Expression of Endogenous Putative TSH Binding Protein in Orbit.

Thyroid stimulating antibodies (TSAB) cause Graves' disease and contribute to Graves' Orbitopathy (GO) pathogenesis. We hypothesise that the presence of TSH binding proteins (truncated TSHR variants (TSHRv)) and/or nonclassical ligands such as thyrostimulin (α2ß5) might provide a mechanism to protect against or exacerbate GO. We analysed primary human orbital preadipocyte-fibroblasts (OF) from GO patients and people free of GO (non-GO). Transcript (QPCR) and protein (western blot) expression levels of TSHRv were measured through an adipogenesis differentiation process. Cyclic-AMP production by TSHR activation was studied using luciferase-reporter and RIA assays. After differentiation, TSHRv levels in OF from GO were significantly higher than non-GO (p = 0.039), and confirmed in ex vivo analysis of orbital adipose samples. TSHRv western blot revealed a positive signal at 46 kDa in cell lysates and culture media (CM) from non-GO and GO-OF. Cyclic-AMP decreased from basal levels when OF were stimulated with TSH or Monoclonal TSAB (M22) before differentiation protocol, but increased in differentiated cells, and was inversely correlated with the TSHRv:TSHR ratio (Spearman correlation: TSH r = -0.55, p = 0.23, M22 r = 0.87, p = 0.03). In the bioassay, TSH/M22 induced luciferase-light was lower in CM from differentiated GO-OF than non-GO, suggesting that secreted TSHRv had neutralised their effects. α2 transcripts were present but reduced during adipogenesis (p < 0.005) with no difference observed between non-GO and GO. ß5 transcripts were at the limit of detection. Our work demonstrated that TSHRv transcripts are expressed as protein, are more abundant in GO than non-GO OF and have the capacity to regulate signalling via the TSHR.

Distinctive Features of Orbital Adipose Tissue (OAT) in Graves' Orbitopathy.

Depot specific expansion of orbital-adipose-tissue (OAT) in Graves' Orbitopathy (GO) is associated with lipid metabolism signaling defects. We hypothesize that the unique adipocyte biology of OAT facilitates its expansion in GO. A comprehensive comparison of OAT and white-adipose-tissue (WAT) was performed by light/electron-microscopy, lipidomic and transcriptional analysis using ex vivo WAT, healthy OAT (OAT-H) and OAT from GO (OAT-GO). OAT-H/OAT-GO have a single lipid-vacuole and low mitochondrial number. Lower lipolytic activity and smaller adipocytes of OAT-H/OAT-GO, accompanied by similar essential linoleic fatty acid (FA) and (low) FA synthesis to WAT, revealed a hyperplastic OAT expansion through external FA-uptake via abundant SLC27A6 (FA-transporter) expression. Mitochondrial dysfunction of OAT in GO was apparent, as evidenced by the increased mRNA expression of uncoupling protein 1 (UCP1) and mitofusin-2 (MFN2) in OAT-GO compared to OAT-H. Transcriptional profiles of OAT-H revealed high expression of Iroquois homeobox-family (IRX-3&5), and low expression in HOX-family/TBX5 (essential for WAT/BAT (brown-adipose-tissue)/BRITE (BRown-in-whITE) development). We demonstrated unique features of OAT not presented in either WAT or BAT/BRITE. This study reveals that the pathologically enhanced FA-uptake driven hyperplastic expansion of OAT in GO is associated with a depot specific mechanism (the SLC27A6 FA-transporter) and mitochondrial dysfunction. We uncovered that OAT functions as a distinctive fat depot, providing novel insights into adipocyte biology and the pathological development of OAT expansion in GO.

Role of Hyaluronan in Human Adipogenesis: Evidence from in-Vitro and in-Vivo Studies.

Hyaluronan (HA), an extra-cellular matrix glycosaminoglycan, may play a role in mesenchymal stem cell differentiation to fat but results using murine models and cell lines are conflicting. Our previous data, illustrating decreased HA production during human adipogenesis, suggested an inhibitory role. We have investigated the role of HA in adipogenesis and fat accumulation using human primary subcutaneous preadipocyte/fibroblasts (PFs, n = 12) and subjects of varying body mass index (BMI). The impact of HA on peroxisome proliferator-activated receptor gamma (PPARγ) expression was analysed following siRNA knockdown or HA synthase (HAS)1 and HAS2 overexpression. PFs were cultured in complete or adipogenic medium (ADM) with/without 4-methylumbelliferone (4-MU = HA synthesis inhibitor). Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, and measurement of a terminal differentiation marker. Modulating HA production by HAS2 knockdown or overexpression increased (16%, p < 0.04) or decreased (30%, p = 0.01) PPARγ transcripts respectively. The inhibition of HA by 4-MU significantly enhanced ADM-induced adipogenesis with 1.52 ± 0.18- (ORO), 4.09 ± 0.63- (foci) and 2.6 ± 0.21-(marker)-fold increases compared with the controls, also increased PPARγ protein expression (40%, (p < 0.04)). In human subjects, circulating HA correlated negatively with BMI and triglycerides (r = -0.396 (p = 0.002), r = -0.269 (p = 0.038), respectively), confirming an inhibitory role of HA in human adipogenesis. Thus, enhancing HA action may provide a therapeutic target in obesity.

