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Biomedical subjects

N G Morgenthaler

Publications and source records attributed to N G Morgenthaler.

At least 19 recordsLinked to original sources
PubMed2007

Improved prediction of relapse of Graves' thyrotoxicosis by combined determination of TSH receptor and thyroperoxidase antibodies.

BACKGROUND: Recently, we and others have demonstrated that high levels of auto-antibodies to the human TSH-receptor (TRAb) predict relapse of hyperthyroidism in Graves' disease (GD). Our objective was to extend the outcome of the prediction by combining TRAb with thyroperoxidase antibody (TPO-Ab) measurement. PATIENTS AND METHODS: One hundred and thirty-one GD patients (118 females, 13 males) were analysed, of whom 94 patients (71.8%) had relapse, whereas 37 (28.2%) went into remission. Second generation TRAb and TPO-Ab assays were performed in GD patients with relapse and remission in mean 4.3 months after initial diagnosis. RESULTS: The mean anti-TPO-Ab levels were similar in all patients with relapse and remission. However, there was a steady decline from 4047 U/ml to 530 U/ml in the remission group that correlated positively with TRAb values (>2 to >10 IU/l). The relapse group remained at consistently high levels. The positive predictive value (PPV) for relapse in patients with TRAbs >6 IU/l and anti-TPO-Abs >5000 U/ml was 100, whereas TRAbs >6 IU/l and anti-TPO-Abs >500 U/ml were associated with a PPV of 93.7 up to 96 (p=0.008). These Ab constellations accounted for about one third of all GD patients. For patients with TRAbs between >2 and <6 IU/l the PPV was 66.7-90.0. CONCLUSION: Our follow-up analysis indicates that the prediction of relapse of GD can be improved by a combined measurement of TRAb and TPO-Ab. In patients with moderately increased Abs, determined about 6 months after initial diagnosis, an ablative therapy can be approached without delay.

Adolescent↗
PubMed2006

Copeptin, a fragment of the vasopressin precursor, as a novel predictor of outcome in heart failure.

BACKGROUND: Natriuretic peptides, particularly brain natriuretic peptide (BNP), are elevated in heart failure and therefore considered to be excellent predictors of outcome. Vasopressin is also known to be related to the severity of heart disease. Copeptin--an inactive fragment of the vasopressin precursor--has not been previously investigated in the context of heart failure. MATERIALS AND METHODS: We prospectively studied 268 patients with advanced heart failure after they had been discharged from the hospital. We investigated the ability of BNP and copeptin to predict death, re-hospitalization due to heart failure, and a combination of the two endpoints. RESULTS: Over a mean follow-up period of 15.8 months (up to 24 months), 83 patients died, 122 patients experienced worsening of heart failure, and 145 patients achieved the combined endpoint. Univariate predictors of death were copeptin, BNP, age and impaired kidney function. In multivariate analysis, copeptin (chi(2) = 16, P < 0.0001) and age (chi(2) = 4, P < 0.05) were independent predictors. Univariate predictors of re-hospitalization due to heart failure were copeptin, BNP, age and impaired kidney function. Furthermore, in multivariate analysis BNP (chi(2) = 18, P < 0.0001), age (chi(2) = 11.8, P < 0.001) and copeptin (chi(2) = 4.2, P < 0.05) were found to be independent predictors. CONCLUSION: Our study is the first to show that copeptin is an excellent predictor of outcome in advanced heart failure patients. Its value is superior to that of BNP in predicting death and a combined endpoint, although BNP is still suitable for predicting chronic heart failure (CHF) re-hospitalization. Our data imply that vasopressin antagonism might be a new target to improve outcome in this population.

Aged↗
PubMed2006

Circulating levels of pro-atrial natriuretic peptide in lower respiratory tract infections.

