RNA species coding for TPO are present in thyroid tissue, including the species coding for a 933-amino acid protein (termed TPO-1) and a second in which exon 10 is deleted and which is 57 residues shorter (termed TPO-2). However, it is not known whether the smaller, TPO-2 isoform is expressed as a protein in thyroid cells. In SDS-PAGE under reducing conditions, TPO appears in the thyroid microsome and purified protein preparations as a closely migrating double band of approximately 105 (larger form) and 100 kilodaltons (smaller form). We investigated the presence of the isoform TPO-2 polypeptide in Graves' thyroid tissue using rabbit antisera to three different synthetic peptides from exon 10 (specific for TPO-1) and a polyclonal rabbit and monoclonal anti-TPO antibody (both of which are specific for the two forms of TPO). The larger and smaller forms of TPO were purified by electroelution after gel electrophoresis of highly purified natural TPO from Graves' thyroid microsomes. Both of the purified forms of TPO react with all three anti-exon 10 peptide antibodies, the polyclonal anti-TPO and the monoclonal antibody anti-TPO. This shows that both forms of TPO contain exon 10-encoded polypeptide of TPO-1. Interestingly, the proportion of the larger and smaller forms of TPO varied in different Graves' thyroid microsome preparations. To investigate the presence of the smaller TPO-2 isoform in the purified natural TPO preparation, affinity depletion of TPO-1 using the anti-exon 10 peptide antibodies was carried out. The binding of anti-exon 10 peptide antibodies to the immunodepleted TPO-1 fraction was considerably diminished in comparison to binding of polyclonal anti-TPO, suggesting the presence of small amounts (< 10%) of TPO-2 expressed as a protein in thyroid cells. Our results extend previous observations by showing that the alternatively spliced form of TPO, in which exon 10 is excised, is expressed at low levels in Graves' thyroid tissue. Furthermore, we confirm that both the larger and smaller forms of TPO observed on gel electrophoresis contain TPO-1, suggesting that the difference is caused by posttranslational modifications. The presence of small amounts of TPO-2 in Graves' thyroid glands argues for its role in thyroid function, which remains to be clarified.

Amino Acid Sequence↗

PubMed1997

Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis.

Antibodies (Ab) to thyroid peroxidase (TPO) are common in patients with autoimmune thyroid disease and may play a role in disease pathogenesis. We have prepared immunoglobulin G kappa (IgG kappa) and IgG lambda phage display combinatorial libraries from the cervical (thyroid-draining) lymph nodes of 2 Hashimoto's thyroiditis patients and from the thyroid of 1 patient. After selection with purified recombinant human TPO, up to 10 high affinity IgG kappa clones from each tissue source were analyzed further. No IgG lambda Fab were detected in the patient with the highest TPO Ab titer. Sequence analysis of the clones showed restricted heavy and light chain usage, similar to that in previously published TPO-reactive Fabs. This was despite the substantially larger sizes of the initial libraries, the use of lymph node tissue to generate libraries, and the analysis of the repertoire in patients with Hashimoto's thyroiditis rather than Graves' disease. There was overall similarity in sequences obtained from lymph node and thyroid libraries, with higher levels of somatic hypermutation in the former. The Fab inhibited binding of serum TPO Ab from five patients by 55-95%. These data together with those from previous reports indicate that although there is no unique Ab gene usage, there is the recurrent presence of certain variable regions in the high affinity TPO Ab response.

Amino Acid Sequence↗

PubMed1997

Characterisation of the antibody response to the extracellular region of recombinant thyrotropin receptor.

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.

Animals↗

PubMed1996

Majority of thyroid peroxidase autoantibodies in patients with autoimmune thyroid disease are directed to a single TPO domain.