The second wave of the Controlled Antenatal Thyroid Screening (CATS II) study: the cognitive assessment protocol.

BACKGROUND: Children whose mothers had low thyroid hormone levels during pregnancy have been reported to have decreased cognitive function. The reported research is part of the follow-on study of the Controlled Antenatal Thyroid Screening Study (CATS I), a randomised controlled trial which investigated the impact of treated vs. untreated low thyroid hormone level in women during pregnancy with the primary outcome being the child's IQ at age 3. No significant differences in IQ were found between the treated and untreated groups. These children are now aged between 7 and 10 years and aspects of their cognitive functioning including their IQ are being reassessed as part of CATS II. METHODS/DESIGN: Cognitive assessments generate an IQ score and further tests administered will investigate long term memory function and motor coordination. The aim is to complete the assessments with 40% of the children born to mothers either in the treated or untreated low thyroid hormone groups (n = 120 per group). Also children born to mothers who had normal thyroid functioning during CATS I are being assessed for the first time (n = 240) to provide a comparison. Assessments are conducted either in the research facility or the participant's home. DISCUSSION: The study is designed to assess the cognitive functioning of children born to mothers with low thyroid hormone levels and normal thyroid functioning during pregnancy. This is the largest study of its type and also is distinguishable in its longitudinal design. The research has the potential to have a significant impact on public health policy in the UK; universal screening of thyroid hormone levels in pregnancy may be the recommendation.

Gut Microbiome Associated With Graves Disease and Graves Orbitopathy: The INDIGO Multicenter European Study.

CONTEXT: Gut bacteria can influence host immune responses but little is known about their role in tolerance-loss mechanisms in Graves disease (GD; hyperthyroidism caused by autoantibodies, TRAb, to the thyrotropin receptor, TSHR) and its progression to Graves orbitopathy (GO). OBJECTIVE: This work aimed to compare the fecal microbiota in GD patients, with GO of varying severity, and healthy controls (HCs). METHODS: Patients were recruited from 4 European countries (105 GD patients, 41 HCs) for an observational study with cross-sectional and longitudinal components. RESULTS: At recruitment, when patients were hyperthyroid and TRAb positive, Actinobacteria were significantly increased and Bacteroidetes significantly decreased in GD/GO compared with HCs. The Firmicutes to Bacteroidetes (F:B) ratio was significantly higher in GD/GO than in HCs. Differential abundance of 15 genera was observed in patients, being most skewed in mild GO. Bacteroides displayed positive and negative correlations with TSH and free thyroxine, respectively, and was also significantly associated with smoking in GO; smoking is a risk factor for GO but not GD. Longitudinal analyses revealed that the presence of certain bacteria (Clostridiales) at diagnosis correlated with the persistence of TRAb more than 200 days after commencing antithyroid drug treatment. CONCLUSION: The increased F:B ratio observed in GD/GO mirrors our finding in a murine model comparing TSHR-immunized with control mice. We defined a microbiome signature and identified changes associated with autoimmunity as distinct from those due to hyperthyroidism. Persistence of TRAb is predictive of relapse; identification of these patients at diagnosis, via their microbiome, could improve management with potential to eradicate Clostridiales.

Molecular, Metabolic, and Nutritional Changes after Metabolic Surgery in Obese Diabetic Patients (MoMen): A Protocol for a Multicenter Prospective Cohort Study.

