R. Sapin

Recovery from Chronic Demyelination by Thyroid Hormone Therapy: Myelinogenesis Induction and Assessment by Diffusion Tensor Magnetic Resonance Imaging

The failure of the remyelination processes in multiple sclerosis contributes to the formation of chronic demyelinated plaques that lead to severe neurological deficits. Long-term cuprizone treatment of C57BL/6 mice resulted in pronounced white matter pathology characterized by oligodendrocyte depletion, irreversible demyelination and persistent functional deficits after cuprizone withdrawal. The use of a combination of in vivo diffusion tensor magnetic resonance imaging (DT-MRI) and histological analyses allowed for an accurate longitudinal assessment of demyelination. Injection of triiodothyronine (T3) hormone over a 3 week interval after cuprizone withdrawal progressively restored the normal DT-MRI phenotype accompanied by an improvement of clinical signs and remyelination. The effects of T3 were not restricted to the later stages of remyelination but increased the expression of sonic hedgehog and the numbers of Olig2+ and PSA-NCAM+ precursors and proliferative cells. Our findings establish a role for T3 as an inducer of oligodendrocyte progenitor cells in adult mouse brain following chronic demyelination.

Free Thyroxine Measured by Equilibrium Dialysis and Nine Immunoassays in Sera with Various Serum Thyroxine-binding Capacities

Despite the predominant role of thyrotropin measurements in the assessment of thyroid status, free thyroxine (FT4) measurements remain useful either when thyrotropin determination is not conclusive or when a diagnosis of thyroid disease must be confirmed (1). Because it represents only a minute fraction (0.02%) of total T4 (TT4), FT4 is more difficult to measure (2). Direct equilibrium dialysis (ED) methods are considered analytically accurate (3) and are the methods against which others are compared (4). Compared with ED, other FT4 immunoassays may show significant biases related to protein-bound T4 or to the serum T4-binding capacity (sBC: concentration × affinity of binding proteins) (4)(5)(6). We assume assays are calibrated to have roughly the same euthyroid range in samples with normal sBC, and we expect that markedly negative biases may be observed in samples with low sBC and that smaller positive biases may be observed in samples with high sBC (7). The aim of our study was to determine, in clinical samples from euthyroid patients classified into three groups as a function of their low, normal, or high sBC, the bias between FT4 measured with ED and that measured with nine frequently used immunoassays. We also studied the specificity of each assay method and the concordance of immunoassays with ED. FT4 was determined with the Nichols ED/RIA assay (Nichols Institute Diagnostics) and the following nine immunoassays: Elecsys (EL) from Roche Diagnostics, VIDAS (VD) from bioMerieux, Vitros ECi (VT) from Ortho-Clinical Diagnostics, GammaCoat 2-step RIA (GC) from DiaSorin, Immulite (IM) from Diagnostic Products Corporation (DPC), Nichols Advantage (AD), AxSYM (AX) from Abbott Diagnostic, ACS (AC) from Bayer Diagnostics, and AIA (AI) from Tosoh Bioscience. All assays were performed in compliance with the manufacturers’ instructions. The sBC was calculated …

Reference range of serum calcitonin levels in humans: influence of calcitonin assays, sex, age, and cigarette smoking

