Continuous intraperitoneal insulin infusion does not increase the risk of organ-specific autoimmune disease in type 1 diabetic patients: results of a multicentric, comparative study.

AIM: The purpose of this national multicenter prospective study by the French EVADIAC group was to investigate the possibility that continuous intraperitoneal insulin infusion using an implanted pump (CIpii) increases the risk of autoimmune disease in type 1 diabetic patients as it increased anti-insulin immunogenicity. METHODS: Prevalence of clinical (Hashimoto's disease, hyperthyroidism, gastric atrophic disease and vitiligo) and subclinical (presence of anti-thyroperoxidase antibodies, anti-intrinsic factor antibodies, abnormal TSH levels) autoimmune diseases was estimated by comparing two groups of patients already treated by either CIpii (n=154) or external pump (CSII) (n=121) for an average of 6 years. Incidence of autoimmune disease was determined by comparing the same measurements one year after inclusion. RESULTS: No significant difference was observed for the total prevalence of clinical and subclinical auto-immune thyroid and gastric di-seases (35.6% and 3.2% respectively in the CIpii group versus 40.4% and 2.6% in the CSII group). No significant difference for the incidence of clinical and subclinical auto-immune diseases was observed: 7.2% and 0% in CIpii and 7.3% and 1.7% in CSII. CONCLUSION: As previously shown AIA (anti-insulin antibodies) levels were higher in CIpii than in CSII (32.9% vs 20.2%, P<0.0001) but no correlation was observed with either clinical or subclinical autoimmune disease. This large-scale study eliminates the possibility that CIpii increases the risk of autoimmune disease.

Changes in serum level and affinity for concanavalin A of human alpha 1-proteinase inhibitor in severe burn patients: relationship to natural killer cell activity.

In serum from five patients with severe burns, alpha 1-proteinase inhibitor (alpha 1-PI) was analyzed and then isolated by immunosorption chromatography. By Con A-Sepharose chromatography alpha 1-PI was separated into two types of fractions: the first containing the Con A-non-reactive isoforms and the second containing the Con A-reactive isoforms. The increase of alpha 1-PI serum level in burn patients is associated on the fifth day after the burn with a significant shift toward species enriched in bi-antennary oligosaccharides (Con A-reactive isoforms). This latter change passed very quickly and ten days after the burn, whereas the alpha 1-PI serum level was still high, the difference in proportions of Con A-reactive and non-reactive isoforms was not statistically significant. With respect to the difference in oligosaccharide structure, it appeared that the glycan moiety was involved in the inhibitory effect on natural killer cell activity. At the same concentration, purified alpha 1-PI and retained alpha 1-PI isoforms had an equal effect, whereas the non-retained alpha 1-PI isoforms were more efficient (P less than or equal to 0.01). Purified alpha 1-PI and its isoforms inhibited the natural killer cell activity in a dose-dependent manner.

Thyroglobulin autoantibodies in iodized subjects: relationship between epitope specificities and longitudinal antibody activity.

INTRODUCTION: We previously reported a high thyroglobulin autoantibodies (TgAb) prevalence in healthy Sri Lankans after iodine supplementation. In the present study 58 TgAb-positive schoolgirls were followed up after 5 years of continued iodination. The objectives were: (1) to observe the longitudinal profile of TgAb epitope specificities and (2) to examine the relationship between these specificities and the course of thyroid autoimmunity in this population. METHODS: Paired subjects' sera (at onset and at 5-year follow-up) were tested for TgAb, thyroid peroxidase antibody (TPOAb), and TgAb epitope-specificity. Epitope reactivity was determined by employing a panel of 10 murine monoclonal antibodies (Tg-mAbs) directed against 6 Tg antigenic clusters (I-VI) in competitive enzyme-linked immunosorbent assay (ELISA) reactions with test sera. RESULTS: The overall pattern of epitope recognition in individual subject's sera remained preserved over the time period. Nine subjects showed restricted specificities while majority of the subjects were broadly heterogeneous. At follow-up, median TgAb concentration in the restricted group was higher than in the unrestricted (1650 versus 110 kIU/L; p < 0.005). Epitope specificity was a stronger determinant of TgAb persistence than the height of the initial TgAb response or the TPOAb status of subjects. CONCLUSION: Tg epitope reactivity pattern in iodised populations may identify subjects at greater risk of developing autoimmune thyroid disease (AITD).