OBJECTIVE: To analyse the mid region of plasma N-terminal pro-atrial natriuretic peptide (MR-proANP) levels in patients with lower respiratory tract infections to evaluate its prognostic use for the severity of disease and outcome. DESIGN: Prospective observational study. Setting. Emergency department of a university hospital. SUBJECTS: A total of 545 consecutive patients with lower respiratory tract infections and 50 healthy controls. Interventions. MR-proANP was measured in serum from all patients using a new sandwich immunoassay. RESULTS: MR-proANP levels (median [IQR], in pmol L(-1)) were significantly higher in patients with lower respiratory tract infections when compared with controls (138.0 [74.1-279.0] vs. 72.7 [62.5-89.5], P < 0.001), with highest levels in patients with community-acquired pneumonia (CAP). MR-proANP, but not C-reactive protein (CRP) levels, gradually increased with increasing severity of CAP, classified according to the pneumonia severity index (PSI) score (P < 0.001). On admission, MR-proANP levels were significantly higher in nonsurvivors when compared with survivors (293.0 [154.0-633.0] vs. 129.0 [71.4-255.0], P < 0.001). In a receiver operating characteristic (ROC) analysis for the prediction of survival of patients with CAP the area under the ROC curve (AUC) for MR-proANP was 0.69, similar when compared with the PSI (AUC 0.74, P = 0.31), and better when compared with other biomarkers, i.e. procalcitonin (AUC 0.57, P = 0.08), CRP (AUC 0.52, P = 0.02), and leucocyte count (AUC 0.56, P = 0.07). CONCLUSIONS: MR-proANP levels are increased in lower respiratory tract infections, especially in CAP. Together with other clinical, radiographic and laboratory findings, MR-proANP levels might be helpful for the risk stratification in CAP.

Acute Disease↗
PubMed2006

Course of vasopressin and copeptin plasma concentrations in a patient with severe septic shock.

In this case report, the course of arginine vasopressin and copeptin, the stable C-terminal part of the arginine vasopressin precursor, is described during the period of critical illness in a septic shock patient. Arginine vasopressin and copeptin concentrations were substantially increased during the initial 36 hours of shock. Subsequently, both hormones continuously decreased, but exhibited another peak in response to stress during extubation. During restoration of cardiovascular stability, endogenous arginine vasopressin levels further decreased and obviously did not contribute to haemodynamic improvement. In contrast, the decrease in arginine vasopressin and copeptin can be at least partly explained by an improvement of cardiovascular function.

Arginine Vasopressin↗
PubMed2006

Graves' disease and Hashimoto's thyroiditis in monozygotic twins: case study as well as transcriptomic and immunohistological analysis of thyroid tissues.

OBJECTIVE: To report on the rare simultaneous occurrence of Graves' disease (GD) and Hashimoto's thyroiditis (HT) in monozygotic twins. DESIGN: We compared the pattern of thyroid tissue-derived cDNAs to gain insight into previous and ongoing immune destruction and reconstruction processes using microarrays. The results were confirmed by immunohistology and real-time PCR. RESULTS: Destruction of thyroid tissue in HT reduced levels of thyrocyte-related cDNAs and cDNAs encoding extracellular matrix components, but increased levels of proteases involved in extracellular matrix degradation compared with GD. Lymphocytic infiltrates forming ectopic follicles replaced the thyroid tissue almost completely in HT. Thus, lymphocyte-related cDNA levels were higher in HT than in GD. The same was true for many chemokines and their receptors, which not only enable migration towards the thyroid but also maintain the lymphocytic infiltrate. HT also showed increased levels of cDNAs encoding molecules related to apoptosis than did GD. Surprisingly, the Th1- and Th2-specific cytokine profiles suggested for HT and GD respectively could not be confirmed. cDNAs encoding factors and receptors involved in angiogenesis were increased in GD compared with HT. CONCLUSIONS: Comparison of gene expression reflects the cellular differences between the two types of autoimmune thyroid disease in twins with identical genetic and similar environmental background.

Adolescent↗
PubMed2005

Pro-A-type and N-terminal pro-B-type natriuretic peptides in different thyroid function states.

