Danila Covelli

Rituximab treatment in a patient with severe thyroid-associated ophthalmopathy: Effects on orbital lymphocytic infiltrates

Rituximab (RTX) has been shown in previous work to improve thyroid-associated ophthalmopathy (TAO), but very little data is available on the effects of RTX in the target tissues. We studied the effects of RTX on peripheral lymphocytes and on the intra-orbital infiltrates in one patient with severe TAO who was treated with two cycles of therapy. Intra-orbital tissues derived at decompression from 3 patients with moderate-severe and 1 with severe TAO, treated with standard immunosuppression, were studied as controls. Peripheral blood lymphocytes were analyzed throughout the study period, while intra-orbital tissue lymphocytes at decompression. In the patient treated with RTX visual field and acuity improved in response to peripheral CD 20+ cell depletion, although there was a proportion of persisting CD 19+ cells. After RTX re-treatment the patients optic nerve function improved only transiently. The number of CD 20+ cells was lower in orbital tissues (0-1%) than in the peripheral blood (3%). A greater percentage of CD 19+ was observed in the orbits compared to the periphery, most of which were CD 19+5+ (80%). By immunohistochemistry, orbital tissues from all control patients showed CD 20+ and CD 3+ cells, independently of the duration of TAO and of the treatment with either steroids or radiotherapy. This is the first report on the therapeutic effect of RTX in active, severe TAO associated to the depletion of intra-orbital CD 20+ lymphocytes. After RTX, CD 19+5+ lymphocytes were shown to be 2-3 times more prevalent in the orbital infiltrates, compared to CD 20+ cells. Persistence of autoreactive cells is believed to be related to TAO relapse.

Small Dose of Rituximab for Graves Orbitopathy: New Insights Into the Mechanism of Action

citing role as increased levels of dopamine have been shown to cause choroidal vasodilation. The time course of effusion development in this case is similar to that with topiramate. This suggests that if bupropion use were causative, one of its major active metabolites, hydroxybupropion or theobupropion (both with half-lives similar to that of topiramate), may be responsible. Both metabolites inhibit dopamine and norepinephrine reuptake; thus, norepinephrine could also have a role. Bilateral effusions have been reported with venlafaxine hydrochloride, a norepinephrine and serotonin reuptake inhibitor. Bupropion is a common medication, and it is unclear why other cases have not been reported. If bupropion use were causative in this case, the absence of other similar reports may reflect underreporting; alternatively, this patient may harbor a rare, private polymorphism that causes bupropion or one of its metabolites to become a particularly potent choroidal vasodilator.

PREGO (presentation of Graves’ orbitopathy) study: changes in referral patterns to European Group On Graves’ Orbitopathy (EUGOGO) centres over the period from 2000 to 2012

Background/aims The epidemiology of Graves’ orbitopathy (GO) may be changing. The aim of the study was to identify trends in presentation of GO to tertiary centres and initial management over time. Methods Prospective observational study of European Group On Graves’ Orbitopathy (EUGOGO) centres. All new referrals with a diagnosis of GO over a 4-month period in 2012 were included. Clinical and demographic characteristics, referral timelines and initial decisions about management were recorded. The data were compared with a similar EUGOGO survey performed in 2000. Results The demographic characteristics of 269 patients studied in 2012 were similar to those collected in the year 2000, including smoking rates (40.0% vs 40.2%). Mild (60.5% vs 41.2%, p<0.01) and inactive GO (63.2% vs 39.9%, p<0.01) were more prevalent in 2012. The times from diagnosis of thyroid disease to being seen in EUGOGO centres (6 vs 16 months) and from first symptoms of GO (9 vs 16 months) or from diagnosis of GO (6 vs 12 months) to first consultation in EUGOGO centres were shorter in 2012 (p<0.01). The initial management plans for GO were no different except surgical treatments for patients with mild inactive disease were more frequently offered in the 2012 cohort than in 2000 (27.3% vs 17%, p<0.05), and selenium supplements were offered only in the 2012 cohort (21.2% vs 0%, p<0.01). Conclusions These findings suggest that the clinical manifestations of patients with GO may be changing over time in Europe.