Characterization of a hormonogenic domain from human thyroglobulin.

A polypeptide domain of molecular mass near 22 kDa was purified from CNBr-digest of iodine poor human thyroglobulin (hTgb). This fragment represents the N-terminal part of the hTgb molecule and consequently contains the preferential hormonogenic tyrosine 'acceptor' of the protein. This fragment could correspond to the non-iodinated and unreduced form of the thyroxinyl-containing 26 kDa peptide previously purified from reduced and iodinated hTgb. This 22 kDa fragment is capable by itself, i.e. independently of the remaining hTgb molecule, of synthesizing thyroxine with a high efficiency after in vitro iodination. Its study should constitute a valuable way to identify at least one of the hormonogenic tyrosine 'donor' residues of hTgb.

In vitro and in vivo iodination of human thyroglobulin in relation to hormone release.

Reduced and S-alkylated thyroglobulin (Tgb) from different species were shown by SDS-PAGE to contain small peptides (from 45-9 kDa) rich in thyroxine. Several hypotheses were proposed to explain their origin. The polypeptide composition of iodine-poor (Tgb A) and normally iodinated (Tgb B) human Tgb prepared by two different procedures (one minimizing and the other favoring post-mortem proteolysis) was compared in the native state and after in vitro iodination. Results show that one of the hormonogenic sites of human Tgb is part of a domain of the molecule most susceptible to proteolysis, especially when it is very iodinated.

Precursor-product relationship between the 26-kDa and 18-kDa fragments formed by iodination of human thyroglobulin.

At moderate iodination levels (20 iodine at atoms/molecule), human thyroglobulin (hTgb) produces after reduction a thyroxinyl-peptide of 26 kDa which represents the N-terminal part of the protein. At higher iodination levels, the 26-kDa peptide is accompanied by another T4-containing peptide of 18 kDa. A precursor-product relationship between the 26- and 18-kDa fragments was demonstrated by the study of the tryptic fragments of both hormonopeptides. In addition, comparison with the protein sequence deduced from the nucleotide sequence of the 5'-end of hTgb mRNA demonstrated that the N-terminal region of Htgb from which are issued the 26-kDa peptide and its 18-kDa derivative is especially sensitive to proteolysis. This character is possibly related with a facilitated release of thyroid hormones in vivo.

Hormone synthesis in human thyroglobulin: possible cleavage of the polypeptide chain at the tyrosine donor site.

At moderate iodination levels (20 iodine atoms/mol) human thyroglobulin (hTg) produces after reduction a hormone-rich peptide of 26 kDa which contains the preferential hormonogenic 'acceptor' tyrosine (Tyr 5) of the protein. The site of cleavage of the hTg chain was demonstrated by analysis of the 26 kDa tryptic hydrolysis products. It consistently yielded the peptide Gln-82-Val-129 which consequently made it possible to localize the hTg chain cleavage at tyrosine residue 130. Evidence for tyrosine involvement in hTg cleavage during thyroid hormone formation supports the hypothesis that peptide bond cleavage would occur at the 'donor' tyrosine residue and suggests that tyrosine 130 would be the donor site reacting with the major hormone-forming acceptor site (Tyr 5) of hTg.

Dityrosine bridge formation and thyroid hormone synthesis are tightly linked and are both dependent on N-glycans.

Formation of dityrosine bridges is a ubiquitous process mainly attributed to oxidative stress leading to protein degradation and cellular damages. Here we show that dityrosine formation is involved in a physiological process, thyroid hormone synthesis, and is strictly dependent on structural characteristics, namely N-glycans, presented by the protein acting as the prothyroid hormone. We used two isoforms of the N-terminal thyroid hormone forming domain (NTD) of human thyroglobulin: one without N-glycan (19 kDa isoform) and the other with high mannose type structures (25 kDa isoform). Both isoforms were able to form iodotyrosines after in vitro iodination. However, iodotyrosine coupling to form thyroxine did not occur with the unglycosylated 19 kDa NTD. In contrast, the 25 kDa isoform formed thyroxine. Strikingly, thyroxine synthesis was accompanied by dimerization of the 25 kDa isoform and formation of a dityrosine bridge; none of this was observed with the 19 kDa isoform. Taken as a whole, our results indicate that dimerization through dityrosine bridging accompanies and could have a role in thyroid hormone synthesis.