QUESTIONS UNDER STUDY: Natriuretic peptides are produced predominantly in the heart and secreted in response to volume expansion and pressure overload. A wide spectrum of cardiac changes is observed in thyroid dysfunctions. This study investigates mid regional pro A-type (proANP) and N-terminal pro-B-type natriuretic peptide (NTproBNP) levels in different thyroid states and evaluates the effect of L-thyroxine treatment on natriuretic peptides in patients with subclinical hypothyroidism. METHODS: Case-control and double-blind, placebo-controlled trial. Sera from 161 female patients (35 with overt, 63 with subclinical hypothyroidism; 10 with overt, 14 with subclinical hyperthyroidism; 40 euthyroid controls) were analysed. ProANP and NT-proBNP were measured at baseline and 48 weeks after L-thyroxine treatment in subclinical hypothyroidism. RESULTS: Circulating proANP and NT-proBNP levels were higher in hyperthyroid patients than in hypothyroid and euthyroid patients (p <0.001). Plasma proANP levels tended to be lower in overt hypothyroidism than in subclinical hypothyroidism. ProANP and NT-proBNP levels correlated weakly to thyroid stimulating hormone (TSH) (r = -0.3 and -0.2, respectively). The natriuretic peptide levels of subclinical and overt hypothyroid subjects showed no difference with those of euthyroid subjects. L-thyroxine treatment had no effect on natriuretic peptide levels in subclinical hypothyroidism. CONCLUSION: Natriuretic peptide levels are altered in different thyroid states with a more pronounced effect in hyperthyroidism than in hypothyroidism. Hyperthyroidism should be considered in patients presenting with unclear symptoms and mildly elevated natriuretic peptide levels, as overt hyperthyroidism results in increased serum A- and B-type natriurectic peptide levels, typically seen in mild heart failure.

Atrial Natriuretic Factor↗
PubMed2005

Relevance of TSH receptor stimulating and blocking autoantibody measurement for the prediction of relapse in Graves' disease.

Recently, we demonstrated that higher levels of autoantibodies to the human TSH receptor (TBII) predict relapse of hyperthyroidism in Graves' disease (GD). The aim of this study was to extend this outcome prediction by dividing TBII into stimulating (TSAb) and blocking (TBAb) TSH receptor autoantibodies. Altogether, ninety patients (81 female, 9 male) were retrospectively analyzed; sixty-four patients (71 %) did not go into remission or relapsed, whereas twenty-six patients (29 %) went into remission (median follow-up: 17.5 months). TSAb and TBAb measurement was performed in a CHO cell bioassay with cAMP readout at the time of their first visit in our outpatient clinic (single point measurement in median 6.5 months after initial diagnosis). In the remission group, eighteen of twenty-six patients (69 %) were TSAb-positive, whereas fifty-three of sixty-four patients (83 %) were TSAb-positive in the relapse group (p = ns). The mean stimulation indices (SI) were 4.1 in the remission group and 12.9 in the relapse group, respectively (p = 0.015). By using a threshold of 10 SI, the specificity for relapse was 96.0 %, as only one in twenty patients with an SI above 10 went into remission during follow-up (PPV 95 %). Most TSAb-positive patients also had high levels of TBII. Neither group showed any difference with respect to blocking type autoantibodies, which were mostly negative in both groups. In summary, high TSAb levels are similar but not superior to TBII for predicting relapse in GD patients. In contrast, TBAb measurement does not add any valuable information in this context. In the clinical routine, TSAb/TBAb measurement may not play an important role for diagnosis or outcome prediction of GD, since sensitive 2 (nd) generation TBII assays are easier to perform and offer similar information to the clinician. Bioassays should be reserved for special clinical questions such as Graves' disease in pregnancy.

Adolescent↗
PubMed2005

Use of pro-atrial natriuretic peptide in the detection of myocardial ischaemia.

BACKGROUND: Because of its unique storage and release mechanisms allowing a very rapid response to haemodynamic changes, pro-atrial natriuretic peptide (proANP) may be a helpful cardiac marker in the detection of myocardial ischaemia. MATERIALS AND METHODS: A total of 260 consecutive patients with suspected myocardial ischaemia referred for rest/ergometry myocardial perfusion single-photon emission computed tomography (SPECT) were enrolled. Levels of plasma proANP were determined before and 1 min after maximal exercise. RESULTS: Baseline proANP and peak exercise proANP were significantly higher in patients with myocardial ischaemia as compared to those without ischaemia (median, 82 [IQR, 57-112] vs. 67 [IQR, 50-106] pmol L(-1), P = 0.007; and 89 [IQR, 65-121] vs. 78 [IQR, 57-116] pmol L(-1), P = 0.033). The area under the ROC curve for baseline proANP was 0.597 (95% CI, 0.527-0.667), as compared to 0.577 (95% CI, 0.507-0.648) for peak exercise proANP. Exercise-induced changes in proANP were similar in patients with and without myocardial ischaemia, and showed no correlation with the extent of myocardial ischaemia. CONCLUSIONS: Baseline proANP and peak exercise proANP are significantly higher in patients with myocardial ischaemia. However, because of considerable overlap in proANP levels between patients with and without myocardial ischaemia, neither measurement seems helpful in the detection of myocardial ischaemia in clinical practice.