Metabolic surgery is an essential option in the treatment of obese patients with type 2 diabetes (T2D). Despite its known advantages, this surgery still needs to be introduced in Malaysia. In this prospective study, the pathophysiological mechanisms at the molecular level will be studied and the metabolomics pathways of diabetes remission will be explored. The present study aims to evaluate the changes in the anthropometric measurements, body composition, phase angle, diet intake, biochemistry parameters, adipokines, microRNA, and metabolomics, both pre- and post-surgery, among obese diabetic patients in Malaysia. This is a multicenter prospective cohort study that will involve obese patients (n = 102) with a body mass index (BMI) of ≥25 kg/m2 (Asian BMI categories: WHO/IASO/IOTF, 2000) who will undergo metabolic surgery. They will be categorized into three groups: non-diabetes, prediabetes, and diabetes. Their body composition will be measured using a bioimpedance analyzer (BIA). The phase angle (PhA) data will be analyzed. Venous blood will be collected from each patient for glycated hemoglobin (HbA1c), lipids, liver, renal profile, hormones, adipokines, and molecular and metabolomics analyses. The serum microRNA will be measured. A gene expression study of the adipose tissue of different groups will be conducted to compare the groups. The relationship between the 1HNMR-metabolic fingerprint and the patients' lifestyles and dietary practices will be determined. The factors responsible for the excellent remission of T2D will be explored in this study.

Changes in the Tibial Strain After Proximal Fibular Osteotomy: A Biomechanical Cadaveric Study.

Total knee arthroplasty surgery is an increasingly common procedure for the treatment of uni- and tricompartmental knee osteoarthritis, particularly in advanced stages and in the older population. Its usage is being extended to younger patients, where implant longevity is of concern. In the younger age group, especially with early disease, other options merit consideration. On the other hand, it may not be possible for elderly patients with medical comorbidities to undergo joint replacement surgery. Proximal fibular osteotomy (PFO) has recently been advocated to treat medial knee osteoarthritis. Although there have been clinical reports showing promising outcomes, the biomechanical basis of this procedure is still unclear. We performed a cadaveric study to investigate the effect of PFO on proximal tibial strain. Eight unpaired cadaveric lower limb specimens were loaded in compression at 2 times body weight. Strain gauges were mounted on various sites on the proximal tibia and fibula. After PFO, there was a significant increase in the lateral tibial strain adjacent to the proximal tibiofibular joint (P<.05). There was moderate effect size reduction in the anteromedial tibial strain as well as moderate effect size increase in the posterior tibial strain. The strain reduction seen at the anteromedial tibia can offer a possible explanation for symptomatic relief after PFO. However, the increase in the lateral and posterior tibial strain raises concern about long-term accelerated wear in these regions. [Orthopedics. 2022;45(5):314-319.].

Orbital Signaling in Graves' Orbitopathy.

Graves' orbitopathy (GO) is a complex and poorly understood disease in which extensive remodeling of orbital tissue is dominated by adipogenesis and hyaluronan production. The resulting proptosis is disfiguring and underpins the majority of GO signs and symptoms. While there is strong evidence for the thyrotropin receptor (TSHR) being a thyroid/orbit shared autoantigen, the insulin-like growth factor 1 receptor (IGF1R) is also likely to play a key role in the disease. The pathogenesis of GO has been investigated extensively in the last decade with further understanding of some aspects of the disease. This is mainly derived by using in vitro and ex vivo analysis of the orbital tissues. Here, we have summarized the features of GO pathogenesis involving target autoantigens and their signaling pathways.

The Role of Mitochondria-Linked Fatty-Acid Uptake-Driven Adipogenesis in Graves Orbitopathy.

CONTEXT: Depot-specific expansion of orbital adipose tissue (OAT) in Graves orbitopathy (GO; an autoimmune condition producing proptosis, visual impairment and reduced quality of life) is associated with fatty acid (FA)-uptake-driven adipogenesis in preadipocytes/fibroblasts (PFs). OBJECTIVE: This work sought a role for mitochondria in OAT adipogenesis in GO. METHODS: Confluent PFs from healthy OAT (OAT-H), OAT from GO (OAT-GO) and white adipose tissue in culture medium compared with culture medium containing a mixed hormonal cocktail as adipogenic medium (ADM), or culture-medium containing FA-supplementation, oleate:palmitate:linoleate (45:30:25%) with/without different concentration of mitochondrial biosubstrate adenosine 5'-diphosphate/guanosine 5'-diphosphate (ADP/GDP), AICAR (adenosine analogue), or inhibitor oligomycin-A for 17 days. Main outcome measures included oil-red-O staining and foci count of differentiated adipocytes for in vitro adipogenesis, flow cytometry, relative quantitative polymerase chain reaction, MTS-assay/106 cells, total cellular-ATP detection kit, and Seahorse-XFe96-Analyzer for mitochondria and oxidative-phosphorylation (OXPHOS)/glycolysis-ATP production analysis. RESULTS: During early adipogenesis before adipocyte formation (days 0, 4, and7), we observed OAT-specific cellular ATP production via mitochondrial OXPHOS in PFs both from OAT-H and OAT-GO, and substantially disrupted OXPHOS-ATP/glycolysis-ATP production in PFs from OAT-GO, for example, a 40% reduction in OXPHOS-ATP and trend-increased glycolysis-ATP production on days 4 and 7 compared with day 0, which contrasted with the stable levels in OAT-H. FA supplementation in culture-medium triggered adipogenesis in PFs both from OAT-H and OAT-GO, which was substantially enhanced by 1-mM GDP reaching 7% to 18% of ADM adipogenesis. The FA-uptake-driven adipogenesis was diminished by oligomycin-A but unaffected by treatment with ADP or AICAR. Furthermore, we observed a significant positive correlation between FA-uptake-driven adipogenesis by GDP and the ratios of OXPHOS-ATP/glycolysis-ATP through adipogenesis of PFs from OAT-GO. CONCLUSION: Our study confirmed that FA uptake can drive OAT adipogenesis and revealed a fundamental role for mitochondria-OXPHOS in GO development, which provides potential for therapeutic interventions.