In vitro synthesis of thyroxine in a low molecular weight polypeptide fragment from human thyroglobulin.

A peptide fragment of Mr 16 K was purified from the cyanogen bromide digest of human thyroglobulin either normally iodinated in vivo (0.21 % I) or highly iodinated in vitro (1.40 % I). This peptide segment represents in the native molecule a zone in which tyrosine residues are not or poorly accessible to iodination and consequently do not produce thyroxine. In contrast, after isolation from thyroglobulin and iodination in vitro, the peptide is capable of synthesizing thyroxine with a high efficiency. It is concluded that the peptide described which probably represents a potential hormone forming site in the whole thyroglobulin molecule should constitute a valuable model to study the mechanism of thyroxine formation in vitro.

Thyroglobulin structure and function: recent advances.

Thyroglobulin is a large-size iodoglycoprotein specific to thyroid tissue and is the substrate for the synthesis of thyroid hormones, thyroxine and 3,5,3'-triiodothyronine. Recent studies, which greatly benefited from recombinant DNA methodologies, improved the knowledge of several structural features of this dimeric protein and permitted insights into some structure-function relationships. Analysis-function of the primary structure of the human thyroglobulin monomer revealed several main characteristics: 1) 3 types of internal homologies; 2) extensive homology with the bovine thyroglobulin monomer and known partial sequences in the thyroglobulins of other mammalian species; 3) significant homologies with 2 other non-thyroid proteins (acetylcholinesterase and the invariant chain of the Ia class II histocompatibility antigen); 4) a terminal localization of the hormonogenic sites at both ends of the monomer. Current studies aim at determining conformational characteristics, understanding the molecular mechanisms of thyroid hormone formation and unraveling those interactions which in the thyroid cell and the thyroid follicle will permit this large pro-hormone to synthesize and release a few small thyroid hormone molecules. A more precise knowledge of this molecule in higher vertebrates and during evolution would impart valuable information concerning thyroid pathology, since thyroglobulin has been implicated in some genetic and in autoimmune thyroid diseases.

A micromethod for quantitative determination of iodoamino acids in thyroglobulin.

We describe a new method for quantification of iodoamino acids after enzymatic hydrolysis of thyroglobulin. The procedure involves separation of monoiodotyrosine (MIT), di-iodotyrosine, tri-iodothyronine and thyroxine by reverse phase HPLC with a Vydac C18 stationary phase and a mobile phase of water-acetonitrile-acetic acid. The separation is monitored by sensitive spectrophotometric detection through a 96-well microplate system based on the catalytic Sandell-Kolthoff reaction of iodide on the oxidation of arsenic(III) by cerium(IV). This new microassay is particularly convenient because of its high sensitivity and its rapidity (less than 2 h). It can detect 1 pmol MIT and 0.5 pmol of the other three iodoamino acids with a recovery higher than 96%. Moreover, the 96-well microplate system allows many samples to be tested simultaneously and avoids the use of radiolabeled iodine.

Hormone formation in the isolated fragment 1-171 of human thyroglobulin involves the couple tyrosine 5 and tyrosine 130.