Aged↗
PubMed2004

Levels of autoantibodies against human TSH receptor predict relapse of hyperthyroidism in Graves' disease.

The aim of this study was to evaluate the ability of the more sensitive second-generation TSH receptor (TRAb) assay to predict recurrent Graves' disease (GD) vs. remission depending on TRAb levels. 93 patients with active GD were included in the study. By using a cut-off limit of 1.0 IU/l, all 93 patients were positive for TRAb (median: 4.6 IU/l) at the time of their first visit (single point measurement in median 5.1 months after initial diagnosis). Subsequently, 33 patients went into remission and were euthyroid during follow-up (median follow-up: 21.7 months), whereas 60 patients did not go into remission or developed relapse over the following 24 months. Median TRAb levels in the group of remission were significantly (p < 0.0001) lower than TRAb values in the relapse group (2.1 compared to 8.6 IU/l). Applying ROC plot analysis to compare different TRAb thresholds, a cut-off of 10 IU/l was established. Here, the specificity for relapse was 97 % as only 1 of 29 patients with TRAb values above 10 IU/l went into remission during follow-up, whereas all other 28 patients developed a relapse (positive predictive value for relapse: 96.4 %). In contrast, TRAb values lower than 10 IU/l had no impact on the prediction of remission. In conclusion, our data clearly indicate that TRAb measurement is useful for identifying patients that will not benefit from long-term antithyroid drug treatment.

Adolescent↗
PubMed2004

Antibodies to TSH-receptor in thyroid autoimmune disease interact with monoclonal antibodies whose epitopes are broadly distributed on the receptor.

The hyperthyroidism of Graves' disease (GD) is caused by TSH-receptor (TSH-R) stimulating autoantibodies (TSAb), leading to overproduction of thyroid hormones. We present evidence for TSAb interaction with three distinct regions of the TSH-R, one in immediate vicinity of the carboxy terminal serpentine. Three murine monoclonal antibodies (MoAbs 28.1, A9 and 31.7) directed to amino acids 36-40, 147-228 and 382-415 were labelled and tested for their binding to human recombinant TSH-R on solid phase. All MoAbs bound to TSH-R with a K(d) of 8-12 nm and showed no competition among themselves. We tested 114 sera from euthyroid controls, 118 TBII positive sera from patients with GD (containing TSAb confirmed by bioassays), 16 TBII positive sera from patients with autoimmune thyroid disease (AIT), who were hypothyroid and had TSH blocking antibodies (TBAb), and 20 patients with AIT, who were hypothyroid but negative for all TRAb. Mid-regional MoAb A9 tracer achieved the highest sensitivity in the GD group (72.0%), whereas C-terminal MoAb 31.7 found most sera positive in the AIT group (87.5%). Surprisingly, the N-terminal MoAb 28.1 had the lowest sensitivity in the GD (10.4%) and AIT group (43.8%). Using a mixture of all three tracer MoAbs did not increase the sensitivity in the GD or AIT group, compared to the best single MoAb alone. Median inhibition of MoAb A9 was significantly (P < 0.001) higher than inhibition of MoAbs 28.1 or 31.7 in the group of GD patients but not in other groups. Almost all patient sera with positive reactivity in the MoAb tracer assays had TBII values in the higher range. However, there were many highly TBII positive sera, which did not show a displacement of the MoAb tracers. We conclude that, contrary to some reports, the binding of TSAb and TBAb to the TSH-R is not restricted to distinct and distant epitopes. The middle part of the TSH-R seems to be more relevant for TSAb binding than the N-terminal part, while a proportion of TSAb autoantibodies also binds to a C-terminal epitope of the TSH-R. The method described here is a TSH independent competitive assay for the detection of TSH-R autoantibodies.

Antibodies, Monoclonal↗
PubMed2003

Production of procalcitonin (PCT) in non-thyroidal tissue after LPS injection.

