I. Ionni

Action of three bioavailable antioxidants in orbital fibroblasts from patients with Graves? orbitopathy (GO): a new frontier for GO treatment?

ObjectiveOxidative stress is involved in the pathogenesis of Graves’ orbitopathy (GO) and an antioxidant approach has been advocated for GO treatment. Here, we investigated the action of three antioxidants in orbital fibroblasts, namely, vitamin C, N-acetyl-l-cysteine, and melatonin.MethodsPrimary cultures of orbital fibroblasts from six GO patients and six control subjects were established. Cells were treated with H2O2 to induce oxidative stress. Cell vitality assays were performed to determine the non-cytotoxic dose of each antioxidant. The following assays were performed: glutathione disulfide (GSSG), as a measure of oxidative stress, cell proliferation, hyaluronic acid (HA), TNFα, IFNγ, and IL1β.ResultsH2O2 induced oxidative stress (augmented GSSG), increased cell proliferation as well as cytokine release, but did not affect HA release. All of the three antioxidant substances reduced H2O2-dependent oxidative stress. Vitamin C reduced proliferation in GO, but not in control fibroblasts. N-acetyl-l-cysteine reduced proliferation and IFNγ in GO, and HA and IL1β in both GO and control fibroblasts. Melatonin reduced IL1β and HA in GO and control fibroblasts, and IFNγ only in GO fibroblasts.ConclusionsOur study provides evidence in support of an antioxidant role of vitamin C, N-acetyl-l-cysteine and melatonin in orbital fibroblasts. Some of the effects of these compounds are exclusive to GO fibroblasts, whereas some other are observed also in control fibroblasts. Our observations provide a basis for a possible clinical use of these substances in patients with GO.

VARIABLES AFFECTING THE LONG-TERM OUTCOME OF GRAVES ORBITOPATHY FOLLOWING HIGH-DOSE INTRAVENOUS GLUCOCORTICOID PULSE THERAPY IN PATIENTS NOT TREATED WITH ORBITAL RADIOTHERAPY.

OBJECTIVE Intravenous (iv) glucocorticoids (GC) (ivGC) are used for active Graves orbitopathy (GO), but factors affecting GO outcome are poorly understood. We performed a retrospective study to investigate the variables affecting GO after ivGC. METHODS We evaluated 83 consecutive GO patients treated with ivGC but not orbital radiotherapy (ORT) and re-examined them after a median of 47 months. The endpoints were the relationships between GO outcome or additional treatments with age, sex, smoking habits, thyroid volume, thyroid treatment, time since thyroid treatment, antithyroid-stimulating hormone receptor antibodies (TRAb), GO duration, GO features, and follow-up time. RESULTS GO features improved after treatment, resulting in moderate and marked amelioration in ~75% and ~41% of patients respectively. By multivariate analysis, a moderate GO improvement correlated with diplopia at first observation, which was more severe in responders. A marked GO improvement correlated with time between first and last observation and time after thyroid treatment, which were longer in responders. This likely reflected the combination of an early effect of GC and a late, spontaneous improvement of GO, as shown by analyses of GO outcome at various time points. Additional treatments after ivGC correlated by multivariate analysis with eyelid aperture, diplopia and NOSPECS score (NOSPECS stands for no GO signs [N], only eyelid sign [O], soft tissue involvement [S], proptosis [P], extraocular motility restriction [E], corneal involvement [C], and sight loss [S]) at first observation, which were more severe in responders. CONCLUSION Our study shows that response to ivGC increases with time, likely reflecting the known tendency of GO to improve spontaneously, and is more pronounced when GO is more severe to begin with, which is associated with more additional treatments. ABBREVIATIONS ANOVA = analysis of variance CAS = clinical activity score GC = glucocorticoids GO = Graves orbitopathy 131I = radioactive iodine iv = intravenous ivGC = high-dose intravenous glucocorticoid pulse therapy MMI = methimazole OD = orbital decompression ORT = orbital radiotherapy TRAb = antithyroid-stimulating hormone receptor antibodies.