Controlled Antenatal Thyroid Screening II: Effect of Treating Maternal Suboptimal Thyroid Function on Child Behavior.

CONTEXT & OBJECTIVES: The Controlled Antenatal Thyroid Screening (CATS) study was the first randomized controlled trial to investigate effects of treating suboptimal gestational thyroid function (SGTF) on child cognition. Since observational studies indicated that SGTF may also increase symptoms of autism and attention-deficit/hyperactivity disorder (ADHD), the CATS cohort was used to investigate whether treatment of mothers affected their children's behavior. DESIGN & PARTICIPANTS: Mothers (N = 475) completed 3 questionnaires: the Strengths and Difficulties Questionnaire (SDQ), the Child ADHD Questionnaire, and the Social Communication Questionnaire (SCQ, used as a screen for autism spectrum disorder [ASD]), about their children (mean age 9.5 years). Group comparisons of total scores, numbers of children above clinical thresholds, and association between high maternal free thyroxine (FT4) (> 97.5th percentile of the UK cohort, "overtreated") and child neurodevelopment were reported. RESULTS: There were no differences in total scores between normal gestational thyroid function (GTF) (n = 246), treated (n = 125), and untreated (n = 104) SGTF groups. More children of treated mothers scored above clinical thresholds, particularly the overtreated. Scores were above thresholds in SDQ conduct (22% vs 7%), SCQ total scores (7% vs 1%), and ADHD hyperactivity (17% vs 5%) when comparing overtreated (n = 40) and untreated (N = 100), respectively. We identified significantly higher mean scores for SDQ conduct (adjusted mean difference [AMD] 0.74; 95% confidence interval [CI], 0.021-1.431; P = 0.040, effect size 0.018) and ADHD hyperactivity (AMD 1.60, 95% CI, 0.361-2.633; P = 0.003, effect size 0.028) comparing overtreated with normal-GTF children. CONCLUSIONS: There was no overall association between SGTF and offspring ADHD, ASD, or behavior questionnaire scores. However, children of "overtreated" mothers displayed significantly more ADHD symptoms and behavioral difficulties than those of normal-GTF mothers. Thyroxine supplementation during pregnancy requires monitoring to avoid overtreatment.

CATS II Long-term Anthropometric and Metabolic Effects of Maternal Sub-optimal Thyroid Function in Offspring and Mothers.

CONTEXT AND OBJECTIVES: The Controlled Antenatal Thyroid Screening Study I (CATS-I) was a randomized controlled trial investigating the effects of levothyroxine therapy for suboptimal gestational thyroid function (SGTF), comparing outcomes in children of treated (SGTF-T) with untreated (SGTF-U) women during pregnancy. This follow-up study, CATS-II, reports the long-term effects on anthropometric, bone, and cardiometabolic outcomes in mothers and offspring and includes a group with normal gestational thyroid function (NGTF). DESIGN & PARTICIPANTS: 332 mothers (197 NGTF, 56 SGTF-U, 79 SGTF-T) aged 41.2±5.3 years (mean±SD) and 326 paired children assessed 9.3±1.0 years after birth for (i) body mass index (BMI); (ii) lean, fat, and bone mass by dual-energy X-ray absorptiometry; (iii) blood pressure, augmentation index, and aortic pulse-wave-velocity; and (iv) thyroid function, lipids, insulin, and adiponectin. The difference between group means was compared using linear regression. RESULTS: Offspring's measurements were similar between groups. Although maternal BMI was similar between groups at CATS-I, after 9 years (at CATS-II) SGTF-U mothers showed higher BMI (median [interquartile ratio] 28.3 [24.6-32.6] kg/m2) compared with NGTF (25.8 [22.9-30.0] kg/m2; P = 0.029), driven by fat mass increase. At CATS-II SGTF-U mothers also had higher thyroid-stimulating hormone (TSH) values (2.45 [1.43-3.50] mU/L) than NGTF (1.54 [1.12-2.07] mU/L; P = 0.015), since 64% had never received levothyroxine. At CATS-II, SGTF-T mothers had BMI (25.8 [23.1-29.8] kg/m2, P = 0.672) and TSH (1.68 [0.89-2.96] mU/L; P = 0.474) values similar to NGTF mothers. CONCLUSIONS: Levothyroxine supplementation of women with SGTF did not affect long-term offspring anthropometric, bone, and cardiometabolic measurements. However, absence of treatment was associated with sustained long-term increase in BMI and fat mass in women with SGTF.