Procalcitonin (PCT) is one of the precursors in the synthesis of calcitonin in thyroidal C-cells and other neuroendocrine cells. PCT, among other calcitonin precursors, is elevated in the serum of many conditions associated with a systemic inflammatory response syndrome, even in the absence of the thyroid gland. The aim of our study was to identify PCT-producing extrathyroidal tissues in primate sepsis. In order to induce PCT production, we treated four olive baboons ( papio cynocephalus anubis) with the endotoxin lipopolysaccharide (LPS) from s. typhimurium. We found an increase in serum PCT 3 to 5 hours after LPS injection to levels of 0.2 ng/ml, attaining a peak above 4 ng/ml PCT at 10 hours. In contrast, the untreated baboon had no detectable circulating PCT in the serum. In one animal, additional LPS boosting after 24 hours did not increase serum PCT further. Soluble proteins were extracted from different organs, fractionated by C18 extraction, and PCT was measured in an immunoluminometric assay (ILMA), which was specifically developed for this study. PCT concentrations above 0.2 ng/g of wet tissue were found in a variety of organs in LPS treated baboons, but not in the control baboon. Organs and tissues with the highest PCT concentration included liver, kidney, aorta, fat, ovaries, bladder and adrenal gland. RT-PCR confirmed an extrathyroidal origin of PCT. Importantly, CT-mRNA expression was found in liver, lung, kidney, adrenal, colon, skin, spleen, brain and pancreas. In conclusion, our data confirm previous findings in hamsters, indicating an extrathyroidal origin for PCT in sepsis. Our primate model offers a valuable tool for further investigation of PCT's pathophysiological role and its immunoneutralization as a therapy for sepsis.

Animals↗
PubMed2002

High concentrations of procalcitonin but not mature calcitonin in normal human milk.

Procalcitonin (PCT) is one of the precursors in the synthesis of calcitonin in thyroidal C-cells and other neuroendocrine cells. PCT and other calcitonin precursors in serum are present at less than 50 pg/ml in healthy individuals, but are highly elevated in serum where conditions leading to systemic inflammatory response syndrome or sepsis prevail. We measured PCT concentrations in milk and serum samples taken from 9 healthy women after delivery. PCT concentrations were below 10 pg/ml in serum samples, but were more than 100 times as high in the corresponding milk samples. PCT in milk reached a maximum within the early days after delivery, with a median peak concentration of 2310 pg/ml (range 223 - 4224 pg/ml) at day one and 2442 pg/ml (range 952 - 4488 pg/ml) at day two, then declining over the next days to a median concentration of 747 pg/ml (range 443 - 1656 pg/ml) at day 10 (p = 0.012, by Friedman ANOVA). PCT values reached a steady state of 504 pg/ml median value. Mature calcitonin values measured in parallel with a specific assay were not above the normal range of 10 pg/ml in any samples measured. The strong discrepancy between serum and milk PCT suggests that PCT (but not mature calcitonin) is synthesised in the breast of healthy mothers after delivery. The precise mechanism and the physiological relevance are unclear. Since PCT levels increase drastically in serum from patients suffering from sepsis and related conditions, and since PCT has been ascribed a pro-inflammatory function, we propose that milk PCT might contribute to the activation of the developing neonatal immune system. Similar speculations were proposed for a variety of other pro-inflammatory cytokines, which had comparable kinetics in human milk.

Adult↗
PubMed2001

Isolation and characterization of serum procalcitonin from patients with sepsis.

Procalcitonin (PCT) is one of the precursors in the synthesis of calcitonin in thyroidal C-cells and other neuroendocrine cells. PCT and other calcitonin precursors are elevated in the serum of many conditions leading to systemic inflammatory response syndrome. The measurement of PCT in patients suffering from severe bacterial infections is a useful tool for the diagnosis of sepsis. Furthermore, therapeutic decisions are often based on the increase or decline of serum PCT levels. PCT was reported to have 116 amino acids. The aim of our study was the determination of the primary structure of serum PCT from septic patients. Sera containing high PCT-concentrations (>100 ng/ml) were collected from 22 patients with severe sepsis and were pooled for further purification (12.7 microg total concentration of PCT). Pooled PCT was purified on a CT 21-immunoaffinity column, further purified by reversed phase HPLC, and the resulting pure PCT was digested with endoproteinase Asp-N. N-terminal Edman sequencing showed that the first two amino acids (Ala-Pro) of the proposed pro-peptide were missing. Further analyses by MALDI-TOF mass spectroscopy resulted in a distinct mass signal of 12640 Da +/- 0.1%, which is in concordance with the theoretical molecular weight of the N-terminal truncated form (12628 Da). As opposed to previous suggestions, we could not detect any chemical modifications of PCT. In summary, we could demonstrate that PCT in the serum of septic patients is a peptide of only 114 amino acids, instead of the predicted 116 amino acids, lacking the N-terminal dipeptide Ala-Pro. This information on the primary structure of PCT might help in further studies on the physiological role of PCT during sepsis.