Murine monoclonal antibodies (mAb) produced against native human thyroid peroxidase (TPO) are powerful tools for analyzing the autoantibody (Aab) epitopes on TPO. Binding sites of thirteen mAbs cover all or most antigenic regions on TPO. We determined the competition between Aabs from 75 AITD patients and 13 mAbs in binding to TPO. Autoantibodies recognize predominantly the TPO area close or identical to mAb#9 epitope. All sera tested inhibited this mAb binding by 92.9 +/- 14.8 (mean +/- SD), range from 69-100%. AITD patients' sera with low Aabs titer up to 1/2,000 inhibited mAb#9 binding to TP0 by 85 +/- 11.5% (mean +/- SD) and did not influence remaining mAbs binding to TPO. With elevated Aab levels the inhibition of other mAbs binding was higher, but never exceeded 35%. The amount of Aabs yielding 50% inhibition of mAbs binding was lowest for mAb#9. In order to obtain this degree of inhibition for other mAbs 5 to 25 times more Aabs were needed. Our results demonstrate that the majority of autoantibodies in sera of patients with AITD recognize a single immunodominant region on the TPO mapped by mAb#9. They account for about 80-90% of serum TPO autoantibodies. The autoimmune response to other regions on TPO molecule is directed to several other epitopes, but represents quantitatively a minority of autoantibodies. This response intensifies with increasing Aabs level in the serum.

Adolescent↗

PubMed1996

[Hyperthyreosis during pregnancy. Case reports].

Explore the source record for details and available documents.

Adult↗

PubMed1993

Iodine induced splitting of peptide bonds in human thyroglobulin.

Human thyroglobulin pretreated with iodine (1mM) at alkaline pH is split to small molecular weight fragments after reduction with dithiothreitol. Iodine pretreatment alone did not induce any changes in the thyroglobulin molecular weight.

Chromatography, High Pressure Liquid↗

PubMed1993

Iodine induced alteration in immunological and biochemical properties of thyroglobulin.

The influence of iodine-iodide solution on the biochemical and immunological properties of human thyroglobulin (hTg) were studied. Human Tg preincubated with the iodine-iodide solution is split to small molecular mass fragments after disulphide bridge reduction with dithiothreitol. The peptide bond cleavage by iodine pretreatment and reduction is possibly linked with the coupling reaction of diiodotyrosyl residues. Pretreatment of hTg with iodine-iodide solution at 1-10 microM decreased the binding of autoantibodies to hTg. The iodine-iodide induced inactivation of hTg autoepitopes is pH dependent and is possibly caused by iodination of tyrosyl residues present in the epitope structure.

Chromatography, High Pressure Liquid↗

PubMed1991

The influence of iodine on the immunological properties of thyroglobulin and its immunological complexes.

Several papers described different immunological properties of thyroglobulin (Tg) after iodination. The influence of iodine-iodide solution on the immunological properties of hTg and its immunological complexes with autoantibodies (aAbs) were studied. Human Tg coated to polystyrene plates, incubated for 30 min with iodine-iodide solution at concentration from 1 to 200 microM at pH 9.0 lost its ability to bind aAbs. Preincubation with iodine (2 microM), decreased aAbs binding by 50%. Tg epitope inactivation induced by iodine depended on the buffer pH and the presence of carbonate ions. The binding of rabbit Tg-antibodies to iodine pretreated Tg was only slightly changed. Thyroglobulin preincubation with iodine solutions decreased aAbs binding from all tested sera (67) of patients with autoimmune thyroid disease (AITD). Excess of iodide (0.2 M KJ) or equimolar concentration of diiodotyrosine protects the Tg molecule from iodine induced inactivation. Immunological complexes of Tg with aAbs dissociate at low iodine concentrations. The results suggest that a product of iodine disproportionation reaction induces changes in the Tg molecule and Tg-aAb's complexes leading to complex dissociation or epitope inactivation.

Antigen-Antibody Complex↗

PubMed1991

[Laboratory tests: level of T3, T4, TSH and antithyroid gland autoantibodies in the Polish population].

In 1986-1990 epidemiologic studies on the thyroid diseases frequency in the polish population were undertaken. During this studies the serum levels of TSH, T4, T3 and autoantibodies were estimated in more than 30 thousand people. TSH was estimated in 30749, T4 in 30928, T3 in 30621 and autoantibodies in 31265 randomly chosen people in 5 districts. We have arbitrarily established the normal range for TSH to be between 0.4-3.8 microIU/ml, for T4 4.4-12.5 micrograms% and for T3 0.9-1.95 ng/ml. The tested group has been divide in 4 subgroup: girls and boys from 1 to 15 years of age the year of the Chernobyl accident, men and women from 15 to 50 years of age at the date of the accident. TSH values in the normal range were found in 85 to 92% of the tested population, depending on the subgroup. T4 vales in the normal range were found in 85 to 92% of the tested groups. T3 vales in the normal range were found in 86 to 93% of the tested groups. The absence of any kind of autoantibodies was established in 89.7% of the tested population. TSH values was above the normal range (above 3.8 microIU/ml) in 3.4% of boys, 4.3% girls, 3.2% men and 3.8% women. TSH vales below the normal range (less the 0.4 microIU/ml) were found in 4.3% boys, 5.0% girls, 10% men and 11.2% women. T4 values above the normal range (higher then 12.5 micrograms%) were found in 12.9% boys, 12.6% girls, 4.6% men and 5.9% women.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1991

[Immunoenzymatic method for determining thyroglobulin levels in human blood serum].