Primary therapy of Graves' disease and cardiovascular morbidity and mortality: a linked-record cohort study.

BACKGROUND: Graves' disease is routinely treated with antithyroid drugs, radioiodine, or surgery, but whether the choice of initial therapy influences long-term outcomes is uncertain. We evaluated cardiovascular morbidity and mortality according to the method and effectiveness of primary therapy in Graves' disease. METHODS: In this retrospective cohort study, we identified patients with hyperthyroidism, diagnosed between Jan 1, 1998, and Dec 31, 2013, from a thyroid-stimulating hormone (TSH)-receptor antibody (TRAb) test register in south Wales, UK, and imported their clinical data into the All-Wales Secure Anonymised Information Linkage (SAIL) Databank (Swansea University, Swansea, UK). Patients with Graves' disease, defined by positive TRAb tests, were selected for the study, and their clinical data were linked with outcomes in SAIL. We had no exclusion criteria. Patients were matched by age and sex to a control population (1:4) in the SAIL database. Patients were grouped by treatment within 1 year of diagnosis into the antithyroid drug group, radioiodine with resolved hyperthyroidism group (radioiodine group A), or radioiodine with unresolved hyperthyroidism group (radioiodine group B). We used landmark Kaplan-Meier and Cox regression models to analyse the association of treatment with the primary outcome of all-cause mortality and the secondary outcome of major adverse cardiovascular events (myocardial infarction, heart failure, ischaemic stroke, or death) with the landmark set at 1 year after diagnosis. We analysed the association between outcomes and concentration of TSH using Cox regression and outcomes and free thyroxine (FT4) concentration using restricted cubic-spline regression models. FINDINGS: We extracted patient-level data on 4189 patients (3414 [81·5%] females and 775 [18·5%] males) with Graves' disease and 16 756 controls (13 656 [81·5%] females and 3100 [18·5%] males). In landmark analyses, 3587 patients were in the antithyroid drug group, 250 were in radioiodine group A, 182 were in radioiodine group B. Patients had increased all-cause mortality compared with controls (hazard ratio [HR] 1·22, 95% CI 1·05-1·42). Compared with patients in the antithyroid drug group, mortality was lower among those in radioiodine group A (HR 0·50, 95% CI 0·29-0·85), but not for those in radioiodine group B (HR 1·51, 95% CI 0·96-2·37). Persistently low TSH concentrations at 1 year after diagnosis were associated with increased mortality independent of treatment method (HR 1·55, 95% CI 1·08-2·24). Spline regressions showed a positive non-linear relationship between FT4 concentrations at 1 year and all-cause mortality. INTERPRETATION: Regardless of the method of treatment, early and effective control of hyperthyroidism among patients with Graves' disease is associated with improved survival compared with less effective control. Rapid and sustained control of hyperthyroidism should be prioritised in the management of Graves' disease and early definitive treatment with radioiodine should be offered to patients who are unlikely to achieve remission with antithyroid drugs alone. FUNDING: National Institute for Social Care and Health Research, Wales.

Prostaglandin F2-Alpha Eye Drops (Bimatoprost) in Graves' Orbitopathy: A Randomized Controlled Double-Masked Crossover Trial (BIMA Trial).