The 22 kDa fragment (Asn1-Met171) purified from iodine-poor human thyroglobulin (hTg) is capable by itself to synthesize thyroxine at Tyr5, the preferential hormonogenic acceptor site of the protein, after iodination in vitro. To identify the corresponding donor site in this model we studied the fate of the six Tyr residues present in the 22 kDa peptide after in vitro hormone synthesis. Structural studies of the tyrosyl peptides showed that Tyr5 was the only thyroxine-forming site, the other tyrosines (29, 89, 97 and 107) were noniodinated and Tyr130 was recovered in alanine form after CNBH4 treatment of the Tyr130-containing peptide. Taking into account that alanine could arise from aminoreduced pyruvate species, these results showed that in the 22 kDa fragment (1) hormone formation involves the couple Tyr5 (acceptor)-Tyr130 (donor), and (2) dehydroalanine, the resultant product of donor tyrosine after hormone synthesis, has evolved in pyruvoyl form. To test whether Tyr130 could also act as donor in hTg hormone synthesis, the 22 kDa peptide was isolated from hTg iodinated under conditions leading to iodotyrosine formation followed or not by hormone formation and the tyrosyl peptides were analyzed. After hTg iodination and before coupling (i.e. hormone synthesis) only Tyr5 and Tyr130 were recovered in iodotyrosine form; after coupling thyroxine was found at Tyr5 whereas Tyr130 disappeared. Taken together these results, correlated with the previously reported cleavage of hTg chain at Tyr130 occurring during in vivo hormone synthesis, support the theory that the couple Tyr5 (acceptor)-Tyr130 (donor) would be the preferential hormonogenic site in human Tg.

Tyrosine iodination and iodotyrosyl coupling of the N-terminal thyroid hormone forming site of human thyroglobulin modulate its binding to auto- and monoclonal antibodies.

The present work was aimed at studying the interaction of autoantibodies (aAb) and monoclonal antibodies (mAb) with the N-terminal thyroid hormone forming site of human thyroglobulin (TG). Obtained by CNBr treatment of TG, the peptide (22 kDa) containing the complete major hormonogenic site of human TG was purified in three forms according to the degree of iodination and iodotyrosine coupling: the native, poorly iodinated form (n-22K), the iodinated form containing iodotyrosine but not hormone residues (i-22K) and the form containing thyroid hormone (t-22K). We report that aAb from some patients with autoimmune thyroid diseases showed significant binding to both iodinated 22 kDa forms. Furthermore, a detailed study using mAb evidenced that iodination and coupling induced changes in the antigenicity of the molecule, some occurring without direct implication of iodine or thyroid hormones. The 22 kDa peptide appears as an interesting model to study the antigenic changes induced by the structural modifications in the course of thyroid hormone synthesis. This observation could be relevant to the etiopathogenic process of thyroid autoimmune diseases.

Abnormal calcitonin basal levels and pentagastrin response in patients with chronic renal failure on maintenance hemodialysis.

Hypercalcitoninemia has been reported in renal failure. Using a specific monomeric calcitonin (CT) immunoassay, we measured CT levels in 154 hemodialyzed patients. The relationship between CT and serum intact parathyroid hormone (PTH), gastrin, alkaline phosphatases, phosphate and calcium was studied. The pentagastrin test was performed in 26 patients exhibiting basal hypercalcitoninemia. Basal CT levels over 5.7 pmol/l (20 ng/l) were found in 25.3% of the patients and values higher than 26 pmol/l (90 ng/l) in 7.8%. Although CT is cleared by hemodialysis, post-dialysis CT levels either were unchanged or increased as compared with pre-dialysis values. This suggests that hypercalcitoninemia is not related to a decreased renal clearance, and that hemodialysis induces a specific regulatory pathway. None of the parameters studied were found to explain high CT levels. Of the patients with hypercalcitoninemia, 11.5% exhibited abnormal CT response to pentagastrin but no relationship between CT and phosphate, calcium and PTH levels was evidenced. Our findings confirm high CT monomer levels in renal failure. As there was no correlation with parameters classically involved in CT regulation, its physiological significance remains unclear. Abnormal CT response to pentagastrin raises the problem of its specificity as a tumoral marker with regard to medullary thyroid carcinoma.

Multicenter study on TGPO autoantibody prevalence in various thyroid and non-thyroid diseases; relationships with thyroglobulin and thyroperoxidase autoantibody parameters.