Reference Range of Serum Calcitonin in Pediatric Population

BACKGROUND Children belonging to the multiple endocrine neoplasia type 2 (MEN 2) pedigree and carrying germline RET mutations are candidates for prophylactic thyroidectomy, the timing of which is based on the mutation-associated risk and the calcitonin (CT) levels. DESIGN The aim of this study was to establish the reference range for serum CT in a pediatric population. The study included 2740 subjects (1339 females and 1401 males) ranging in age from 1 day to 16 years and undergoing blood testing for any medical condition not affecting serum CT. RESULTS Overall, serum CT was undetectable in 61.5% of the samples and detectable in 38.5%. Detectable samples were more frequent in the first 2 years of life. Thereafter, undetectable samples became more frequent, particularly in females. Mean serum CT concentrations were higher in the first year of life (9.81 ± 8.8 pg/mL; range, 2.0-48.9 pg/mL) and the second year of life (4.56 ± 2.64 pg/mL; range, 2.0-14.7 pg/mL). A significant decrease of serum CT levels was observed thereafter (P < .001), and starting from the third year of life serum CT levels were similar to those found in adults. No gender difference was found in any age group. Based on these results, age-specific CT reference ranges are needed in the pediatric population, and especially in the first 2 years of life. CONCLUSIONS This is the first study defining the reference range for serum CT in the pediatric population and large enough to be statistically meaningful. Our proposal may facilitate the process of decision making when dealing with gene carriers of MEN 2.

The thyroid, the eyes and the gut: a possible connection.

IntroductionGraves’ disease (GD) is an autoimmune disorder responsible for 60–90% of thyrotoxicosis, with an incidence of 1 to 2 cases per 1000 population per year in England. Graves’ orbitopathy (GO) is the most frequent extrathyroidal manifestation, not provoked directly by abnormal thyroid hormone levels, but by the consequence of the underlying autoimmune process. The aetiology of autoimmune disorders is due to an interplay between susceptibility genes and environmental factors, such as infections and stress. What triggers the autoimmune reaction to a specific site of the body is not yet clearly understood. The lack of knowledge in GD and GO pathogenesis implicates therapies that only limit damage but do not prevent disease onset.Material and methodsWe performed on PubMed and the Cochrane Library a literature search for the articles published until July 2016 by using the search terms ‘graves disease’ and ‘microbiome’, ‘orbitopathy’ and ‘autoimmune pathogenesis’. Reference lists of relevant studies were hand-searched for additional studies.ConclusionIn this scenario, a Marie Sklodowska–Curie funded project INDIGO (http://www.indigo-iapp.eu/) is investigating the role of the gut bacteria in GD and GO pathogenesis. The gut is the first and the widest area of bacteria access, with the highest concentration of T cells in the human body and trained to react to microorganisms. Interestingly, all the environmental factors involved in GD and GO pathogenesis can alter the balance within the microorganisms located in the gut, and influence the immune system, in particular the proportions of regulatory Treg and inflammatory TH17 cells. It is hoped that investigating GD and GO pathogenesis from this novel aspect will identify new targets for prevention and treatment.

Diabetes mellitus in a girl with thyroid hormone resistance syndrome: a little recognized interaction between the two diseases

The syndrome of resistance to thyroid hormone (RTH) is characterized by elevated serum free thyroid hormones (FT4 and FT3) in the presence of unsuppressed TSH levels, reflecting resistance to the normal negative feedback mechanisms in the hypothalamus and pituitary. The degree of resistance within peripheral tissues determines whether thyrotoxic clinical features are associated with this condition. Classic features include attention deficit hyperactivity disorder, growth delay, tachycardia, and goiter. However, other features, such as frequent ear, nose and throat infections, hearing deficit, and decreased bone mass have recently been recognized. The phenotype of RTH is variable, with most patients presenting with mild to moderate symptoms. In this report we describe a girl with familiar RTH and diabetes mellitus. This is, to our knowledge, the first report regarding this association. Nearly one year after long-term triiodothyroacetic acid (Triac) therapy, we observed a reduction of thyroid hormone levels with an amelioration of insulin resistance. The possible interactions between these disorders are discussed.

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.

Gut microbiota in experimental murine model of Graves' orbitopathy established in different environments may modulate clinical presentation of disease.