BACKGROUND: Previous in vitro experiments have demonstrated that prostaglandin F2-alpha (PF2α) reduced proliferation and adipogenesis in a murine cell line and human orbital fibroblasts derived from subjects with inactive Graves' orbitopathy (GO). The objective of this study was to determine if the PGF2α analogue bimatoprost is effective at reducing proptosis in this population. METHODS: A randomized controlled double-masked crossover trial was conducted in a single tertiary care academic medical center. Patients with long-standing, inactive GO but persistent proptosis (>20 mm in at least one eye) were recruited. Allowing for a 15% dropout rate, 31 patients (26 females) were randomized in order to identify a treatment effect of 2.0 mm (p = 0.05; power 0.88). Following informed consent, participants were randomized to receive bimatoprost or placebo for three months, after which they underwent a two-month washout before switching to the opposite treatment. The primary outcome was the change in exophthalmometry readings over the two three-month treatment periods. RESULTS: The mean exophthalmometer at baseline was 23.6 mm (range 20.0-30.5 mm), and the mean age of the patients was 55 years (range 28-74 years). The median duration of GO was 7.6 years (interquartile range 3.6-12.3 years). The majority were still suffering from diplopia (61.3%) with bilateral involvement (61.3%). Using multi-level modeling adjusted for baseline, period, and carry-over, bimatoprost resulted in a -0.17 mm (reduction) exophthalmometry change ([confidence interval -0.67 to +0.32]; p = 0.490). There was a mean change in intraocular pressure of -2.7 mmHg ([confidence interval -4.0 to -1.4]; p = 0.0070). One patient showed periorbital fat atrophy on treatment, which resolved on stopping treatment. Independent analysis of proptosis by photographic images (all subjects) and subgroup analysis on monocular disease (n = 12) did not show any apparent benefit. CONCLUSIONS: In inactive GO, bimatoprost treatment over a three-month period does not result in an improvement in proptosis.

Central pontine myelinolysis complicating treatment of the hyperglycaemic hyperosmolar state.

Central pontine myelinolysis (CPM) is well recognized to occur in a variety of clinical settings, but particularly following rapid correction of severe hyponatraemia. The development of CPM as a result of rapid shifts in plasma osmolality during the treatment of the hyperglycaemic hyperosmolar state (HHS) has hitherto been described in only one case. We report a second case in which this complication occurred in association with treatment of the HHS. The patient was a 49-year-old woman who presented to another hospital with drowsiness and a plasma glucose of 106 mmol/L. Her admission plasma sodium was 135 mmol/L. She received treatment with intravenous insulin and 0.9% normal saline, and there was a rapid drop in plasma glucose by 60 mmol/L within 6 h, which was associated with a rebound rise of plasma sodium to 159 mmol/L. Her plasma glucose and sodium were later stabilized. When the patient was transferred to our hospital a few days later, she was noted to have flaccid quadraparesis and pseudobulbar palsy. A magnetic resonance imaging scan revealed a pontine lesion consistent with CPM. She made a gradual recovery over several months with intensive rehabilitation and eventually returned to near normal functional capacity. This is the second case report in the literature of CPM complicating the management of HHS and highlights the importance of the judicious and measured correction of hyperglycaemia and the appropriate management of fluid replacement and electrolyte balance when treating this condition.

Longitudinal Characterization of Autoantibodies to the Thyrotropin Receptor (TRAb) During Alemtuzumab Therapy: Evidence that TRAb May Precede Thyroid Dysfunction by Many Years.

BACKGROUND: Thyroid autoimmunity, especially Graves' disease or hypothyroidism with positive autoantibodies (TRAb) to the thyrotropin receptor (TSHR), occurs in 30-40% of patients with relapsing multiple sclerosis following treatment with alemtuzumab (ALTZ). ALTZ therapy therefore provides a unique opportunity to study the evolution of TRAb prior to clinical presentation. TRAb can stimulate (TSAb), block (TBAb), or not affect ("neutral") the TSHR function, causing hyperthyroidism, hypothyroidism, or euthyroidism, respectively. METHODS: A longitudinal retrospective analysis was conducted of TRAb bioactivity over a period of nine years in 45 multiple sclerosis patients receiving ALTZ using available stored serum. Of these 45 patients, 31 developed thyroid dysfunction (TD) and 14 remained euthyroid despite being followed for a minimum of five years (NO-TD). The presence of TRAb was evaluated at standardized time points: (i) before ALTZ, (ii) latest time available following ALTZ and before TD onset, and (iii) following ALTZ during/after TD onset. Serum TRAb were detected by published in-house assays (ihTRAb): flow cytometry detecting any TSHR-binding TRAb, and luciferase bioassays detecting TSAb/TBAb bioactivity. Purified immunoglobulin G was used to verify TSAb/TBAb in selected hypothyroid cases. Standard clinical automated measurements of TRAb, antithyroid peroxidase autoantibodies (TPOAb), thyrotropin, free thyroxine, and free triiodothyronine were also collected. RESULTS: Before ALTZ, combined ihTRAb (positive with flow cytometry and/or luciferase bioassay) but not automated TRAb were present in 5/16 (31.2%) TD versus 0/14 (0%) NO-TD (p = 0.017). Detectable ihTRAb preceded TD development in 9/28 (32.1%) and by a median of 1.2 years (range 28 days-7.3 years). Combination testing of ihTRAb and TPOAb at baseline predicted 20% of subsequent cases of hyperthyroidism and 83% of hypothyroidism. CONCLUSIONS: Evidence is presented that TRAb measured with custom-made assays can be detected prior to any change in thyroid function in up to a third of cases of ALTZ-related TD. Furthermore, the presence of ihTRAb prior to ALTZ treatment was strongly predictive of subsequent TD. The findings suggest that a period of affinity maturation of TRAb may precede clinical disease onset in some cases. Combined testing of TPOAb and ihTRAb may increase the ability to predict those who will develop TD following ALTZ.