OBJECTIVE: TGPO autoantibodies (aAbs) that bind simultaneously to thyroglobulin (Tg) and thyroperoxidase (TPO) are present in the serum of patients with autoimmune thyroid diseases (AITD) and have been found to differ from monospecific Tg and TPO aAbs. To obtain further insights on the prevalence defined as the rate of occurrence and significance of TGPO aAbs in a large population, we carried out a collaborative study involving 15 European teams. METHODS: Serum samples from 3122 patients with various thyroid and non-thyroid diseases and normal subjects were assayed using a novel TGPO aAb detection kit. This test was designed so that TGPO aAbs are trapped between the Tg-coated solid phase and the soluble TPO labeled with a radioiodinated monoclonal antibody. RESULTS: Only three out of the 220 normal subjects (prevalence of 1.4%) were found to have positive TGPO aAb levels, which were mainly observed in the patients with AITD: the group of patients suffering from Hashimoto's thyroiditis had a TGPO aAb prevalence of 40.5% (n=437 patients), those with Graves' disease, a prevalence of 34.6% (n=645) and those with post-partum thyroiditis, 16.0% (n=243). Among the non-AITD patients with positive TGPO aAb levels, the TGPO aAb prevalence ranged from 20.7% among those with thyroid cancer (n=246) to 0% among those with toxic thyroid nodules (n=47). Among the patients with non-thyroid diseases, the TGPO aAb prevalence ranged from 9.8% in the case of Biermer's pernicious anemia (n=78) to 0% in that of premature ovarian failure (n=44). It is worth noting that the groups showing the highest TGPO aAb prevalence also contained the patients with the highest TGPO aAb titers. Statistical comparisons between the TGPO aAb prevalences in the various groups showed that TGPO aAb could be used as a parameter to distinguish between the groups of Hashimoto's and Graves' patients and between the women with post-partum thyroiditis and the post-partum women with only Tg and/or TPO aAb established during early pregnancy. Unexpectedly, the correlations between TGPO aAbs and Tg and TPO aAbs were found to depend mainly on the assay kit used. CONCLUSION: High TGPO aAb titers are consistently associated with AITD but the reverse was not found to be true. TGPO aAbs are a potentially useful tool, however, for establishing Hashimoto's diagnosis, and would be worth testing in this respect with a view to using them for routine AITD investigations.

Increased plasma levels of pro-brain natriuretic peptide in patients with cardiovascular complications following off-pump coronary artery surgery.

OBJECTIVE: To compare N-terminal pro-brain natriuretic peptide (NT-pro-BNP), procalcitonin (PCT), and troponin I (Tn I) concentrations during and after coronary artery surgery in patients with or without cardiovascular complications. DESIGN AND SETTING: Prospective, comparative study of 12 months in the cardiovascular intensive care unit in a university hospital. PATIENTS: 60 adult patients undergoing coronary artery bypass grafting with the off-pump technique. MEASUREMENTS AND RESULTS: Plasma NT-pro-BNP, PCT, and Tn I levels were measured before and immediately after the end of operation and on PODs 1, and 2 and 3. We defined complicated postoperative course as myocardial infarction, cardiogenic shock, arrhythmias, congestive heart failure, and death occurring after the fourth postoperative hour. Receiver operating characteristic (ROC) curve cutoff values were used to assess the ability of the three markers to predict future cardiac events. The area under ROC curve (AUC) using NT-pro-BNP to detect a cardiovascular complicated course was 0.780 at the preoperative time and 0.850 at the end of surgery. A preoperative NT-pro-BNP value of 397 pg/ml had a sensitivity of 76%, specificity of 67%, and accuracy of 74% for predicting a subsequent cardiovascular complication. An immediate postoperative NT-pro-BNP value of 430 pg/ml had a sensitivity of 80%, specificity of 77%, and accuracy of 76%. Patients with preoperative NT-pro-BNP levels less than 275 pg/ml had an excellent postoperative prognosis. Other two markers were less appropriate. CONCLUSIONS: NT-pro-BNP levels measured before and immediately after off-pump coronary artery bypass seem to be predictive of postoperative cardiac events.

Red blood cell triiodothyronine uptake in unipolar major depression: effect of a chronic antidepressant treatment.