Amino Acid Sequence↗
PubMed2001

A luminescent bioassay for thyroid blocking antibodies.

OBJECTIVE: Thyroid blocking antibodies (TBAb) have a role in the development of hypothyroidism and in the neonate are responsible for transient hypothyroidism. Specific measurement of TBAb requires a bioassay, but current methods are lengthy and cumbersome. We describe a rapid luciferase-based method for the detection of TBAb using the lulu* cell line which is suitable for the provision of a clinical service PATIENTS AND MEASUREMENTS: Chinese hamster ovary (CHO) cells were transfected with human TSH-R together with G418 resistance and a cAMP responsive luciferase construct. Stable pools of transfected cells were selected and clones identified by limiting dilution. Clone lulu* gave the best response to stimulation by TSH and was used to develop a bioassay for TBAb. The luminescent bioassay conditions have been optimized and validated using 12 serum samples from patients found to be TBAb positive in a bioassay using an established method quantifying cAMP by radioimmunoassay (RIA). The effect of thyroid stimulating antibodies (TSAb) on the calculation of Inhibition Index (InI) using two previously described formulae have been investigated and we have used serum containing both TSAb and TBAb to investigate detection of TBAb in samples containing more than one type of activity. RESULTS: Lulu* displays a dose dependent increase in luciferase expression in response to stimulation with bovine (b) TSH which is more effective in serum free medium than in salt free buffer. TSH stimulated luciferase expression can be inhibited by TBAb in either serum or an immunoglobulin preparation. Using optimized assay conditions, challenging 10% serum against 1 U/l bTSH in culture medium, we have tested 31 euthyroid sera to determine a reference range: InI values >23% were considered positive. Twelve samples previously shown to contain TBAb by an established method quantifying cAMP by RIA were positive by the luciferase-based assay. Of control sera, 20/20 systemic lupus erythematosus, 13/14 rheumatoid arthritis, 12/12 multinodular goitre were negative. We demonstrated that if more complex formulae are used to calculate InI, false positive TBAb results can be obtained in samples containing only TSAb. Finally, when sera contain both TSAb and TBAb, the net activity of stimulating and blocking antibodies is detected in the bioassay. Where TSAb are also present, analysis of serum may be required at several dilutions to detect TBAb. CONCLUSIONS: We describe the production of a new cell line, lulu*, and its use to develop a luminescent bioassay for TBAb suitable for clinical use. Comparing two established methods of calculating TBAb, we found that they do not give identical results. In light of this, the high prevalence reported for TBAb in some studies has to be considered with caution.

Animals↗
PubMed2001

Autoimmunity involving the human sodium/iodide symporter: fact or fiction?

Presence, functional activity and clinical relevance of autoantibodies directed against the human sodium iodide symporter (NIS) in thyroid autoimmune diseases have become the subject of much controversy in recent years. Earlier reports have claimed that NIS may represent a major thyroid autoantigen that elicits formation of functionally relevant autoantibodies in a significant proportion of patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). Moreover, a recent study has extended this notion by reporting detection of NIS-autoantibodies in 22% and 24% of a small number of patients with GD and HT, respectively, but not in patients with other autoimmune diseases. However, in striking contrast to these reports, two independent groups of investigators have now presented convincing evidence that NIS-directed autoantibodies occur with low frequency among a large sample of patients with autoimmune thyroid diseases. Moreover, no evidence of specific iodide uptake inhibiting activity was obtained once sera had been subjected to dialysis and/or IgG extraction. Thus, although the controversy has not been definitively resolved, hNIS does not appear to be a major functionally relevant antigen in autoimmune thyroid diseases. Moreover, when detected in addition to TPO and TSH receptor autoantibodies, NIS-directed autoantibodies do not appear to contribute any diagnostic power for GD and HT.

Animals↗
PubMed2000

Two amino acids in glutamic acid decarboxylase act in concert for maintenance of conformational determinants recognised by Type I diabetic autoantibodies.