OBJECTIVE The objective of this study was to re-evaluate the adult C(T) reference values determined by five different immunoassays and by introducing criteria for selecting control subjects. DESIGN A prospective multicenter study. PATIENTS Three hundred and seventy-five clinically euthyroid subjects. METHODS We used five different C(T) immunoassays. Sera were assayed for the concentration of TSH, gastrin, procalcitonin, urea, calcium, and anti-thyroperoxidase antibodies. RESULTS Screening for the various potential causes of hypercalcitoninemia led to the exclusion of 23% of the sera. Our reference value analysis dealt with 287 subjects (142 men and 145 women). The proportion of samples in which no C(T) was detected varied from 56% (for assay D) to 88% (for assay C). We observed significant correlations (whose magnitude depended on the assay used) between C(T) levels and age or body mass index (BMI) (primarily in men). The distribution of C(T) levels showed that 4.7, 9.8, 2.5, 6.5, and 8.0% of the values were over 10 pg/ml respectively. These values corresponded essentially to samples from 11 male subjects (median age: 55 years), most of whom were smokers. The highest C(T) values were around twice as high in men than women, and were higher in smokers than non-smokers. Conclusion In clinical practice (and after having excluded the usual causes of raised C(T) levels), the interpretation of C(T) assay results must take into account i) the method used; ii) the patients gender, age, and weight; and iii) the potential influence of cigarette smoking.

Developmental and cell type-specific expression of thyroid hormone transporters in the mouse brain and in primary brain cells.

Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is mutated in Allan‐Herndon‐Dudley syndrome, a severe mental retardation associated with abnormal thyroid hormone constellations. Since mice deficient in Mct8 exhibit a milder neurological phenotype than patients, we hypothesized that alternative thyroid hormone transporters may compensate in murine brain cells for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we investigated the expression of three different thyroid hormone transporters, i.e., Mct8 and L‐type amino acid transporters Lat1 and Lat2, in mouse brain. All three thyroid hormone transporters are expressed from corticogenesis and peak around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed Mct8 protein, consistent with delayed myelination in MCT8‐deficient patients. Functional T3‐ and T4‐transport assays into primary astrocytes showed KM values of 4.2 and 3.7 μM for T3 and T4. Pharmacological inhibition of L‐type amino acid transporters by BCH and genetic inactivation of Lat2 reduced astrocytic T3 uptake to the same extent. BSP, a broad spectrum inhibitor, including Mct8, reduced T3 uptake further suggesting the cooperative activity of several T3 transporters in astrocytes.

Clinical Evaluation of Nine Free Thyroxine Assays: Persistent Problems in Particular Populations

Abstract Over the past decade, numerous papers have addressed the various methodological problems encountered with free thyroxine (FT4) assays. We evaluated the clinical performance of nine FT4 assays in five centres, using a panel of 310 sera: 156 from euthyroid controls; 27 from hyperthyroid patients; 34 from untreated hypothyroidism; 22 from patients with renal failure; 30 from women in the last trimester of pregnancy; 23 from patients on thyroid substitutive therapy; and 18 from patients treated with amiodarone. Only three methods showed a Gaussian distribution of FT4 concentrations. Reference ranges were calculated using the 2.5th and 97.5th percentiles. A significant difference was observed between FT4 values in men and women. The areas under the receiver operating characteristic (ROC) curves ranged from 0.996 to 1 for hyperthyroidism and from 0.973 to 1 for hypothyroidism. In sera from patients with renal failure and from pregnant women, method-dependent biases were observed and confirmed with dilution experiments. In conclusion, current FT4 assays show good performance regarding the diagnosis of overt dysthyroidism. Nevertheless, FT4 measurements are still vulnerable to method-dependent artefacts in particular populations such as patients with renal failure and pregnant women.

Prognostic Value of Chromogranin A at Admission in Critically Ill Patients: A Cohort Study in a Medical Intensive Care Unit