Combining micro-RNA and protein sequencing to detect robust biomarkers for Graves' disease and orbitopathy.

Graves' Disease (GD) is an autoimmune condition in which thyroid-stimulating antibodies (TRAB) mimic thyroid-stimulating hormone function causing hyperthyroidism. 5% of GD patients develop inflammatory Graves' orbitopathy (GO) characterized by proptosis and attendant sight problems. A major challenge is to identify which GD patients are most likely to develop GO and has relied on TRAB measurement. We screened sera/plasma from 14 GD, 19 GO and 13 healthy controls using high-throughput proteomics and miRNA sequencing (Illumina's HiSeq2000 and Agilent-6550 Funnel quadrupole-time-of-flight mass spectrometry) to identify potential biomarkers for diagnosis or prognosis evaluation. Euclidean distances and differential expression (DE) based on miRNA and protein quantification were analysed by multidimensional scaling (MDS) and multinomial regression respectively. We detected 3025 miRNAs and 1886 proteins and MDS revealed good separation of the 3 groups. Biomarkers were identified by combined DE and Lasso-penalized predictive models; accuracy of predictions was 0.86 (±0:18), and 5 miRNA and 20 proteins were found including Zonulin, Alpha-2 macroglobulin, Beta-2 glycoprotein 1 and Fibronectin. Functional analysis identified relevant metabolic pathways, including hippo signaling, bacterial invasion of epithelial cells and mRNA surveillance. Proteomic and miRNA analyses, combined with robust bioinformatics, identified circulating biomarkers applicable to diagnose GD, predict GO disease status and optimize patient management.

Controlled Antenatal Thyroid Screening II: Effect of Treating Maternal Suboptimal Thyroid Function on Child Cognition.

Context and Objective: The Controlled Antenatal Thyroid Screening (CATS) study investigated treatment of suboptimal gestational thyroid function (SGTF) on childhood cognition and found no difference in intelligence quotient (IQ) at 3 years between children of treated and untreated SGTF mothers. We have measured IQ in the same children at age 9.5 years and included children from normal gestational thyroid function (normal-GTF) mothers. Design, Setting, and Participants: One examiner, blinded to participant group, assessed children's IQ (Wechsler Intelligence Scale for Children, Fourth Edition UK), long-term memory, and motor function (Developmental Neuropsychological Assessment II) from children of 119 treated and 98 untreated SGTF mothers plus children of 232 mothers with normal-GTF. Logistic regression explored the odds and percentages of an IQ < 85 in the groups. Results: There was no difference in IQ < 85 between children of mothers with normal-GTF and combined SGTF, i.e., treated and untreated (fully adjusted odds ratio [OR] = 1.15 [95% confidence interval (CI) 0.52, 2.51]; P = 0.731). Furthermore, there was no significant effect of treatment [untreated OR = 1.33 (95% CI 0.53, 3.34); treated OR = 0.75 (95% CI 0.27, 2.06) P = 0.576]. IQ < 85 was 6.03% in normal-GTF, 7.56% in treated, and 11.22% in untreated groups. Analyses accounting for treated-SGTF women with free thyroxine > 97.5th percentile of the entire CATS-I cohort revealed no significant effect on a child's IQ < 85 in CATS-II. IQ at age 3 predicted IQ at age 9.5 (P < 0.0001) and accounted for 45% of the variation. Conclusions: Maternal thyroxine during pregnancy did not improve child cognition at age 9.5 years. Our findings confirmed CATS-I and suggest that the lack of treatment effect may be a result of the similar proportion of IQ < 85 in children of women with normal-GTF and SGTF.

The Role of Thyrotropin Receptor Activation in Adipogenesis and Modulation of Fat Phenotype.