The evolution of kinetic parameters (Vmax, maximal velocity, and Km, Michaelis constant) of red blood cell (RBC) triiodothyronine (L-T3) initial uptake was followed in 19 inpatients suffering from unipolar depression after 1 week (D7) and 4 weeks (D28) of a chronic administration of fluvoxamine, in relation with the clinical efficacy of the drug. In a drug-free state (DO), Vmax (in pmol/min/10(8) cells) and Km (in nM) were significantly increased in depressed patients (Vmax +/- S.D.= 1.02 +/- 0.29, p< 0.01 and Km +/- S.D.= 68.8 +/-15.4, p< 0.05; n=19) compared to healthy volunteers matched for age and sex (Vmax +/- S.D.= 0.82 +/- 0.15 and Km S.D.= 58.8 +/- 9.0; n= 19). When patients were dichotomized on the basis of their treatment response, responders had kinetic parameters significantly increased (Vmax +/-S.D.= 1.03 +/- 0.26, p< 0.01 and Km +/- S.D.= 71.7 +/- 18.7, p< 0.05, n= 10) compared to controls, whereas non-responders had not (Vmax +/- S.D.= 1.00 +/- 0.33, NS and Km +/- S.D.= 65.7 +/- 10.9, NS, n= 9). At D7, Vmax differed from the one of controls only in the responders (Vmax +/- S.D.= 1.03 +/-0.26, p< 0.01). In addition, the percentage of variation of the individual Vmax values during the first week of treatment was significantly lower in responders than in non-responders (deltaVmax(D7-D0) +/- S.D. in % = 10.7 +/- 6.0 and 22.0 +/- 11. 1, p< 0.05, respectively). At D28, kinetics of L-T3 uptake normalized only in the responders (Vmax +/- S.D.= 0.91 +/- 0.13, NS; Km+/-S.D.= 65.7 +/- 7.4, NS). The results indicate that both RBC L-T3 uptake at the pretreatment level and its change during the first week of fluvoxamine treatment were related to the further clinical response to the antidepressant. RBC L-T3 uptake seems to be a biological correlate of the depressive symptomatology since the disturbances disappear only with the clinical remission.

[Urinary iodide analysis: critical study of the digestion method]. / Dosage de l'iode urinaire: évaluation critique de la méthode de minéralisation.

Urinary iodine is largely measured in microtiter plates by a colorimetic ceric-arsenic assay based on the Sandell-Kolthoff reaction. However, a preliminary digestion step is necessary and requires a particular care not only to transform all the iodo-compounds into iodide but also to prevent the formation of substances liable to the disturb of the subsequent redox reaction. In the present study we tested three types of digestion processes, among them two conventional methods (ammonium persulfate and chloric acid) and a new one using combined nitric acid/hydrochloric acid. Results showed that important errors may be obtained with the chloric acid and the ammonium persulfate digestions. These discordances were the consequence of either an incomplete transformation of iodo-compounds or an oxidation of iodide into molecular iodine or a colorimetric assay disturbance due to a residual yellow coloring. No problems were evidenced with the combined nitric acid/hydrochloric acid process, which remains the better alternative to evaluate the urinary iodine. It could also provide a particularly useful means of assessing the iodine status in epidemiological studies.

[Hypercalcitoninemia in conditions other than medullary cancers of the thyroid]. / Les hypercalcitoninémies en dehors des cancers médullaires de la thyroïde.

Serum calcitonin (CT) assays are the most useful tumoral marker for the diagnosis and follow up of medullary thyroid carcinoma (MTC). Since 1988 the sensitivity and specificity of CT assays have been considerably improved. Normal basal and pentagastrin (Pg) stimulated CT ranges remain to be established and it appears necessary to determine the pathological circumstances which may be responsible for hypercalcitoninemia in addition to MCT. By reviewing literature and data from the "Groupe d'Etude des Tumeurs à Calcitonine": a/we compared basal and Pg stimulated CT values obtained with two commercially available immunometric CT assays and we observed that CT values measured by the CT-EASIA MEDGE-NIX kit were three fold the values obtained by suing the hGH ELSA CIS BIOINDUSTRIE Kit; b/we determined that hypercalcitoninemia may be observed in isolated C Cell Hyperplasia (HCC) surrounding either lymphocytic thyroiditis or follicular thyroid carcinoma loci, in chronic renal failure on maintenance hemodialysis, and in various neuroendocrine tumors. Surprisingly, the hypercalcitoninemia related to HCC has been found in genetically unaffected members (without any identified gene RET mutation) of both a Multiple Endocrine Neoplasia type 2A and isolated familial hereditary MTC.