An inexpensive enzyme immunoassay method was designed for the determination of thyroglobulin concentration in human blood serum. The range of concentrations of thyroglobulin which can be measured by the method is between 6 and 800 ng/ml. The sensitivity of the method is comparable to that of the commercial test kits. The values of thyroglobulin concentration obtained with the use of the described method are strongly correlated (r = 0.946) with those obtained by using the reference method (IRMA kit of Byk, Sweden). The intraassay coefficient of variation ranged from 5.5 to 10.2% and interassay coefficient of variation from 9.5 to 13.2% depending on the thyroglobulin concentration. The upper limit of blood serum thyroglobulin concentration in healthy subjects was 70 ng/ml. The results of thyroglobulin determination obtained with the described method are falsely lowered in the presence of antithyroglobulin antibodies; simultaneous determination of these antibodies is thus necessary in such a case. It seems that the described method may be used for monitoring the patients after surgical treatment of differentiated thyroid cancer aimed at early detection of metastases.

Enzyme-Linked Immunosorbent Assay↗

PubMed1991

[Determination of antithyroid antibodies by an immunoenzymatic method; an adaptation for expressing results in international units].

Solid phase immunoenzymatic methods (ELISA) have been developed for the determination of antithyroglbulin (ATG), antimicrosomal (AMc) and antimembrane (ATMA) antibodies in blood serum. The results have been expressed in international units (IU). The level of nonspecific reaction was determined on the basis of 30 samples of blood serum obtained from healthy donors. The double standard deviation values amounted to 8 IU for antithyroglobulin antibodies, 17 IU for antimicrosomal antibodies and 53 IU for antimembrane antibodies at the serum dilution of 1:100. The values of double standard deviation obtained for the healthy donors correspond to the borderline between the positive serum samples and those containing no autoantibodies. The level of autoantibodies in patients with autoimmune diseases of the thyroid varied considerably ranging from complete absence to several thousand units per milliliter in single cases. Antithyroglobulin antibodies were determined simultaneously by using the described method and the commercial kit (Walker, Cambridge) and the results obtained by the two methods were compared. A linear correlation with the correlation coefficient r = 0.93, p < 0.001 was obtained. A good but nonlinear correlation was demonstrated with the methods expressing the results in titre values.

Autoantibodies↗

PubMed1990

The effect of dithiotreitol on thyroid peroxidase and microsomal antigen epitopes recognized by auto and monoclonal antibodies.

The effect of disulphide bridges reduction of the microsomal antigen (Mic-Ag) and thyroid peroxidase (TPO) by dithiotreitol (DTT) has been investigated. The reaction of all 67 tested sera from untreated hyperthyroid Graves' and from 22 Hashimoto's patients with high microsomal antibodies (aAb) titer was diminished by 90-95% by DTT, at pH 9.6. The remaining 5-10% of the activity was not destroyed by DTT. The residual Mic-Ag after DTT reduction was able to inhibit the binding of all 45 Graves' and 22 Hashimoto's tested aAb's to the native microsomal antigen by 100% at high concentration. Reaction of affinity purified TPO with two monoclonal antibodies (mAb) were diminished by 80% to 95% by DTT pretreatment, while the reaction of one mAb with TPO was only slightly affected. The reaction of TPO and Mic-Ag with rabbit polyclonal anti-TPO serum (rabbit a TPO) was diminished by 60% by DTT pretreatment. The immunological reactivity of TPO with aAb's was diminished by 65% after DTT pretreatment. The microsomal antigen-aAb's complex was not destroyed by DTT. Results presented in this paper suggest conformational epitope structure of the Mic-Ag recognized by aAb's in patients with thyroid autoimmune disease (AITD).

Animals↗