Evidence from clinical and experimental data suggests that thyrotropin receptor (TSHR) signaling is involved in energy expenditure through its impact on white adipose tissue (WAT) and brown adipose tissue (BAT). TSHR expression increases during mesenchymal stem cell (MSC) differentiation into fat. We hypothesize that TSHR activation [TSHR*, elevated thyroid-stimulating hormone, thyroid-stimulating antibodies (TSAB), or activating mutation] influences MSC differentiation, which contributes to body composition changes seen in hypothyroidism or Graves' disease (GD). The role of TSHR activation on adipogenesis was first investigated using ex vivo samples. Neck fat (all euthyroid at surgery) was obtained from GD (n = 11, TSAB positive), toxic multinodular goiter (TMNG, TSAB negative) (n = 6), and control patients with benign euthyroid disease (n = 11, TSAB negative). The effect of TSHR activation was then analyzed using human primary abdominal subcutaneous preadipocytes (n = 16). Cells were cultured in complete medium (CM) or adipogenic medium [ADM, containing thiazolidinedione (TZD), PPARγ agonist, which is able to induce BAT formation] with or without TSHR activation (gain-of-function mutant) for 3 weeks. Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, qPCR measurement of terminal differentiation marker (LPL). BAT [PGC-1α, uncoupling protein 1 (UCP1), and ZIC1], pre-BAT (PRDM16), BRITE- (CITED1), or WAT (LEPTIN) markers were analyzed by semiquantitative PCR or qPCR. In ex vivo analysis, there were no differences in the expression of UCP1, PGC-1α, and ZIC1. BRITE marker CITED1 levels were highest in GD followed by TMNG and control (p for trend = 0.009). This was associated with higher WAT marker LEPTIN level in GD than the other two groups (p < 0.001). In primary cell culture, TSHR activation substantially enhanced adipogenesis with 1.4 ± 0.07 (ORO), 8.6 ± 1.8 (foci), and 5.5 ± 1.6 (LPL) fold increases compared with controls. Surprisingly, TSHR activation in CM also significantly increased pre-BAT marker PRDM16; furthermore, TZD-ADM induced adipogenesis showed substantially increased BAT markers, PGC-1α and UCP1. Our study revealed that TSHR activation plays an important role in the adipogenesis process and BRITE/pre-BAT formation, which leads to WAT or BAT phenotype. It may contribute to weight loss as heat during hyperthyroidism and later transforms into WAT posttreatment of GD when patients gain excess weight.

Cerebral salt wasting following traumatic brain injury.

SUMMARY: Hyponatraemia is the most commonly encountered electrolyte disturbance in neurological high dependency and intensive care units. Cerebral salt wasting (CSW) is the most elusive and challenging of the causes of hyponatraemia, and it is vital to distinguish it from the more familiar syndrome of inappropriate antidiuretic hormone (SIADH). Managing CSW requires correction of the intravascular volume depletion and hyponatraemia, as well as mitigation of on-going substantial sodium losses. Herein we describe a challenging case of CSW requiring large doses of hypertonic saline and the subsequent substantial benefit with the addition of fludrocortisone. LEARNING POINTS: The diagnosis of CSW requires a high index of suspicion. Distinguishing it from SIADH is essential to enable prompt treatment in order to prevent severe hyponatraemia.The hallmarks of substantial CSW are hyponatraemia, reduced volume status and inappropriately high renal sodium loss.Substantial volumes of hypertonic saline may be required for a prolonged period of time to correct volume and sodium deficits.Fludrocortisone has a role in the management of CSW. It likely reduces the doses of hypertonic saline required and can maintain serum sodium levels of hypertonic saline.

Maturation in Serum Thyroid Function Parameters Over Childhood and Puberty: Results of a Longitudinal Study.

Context: Serum thyroid hormone levels differ between children and adults, but have not been studied longitudinally through childhood. Objective: To assess changes in thyroid-stimulating hormone (TSH) and thyroid hormone levels over childhood and their interrelationships. Design: Cohort study. Setting: The Avon Longitudinal Study of Parents and Children, a population-based birth cohort. Participants: A total of 4442 children who had thyroid function measured at age 7, and 1263 children who had thyroid function measured at age 15. Eight hundred eighty-four children had measurements at both ages. Main Outcome Measures: Reference ranges for TSH, free tri-iodothyronine (FT3), free thyroxine (FT4), their longitudinal stability, and interrelationships. Results: Children at age 7 years had a higher FT3 [6.17 pmol/L, standard deviation (SD) 0.62] than children at age 15 (5.83 pmol/L, SD 0.74); P < 0.0001 with 23.2% of children at age 7 having FT3 above the adult reference range. Higher FT3 levels at age 7 in boys (P = 0.0001) and girls (P = 0.04) were associated with attainment of a more advanced pubertal stage at age 13. TSH was positively associated with FT3 at age 7 and age 15 even after adjusting for confounders. In contrast, TSH was negatively associated with FT4. Conclusions: There are substantial changes in TSH and thyroid hormone levels over childhood, in particular for FT3, which appear to relate to pubertal readiness. Our data provide increased insight into the evolution of the pituitary-thyroid axis over childhood and may have implications for determining optimal ranges for thyroid hormone replacement in children.