BACKGROUND Risk assessments of patients should be based on objective variables, such as biological markers that can be measured routinely. The acute response to stress causes the release of catecholamines from the adrenal medulla accompanied by chromogranin A (CGA). To date, no study has evaluated the prognostic value of CGA in critically ill intensive care unit patients. METHODS We conducted a prospective study of intensive care unit patients by measuring serum procalcitonin (PCT), C-reactive protein (CRP), and CGA at the time of admission. Univariate and multivariate analyses were performed to evaluate the ability of these biomarkers to predict mortality. RESULTS In 120 consecutive patients, we found positive correlations between CGA and the following: CRP (r(2) = 0.216; P = 0.02), PCT (r(2) = 0.396; P < 0.001), Simplified Acute Physiologic Score II (SAPS II) (r(2) = 0.438; P < 0.001), and the Logistic Organ Dysfunction System (LODS) score (r(2) = 0.374; P < 0.001). Nonsurvivors had significantly higher CGA and PCT concentrations than survivors [median (interquartile range): 293.0 microg/L (163.5-699.5 microg/L) vs 86.0 microg/L (53.8-175.3 microg/L) for CGA, and 6.78 microg/L (2.39-22.92 microg/L) vs 0.54 microg/L (0.16-6.28 microg/L) for PCT; P < 0.001 for both comparisons]. In a multivariable linear regression analysis, creatinine (P < 0.001), age (P < 0.001), and SAPS II (P = 0.002) were the only significant independent variables predicting CGA concentration (r(2) = 0.352). A multivariate Cox regression analysis identified 3 independent factors predicting death: log-normalized CGA concentration [hazard ratio (HR), 7.248; 95% confidence interval (CI), 3.004-17.487], SAPS II (HR, 1.046; 95% CI, 1.026-1.067), and cardiogenic shock (HR, 3.920; 95% CI, 1.731-8.880). CONCLUSIONS CGA is a strong and independent indicator of prognosis in critically ill nonsurgical patients.

Intermethod Variability in TSH-Receptor Antibody Measurement: Implication for the Diagnosis of Graves Disease and for the Follow-Up of Graves Ophthalmopathy

BACKGROUND We compared the analytical and clinical performance of 3 porcine thyroid receptor antibody (TRAb) methods (1 second- and 2 new third-generation systems) with the conventional TRAb assay based on the human recombinant TSH receptor (hTRAK). PATIENTS AND METHODS We obtained sera from 86 patients with untreated Graves disease (GD) and 71 healthy controls. We measured TRAb concentrations by radioreceptor assay using the hTRAK (Brahms) or the porcine TSH receptor (pRRA) from Beckman-Coulter, by electrochemiluminescence immunoassay (ECLIA) with the Elecsys/Cobas (Roche), and by ELISA using the Medizym TRAb clone (Medipan). RESULTS Between-run assay imprecision was < or =10% and < or =7.6% for hTRAK and ECLIA, but reached 14% and 14.9% for ELISA and pRRA, respectively. Maximal specificity and sensitivity close to 100% were obtained for hTRAK, ECLIA, and ELISA. pRRA failed to detect positive TRAbs in 5 GD patients. Although calibrated against the same reference standard 90/672, the assays displayed a high intermethod variability. The results were significantly higher by ECLIA and lower by ELISA and pRRA compared with hTRAK. Patients with ophthalmopathy had higher TRAb results by ELISA and pRRA than those without eye disease. CONCLUSIONS Second- and third-generation TRAb assays had similar diagnostic sensitivities in the diagnostic evaluation of GD. Despite the use of the same reference standard for calibration, high intermethod variability in TRAb assay results was seen in untreated GD patients. Assay harmonization is necessary for correct interpretation in the follow-up of Graves ophthalmopathy.

Monocarboxylate transporter 8 deficiency: altered thyroid morphology and persistent high triiodothyronine/thyroxine ratio after thyroidectomy