BackgroundVariation in induced models of autoimmunity has been attributed to the housing environment and its effect on the gut microbiota. In Graves’ disease (GD), autoantibodies to the thyrotropin receptor (TSHR) cause autoimmune hyperthyroidism. Many GD patients develop Graves’ orbitopathy or ophthalmopathy (GO) characterized by orbital tissue remodeling including adipogenesis. Murine models of GD/GO would help delineate pathogenetic mechanisms, and although several have been reported, most lack reproducibility. A model comprising immunization of female BALBc mice with a TSHR expression plasmid using in vivo electroporation was reproduced in two independent laboratories. Similar orbital disease was induced in both centers, but differences were apparent (e.g., hyperthyroidism in Center 1 but not Center 2). We hypothesized a role for the gut microbiota influencing the outcome and reproducibility of induced GO.ResultsWe combined metataxonomics (16S rRNA gene sequencing) and traditional microbial culture of the intestinal contents from the GO murine model, to analyze the gut microbiota in the two centers. We observed significant differences in alpha and beta diversity and in the taxonomic profiles, e.g., operational taxonomic units (OTUs) from the genus Lactobacillus were more abundant in Center 2, and Bacteroides and Bifidobacterium counts were more abundant in Center 1 where we also observed a negative correlation between the OTUs of the genus Intestinimonas and TSHR autoantibodies. Traditional microbiology largely confirmed the metataxonomics data and indicated significantly higher yeast counts in Center 1 TSHR-immunized mice. We also compared the gut microbiota between immunization groups within Center 2, comprising the TSHR- or βgal control-immunized mice and naïve untreated mice. We observed a shift of the TSHR-immunized mice bacterial communities described by the beta diversity weighted Unifrac. Furthermore, we observed a significant positive correlation between the presence of Firmicutes and orbital-adipogenesis specifically in TSHR-immunized mice.ConclusionsThe significant differences observed in microbiota composition from BALBc mice undergoing the same immunization protocol in comparable specific-pathogen-free (SPF) units in different centers support a role for the gut microbiota in modulating the induced response. The gut microbiota might also contribute to the heterogeneity of induced response since we report potential disease-associated microbial taxonomies and correlation with ocular disease.

Diabetes mellitus in a girl with thyroid hormone resistance syndrome: a unlooked feature for this condition?

The syndrome of resistance to thyroid hormone(RTH) is characterized by elevated serum free thyroid hormones (FT4and FT3) in the presence of unsuppressed TSH levels, reflecting resistanceto the normal negative feedback mechanisms within the hypothalamus andpituitary. The degree of resistance within peripheral tissues determineswhether thyrotoxic clinical features are associated with this condition.Classic features include attention deficithyperactivity disorder, growth delay, tachycardia, and goiter. However, otherfeatures, such as frequent ear nose and throat infections, hearing deficit, anddecreased bone mass have recently been recognized. The phenotype of RTH isvariable, with most patients presenting with mild to moderate symptoms.In this report we describe a girlwith familiar RTH and diabetes mellitus. This is, to our acknowledge, the firstreport regarding this association. Near 1 year after long-term triiodothyroaceticacid (Triac) therapy, we evidence a reduction of thyroid hormone levels, with anamelioration of insulin resistance. The possible interactions between these disordersare discussed.