AIMS/HYPOTHESIS: Glutamic acid decarboxylase 65 is a major autoantigen in Type I (insulin-dependent) diabetes mellitus, autoimmune polyendocrine syndrome and stiff-man syndrome. These disorders are characterised by the presence of multiple autoantibodies to the autoantigen which can be distinguished in a variety of different ways. We have investigated the role of single amino-acid mutations in glutamic acid decarboxylase 65 in distinguishing the binding of serum antibodies and a variety of patient-derived human IgG monoclonal antibodies directed to different determinants of the autoantigen. METHODS: We identified a mutant of glutamic acid decarboxylase 65 that contained four single amino-acid mutations from the wild-type molecule. The role of these mutations was investigated by site-directed mutagenesis. We investigated the binding of patient-derived serum antibodies to glutamic acid decarboxylase 65 to a number of single and double amino-acid mutants using immunoprecipitation with labelled, recombinant antigen. To overcome the heterogeneity of different anti-glutamic acid decarboxylase 65 antibodies present in a patient's serum, the binding of a panel of eleven patient-derived human monoclonal antibodies recognising different determinants on the autoantigen was also studied. RESULTS: Two replacements in glutamic acid decarboxylase 65 at Asn247Ser and Leu574Pro were identified that preferentially influence the anti-glutamic acid decarboxylase 65 serum antibodies of Type I diabetic patients, without statistically significantly effecting those recognised in other disorders. Single or double amino-acid replacements Asn247Ser and Leu574Pro in the autoantigen showed differential affects on expression of epitopes recognised by the human monoclonals. The double replacement of Asn247Ser and Leu574Pro in glutamic acid decarboxylase 65 resulted in the loss of binding of all eleven human monoclonal antibodies, irrespective of their epitope recognition. In contrast, single replacement of Leu574Pro statistically significantly reduced the binding of some carboxyl terminal-directed antibodies such as MICA 1, MICA 3 and DP-A without influencing the binding of other monoclonals. Replacement of Asn247Ser did not, however, influence the binding of any patients serum or human monoclonal antibodies. CONCLUSION/INTERPRETATION: Two distantly spaced amino acids, Asn247 and Leu574 in glutamic acid decarboxylase 65 were identified that act in concert to greatly influence the conformational structure of the autoantigen and statistically significantly influence the binding of antibodies present in Type I diabetic sera. The single or double amino-acid mutants can be used to distinguish some anti-glutamic acid decarboxylase-65 autoantibodies and could prove useful in distinguishing Type I diabetic from autoimmune polyendocrine syndrome and stiff-man syndrome patients' sera as well as to study changes in antibody patterns during disease progression.

Amino Acid Sequence↗
PubMed2000

Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2.

IA-2, a member of the tyrosine phosphatase family, has been identified as a dominant autoantigen in type 1 diabetes. To define humoral IA-2 epitopes, we generated a panel of IA-2 deletion mutants and chimeric proteins using the highly homologous tyrosine phosphatase-like protein IA-2beta. Analysis of autoantibody reactivity in 111 IA-2 antibody positive sera from patients with type 1 diabetes revealed that humoral epitopes cluster to several domains of the intracytoplasmic part of IA-2 [IA-2ic, amino acid (aa) 604-979]. Immunodominant epitopes were found in the first N-terminal 73 amino acids (56% positive), in the middle domain residing between residues 699-874 (45% positive) and the C-terminus depending on the presence of aa 931-979 (at least 37% positive). Competition experiments with overlapping peptides revealed that autoantibody binding towards the N-terminus was dependent on residues 621-628. In the C-terminal domain, two novel conformation-dependent epitopes were identified. The first epitope requires the presence of the C-terminal part of IA-2 (aa 933-979) and an IA-2-specific region between residues 771-932. Reactivity against the second epitope was dependent on intact C-terminal domains as well as residues in the middle (aa 887-932) and N-terminal regions (aa 604-771) which are conserved in IA-2 and IA-2beta. We here defined novel autoantigenic determinants in the N-terminus of IA-2 and characterized conformational epitopes residing in the C-terminal region or spanning from C-terminal residues to the N-terminal domain of IA-2ic. The identification of dominant target regions of diabetes-specific autoantibodies may help to elucidate the molecular mechanisms involved in the autoimmunity towards IA-2.

Amino Acid Sequence↗