CONTEXT Thyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma triiodothyronine (T(3)) and low/normal thyroxine (T(4)). MCT8 contributes to hormone release from the thyroid gland. OBJECTIVE To characterise the potential impact of MCT8-deficiency on thyroid morphology in a patient and in Mct8-deficient mice. DESIGN Thyroid morphology in a patient carrying the A224V mutation was followed by ultrasound imaging for over 10 years. After thyroidectomy, a histopathological analysis was carried out. The findings were compared with histological analyses of mouse thyroids from the Mct8(-/y) model. RESULTS We show that an inactivating mutation in MCT8 leads to a unique, progressive thyroid follicular pathology in a patient. After thyroidectomy, histological analysis revealed gross morphological changes, including several hyperplastic nodules, microfollicular areas with stromal fibrosis and a small focus of microfollicular structures with nuclear features reminiscent of papillary thyroid carcinoma (PTC). These findings are supported by an Mct8-null mouse model in which we found massive papillary hyperplasia in 6- to 12-month-old mice and nuclear features consistent with PTC in almost 2-year-old animals. After complete thyroidectomy and substitution with levothyroxine (l-T(4)), the preoperative, inadequately low T(4) and free T(4) remained, while increasing the l-T(4) dosage led to T(3) serum concentrations above the normal range. CONCLUSIONS Our results implicate peripheral deiodination in the peculiar hormonal constellation of MCT8-deficient patients. Other MCT8-deficient patients should be closely monitored for potential thyroid abnormalities.

Determination of Free Triiodothyronine by Six Different Methods in Patients with Non-Thyroidal Illness and in Patients Treated with Amiodarone:

We performed a methodological comparison of free triiodothyronine (FT3) estimates in patients with liver cirrhosis and renal failure. Patients were classified in terms of severity of illness on the basis of their total triiodothyronine, total thyroxine and reverse triiodothyronine profiles. FT3 levels, measured in direct dialysis, microchromatography, labelled analogue and two-step immunoextraction assays were significantly (P < 0·01) lower than the control group in all patient categories. However, FT3 measured by a labelled antibody radioimmunoassay was significantly reduced only in the most severely ill sub-group of patients. In a further group of patients on long-term amiodarone therapy for cardiac disease all FT3 methods, with the exception of the labelled antibody radioimmunoassay and an analogue method, yielded significantly (P < 0·01) reduced levels. A significant negative association between FT3 and subject age was demonstrated for all methods except the labelled antibody radioimmunoassay, and a weak but significant negative correlation between log thyrotropin and FT3 was only seen with this assay. Three methods demonstrated a correlation (P < 0·02) with albumin levels in patients with the ‘low T3 syndrome’. In this group, albumin had a predictive value (P ≤ 0·02) for four out of six assays as determined by stepwise variable selection. Our findings suggest that users of FT3 assays should exercise caution in interpreting results in non-thyroidal illness and amiodarone treated patients, as there are method-related differences in the profiles obtained.

Serum thyroxine binding capacity-dependent bias in five free thyroxine immunoassays: assessment with serum dilution experiments and impact on diagnostic performance

OBJECTIVES To assess the presence and magnitude of a serum thyroxine binding capacity (sBC)-dependent bias in five free thyroxine (FT(4)) immunoassays, compared with equilibrium dialysis (ED). The exhibited bias is confronted with clinical results from previous studies to evaluate its impact on FT(4) determination in sera with various sBC. DESIGN AND METHODS The sera of three pregnant women and three non thyroidal ill (NTI) patients were serially diluted in an inert buffer to progressively decrease the sBC. FT(4) values were measured in diluted and undiluted samples with the six assays. RESULTS As a function of increasing dilution performed on pregnancy sera, except for ED and Vitros ECi FT(4,) the other four FT(4) assay results decreased to different degrees, in the following order: two-step GammaCoat RIA< Elecsys< ADVIA:Centaur< Amerlex-MAB RIA. In sera from NTI patients, the decrease was more marked and found at high dilution with the Vitros ECi assay. Data from previous studies showed that FT(4) measured with biased assays were decreased only in samples from very severely NTI patients with low sBC and that FT(4) results in pregnancy sera with high sBC were not significantly biased. CONCLUSIONS The dilution test is a sensitive alarm to assess sBC-dependent bias in FT(4) assays. For all FT4 assays and particularly when a bias is observed, documentation should be sought on the diagnostic performance of the assay and supported by a detailed clinical study including samples with low sBC. Physicians should still be educated about the limitations of FT(4) assays.