Impact of Resistance to Thyroid Hormone on the Cardiovascular System in Adults

Background: The clinical manifestations of resistance to thyroid hormone (RTH) are highly variable, and the impact of RTH on the cardiovascular system has been poorly investigated. Aim: The objective of the study was to evaluate the cardiovascular characteristics of 16 untreated and asymptomatic patients with RTH compared with 16 euthyroid healthy controls to define the cardiovascular involvement in RTH syndrome. Patients and Methods: Sixteen untreated and asymptomatic RTH patients (eight males; aged 33 12 yr, range 21–45 yr) and 16 controls (nine males; aged 33 5 yr, range 24–42 yr) were enrolled. Clinical data, thyroid status, and echocardiographic results were recorded. Results: Heart rate was comparable with that of controls, whereas arterial pressure was higher than controls. Mean interventricular septum diastolic thickness and mean left ventricular (LV) posterior wall diastolic thickness were significantly lower in RTH patients than controls with a consequent significant decrease of the mean LV mass and LV mass indexed by body surface area. Patients also had abnormalities of myocardial relaxation as indicated by a significant increase of peak A and consequent reduction of the early to late ratio. Finally, systemic vascular resistance was significantly higher in RTH patients than controls. Conclusions: Our results suggest the presence of cardiovascular alterations in asymptomatic and untreated RTH patients similar to those reported in hypothyroid patients. Our strict selection likely created a bias in the inclusion of a particular type of RTH patients, who could represent a minority of patients with RTH. However, no correlation was found between the type of mutation and cardiovascular characteristics of RTH patients. Nerve Growth Factor Induces Vascular Endothelial Growth Factor Expression in Granulosa Cells via a trkA Receptor/Mitogen-Activated Protein Kinase-Extracellularly Regulated Kinase 2-Dependent Pathway Marcela Julio-Pieper, Patricia Lozada, Veronica Tapia, Margarita Vega, Cristian Miranda, David Vantman, Sergio R. Ojeda, and Carmen Romero (J Clin Endocrinol Metab, published May 19, 2009, 10.1210/jc.2009-0542) ABSTRACT Context: Acquisition of ovulatory competence by antral follicles requires development of an adequate vascular supply. Although it iswellestablishedthatovarianangiogenesis is cyclically regulatedbyvascularendothelialgrowthfactor (VEGF), thefactorscontrolling VEGF production by ovarian follicles remain largely unknown. Nerve growth factor (NGF) may be one of these factors, because NGF promotes angiogenesis and synthesis of angiogenic factors in other tissues and is produced by human granulosa cells (hGCs). Objective: The aim of the study was to determine whether NGF influences the production of VEGF by hGCs and to identify a potential signaling pathway underlying this effect. Design: We conducted a prospective experimental study. Patients: hGCs were obtained from 41 women participating in the in vitro fertilization program of our institution. Methods: Changes in VEGF mRNA after exposure to NGF were evaluated in cultured hGCs by PCR and real-time PCR. The effect of NGF on VEGF secretion was determined by ELISA. The involvement of trkA, the high affinity NGF receptor, was examined by inhibiting the receptor’s tyrosine kinase activity with K252a. The contribution of an ERK1/ERK2-mediated signaling pathway was identified by detecting NGF-dependent phosphorylation of these proteins and by blocking their activity with the inhibitor U0126. Results: NGF promotes VEGF production in cultured hGCs. Blockade of trkA receptor tyrosine kinase activity blocks this effect. NGF induces MAPK-ERK2 phosphorylation, and blockade of this signaling pathway prevents the NGF-induced increase in VEGF production. Conclusions: NGF promotes ovarian angiogenesis by enhancing the synthesis and secretion of VEGF from hGCs via a trkAand ERK2-dependent mechanism. Endocrine Reviews, August 2009, 30(5):536–543 edrv.endojournals.org 541Context: Acquisition of ovulatory competence by antral follicles requires development of an adequate vascular supply. Although it iswellestablishedthatovarianangiogenesis is cyclically regulatedbyvascularendothelialgrowthfactor (VEGF), thefactorscontrolling VEGF production by ovarian follicles remain largely unknown. Nerve growth factor (NGF) may be one of these factors, because NGF promotes angiogenesis and synthesis of angiogenic factors in other tissues and is produced by human granulosa cells (hGCs). Objective: The aim of the study was to determine whether NGF influences the production of VEGF by hGCs and to identify a potential signaling pathway underlying this effect. Design: We conducted a prospective experimental study. Patients: hGCs were obtained from 41 women participating in the in vitro fertilization program of our institution. Methods: Changes in VEGF mRNA after exposure to NGF were evaluated in cultured hGCs by PCR and real-time PCR. The effect of NGF on VEGF secretion was determined by ELISA. The involvement of trkA, the high affinity NGF receptor, was examined by inhibiting the receptor’s tyrosine kinase activity with K252a. The contribution of an ERK1/ERK2-mediated signaling pathway was identified by detecting NGF-dependent phosphorylation of these proteins and by blocking their activity with the inhibitor U0126. Results: NGF promotes VEGF production in cultured hGCs. Blockade of trkA receptor tyrosine kinase activity blocks this effect. NGF induces MAPK-ERK2 phosphorylation, and blockade of this signaling pathway prevents the NGF-induced increase in VEGF production. Conclusions: NGF promotes ovarian angiogenesis by enhancing the synthesis and secretion of VEGF from hGCs via a trkAand ERK2-dependent mechanism. Endocrine Reviews, August 2009, 30(5):536–543 edrv.endojournals.org 541