Association of T and B Cells Infiltrating Orbital Tissues With Clinical Features of Graves Orbitopathy

Importance Graves orbitopathy (GO) responds to immunosuppressive treatments when clinically active but poorly when inactive. In other autoimmune diseases, response has been ascribed to a reduction in lymphocytes infiltrating the target organ. It is not known whether active vs inactive GO differs in this regard, which would help in understanding the link between GO immunologic features and clinical behavior. Objective To investigate the association between orbital lymphocytic infiltrate and GO clinical features. Design, Setting, and Participants A cohort study aimed at assessing the extent and immunohistochemical phenotype of orbital lymphocytes and associating it with the ophthalmologic features of GO, especially its clinical activity score (CAS), was conducted at a tertiary referral center. Twenty consecutive patients with GO who underwent orbital decompression were included. The study was conducted from January 1 to May 31, 2017. Exposures Orbital tissue histology and immunohistochemistry testing as well as ophthalmologic evaluation. Main Outcomes and Measures Association between CAS and orbital lymphocytes, analyzed as total number of lymphocytes and main lymphoid subsets. Results The patient population included 8 men and 12 women, all of white race, with a mean (SD) age of 46 (13) years. With an established cutoff value of 300 lymphoid cells per tissue sample, lymphocytes above this value were found in orbital tissues of 9 of 20 patients (45%), often organized into distinct foci. The lymphocytes comprised a mixture of T (CD3-positive) and B (CD20-positive) cells, suggesting a mature, polyclonal autoimmune response. In a simple linear regression model, the total number of lymphocytes, as well as the number of CD3- and CD20-positive subsets, correlated with CAS (R = 0.63; 95% CI, 0.27-0.84; P = .003; R = 0.59; 95% CI, 0.20-0.82; P = .006; and R = 0.65; 95% CI, 0.30-0.85; P = .002, respectively). In a multiple linear regression model, lymphocytes maintained their effect on CAS when adjusted for 2 additional variables that were correlated with CAS—smoking and GO duration—highlighting even more the important role of orbital lymphocytes in affecting CAS (total number: R = 0.58; 95% CI, 0.18-0.82; P = .01; CD3-positive: R = 0.58; 95% CI, 0.17-0.82; P = .01; and CD20-positive: R = 0.59; 95% CI, 0.19-0.83; P = .01). Conclusions and Relevance This study shows a correlation between T and B lymphocytes infiltrating orbital tissues and the activity of GO, possibly enhancing our understanding of the association between GO immunologic features and clinical expression.

Antioxidant effects of β-carotene, but not of retinol and vitamin E, in orbital fibroblasts from patients with Graves’ orbitopathy (GO)

BackgroundOxidative stress is involved in the pathogenesis of Graves’ orbitopathy (GO) and several antioxidant agents, namely, selenium, quercetin, enalapril, vitamin C, N-acetyl-l-cysteine, and melatonin, have been shown to reduce oxidative stress and its consequences in primary culture of orbital fibroblasts. In addition, selenium is effective for the treatment of mild GO. Here, we investigated the action of three additional antioxidants in orbital fibroblasts, namely, retinol, β-carotene, and vitamin E.MethodsPrimary cultures of orbital fibroblasts were established from GO patients and control subjects. To induce oxidative stress, cells were treated with H2O2, after which glutathione disulfide (GSSG) (a parameter of oxidative stress), cell proliferation, hyaluronic acid, TNFα, IFNγ, and IL1β were measured.ResultsH2O2-dependent oxidative stress (augmented GSSG) was associated with increased cell proliferation and cytokine release. All the three antioxidant substances reduced GSSG in both GO and control fibroblasts. β-carotene reduced proliferation in GO, but not in control fibroblasts. IL1β was reduced by all three substances. Retinol reduced IFNγ in GO and control fibroblasts.ConclusionsOur study supports an antioxidant role of retinol, β-carotene, and vitamin E in orbital fibroblasts from patients with GO and provides a basis for a possible clinical use these substances.

Peripheral T and B lymphocytes do not correlate with Graves’ orbitopathy

It is somehow recognized that response of Graves’ orbitopathy (GO) to immunosuppressive treatments parallels its activity [1]. In other autoimmune conditions, such as type 1 diabetes, response has been ascribed to a reduction in lymphocytes infiltrating the target organ [2]. In a recent study we assessed the relationship between the immunohistochemical phenotype of orbital lymphocytes and the ophthalmological features of GO [3]. We found that orbital infiltrating lymphocytes, which were present in ~ 50% of patients and were often organized into distinct foci, comprised a mixture of T (CD3-positive) and B (CD20-positive) cells, suggesting a mature, polyclonal autoimmune response. The total number of infiltrating lymphocytes, as well as the number of CD3and CD20-positive cells correlated with the GO clinical activity score (CAS), both in single and multiple regression models, thereby suggesting a correlation between lymphocytes infiltrating orbital tissues and the activity of GO, and possibly enhancing our understanding of the relationship between GO immunological features and clinical expression [3]. Based on the above mentioned observations, we considered the possibility that not only orbital infiltrating lymphocytes correlate with CAS, and that also peripheral, circulating lymphocytes do so. To investigate this possibility, we conducted a cross-sectional investigation in consecutive patients with Graves’ disease (GD), the results of which we briefly report here. Upon signed informed consent, we studied 81 consecutive subjects with GD (15 men and 66 women, aged 46.9 ± 12.8 years), of whom 48 had a clinically evident GO and 33 had no GO. The total number of circulating lymphocytes was measured in all patients, as well as the number of CD20-positive lymphocytes, the latter assessed by FACS analysis. The primary objective of the study was the relationship between peripheral lymphocytes and CAS within GO patients. The secondary objectives were the relationships between peripheral lymphocytes and: (1) GO severity; (2) presence/absence of GO in GD patients; and (3) anti-thyroid autoantibodies, namely anti-thyroglobulin, antithyroperoxidase, and anti-TSH receptor. In contrast with our hypothesis, the total number of peripheral lymphocytes as well as the number of CD-20-positive lymphocytes did not correlate with GO activity and severity, both by univariate and multivariate analyses, nor with the presence/absence of GO in GD patients, and nor with the levels and prevalence of serum anti-thyroid autoantibodies. Based on our findings, we conclude that although orbital infiltrating lymphocytes seem to play a pathogenetic role in GO and are correlated with GO activity, peripheral lymphocyte have a marginal role, as suggested by the absence of a relationship with the presence and the clinical features of GO, even though they likely comprise the cell subsets that are responsible for orbital infiltration and that likely triggers the pathologic changes of the orbit and ultimately the clinical features of GO.

Occurrence of Graves’ Orbitopathy and Graves’ Hyperthyroidism after a Trauma to the Eye

Background: Graves’ orbitopathy (GO) is believed to be the consequence of autoimmunity against antigens that are present both in the thyroid and orbital tissues. Massive release of thyroid antigens causes the appearance or deterioration of GO in patients with Graves’ hyperthyroidism (GH), as it occurs following radioiodine treatment. In theory, a similar release of autoantigens may occur at the eye level, for example due to an orbital trauma or surgical manipulation. To our knowledge, this is the first report of a case of de novo appearance of GO and then GH after an eye trauma, possibly reflecting spreading of autoantigens and activation of the immune system against shared orbital and thyroid antigens. Case Report: An otherwise healthy, 57-year-old man presented 6 months after the appearance of a monolateral right orbitopathy, which occurred 40 days after a trauma in the ipsilateral eye. His thyroid function was normal, with positive serum anti-TSH receptor autoantibodies. The thyroid was normal on ultrasound. A month later he developed hyperthyroidism and orbitopathy in the left eye. Discussion: The development of GO after an eye trauma may reflect tissue damage with release of autoantigens and consequent autoimmunity in a predisposed individual (our patient had a familial history of autoimmune thyroid disease). The subsequent development of hyperthyroidism is in keeping with the hypothesis that GH and GO are due to autoimmunity against antigens present both in the thyroid and in orbital tissues.