Francesca Coperchini

Interferon-γ and tumor necrosis factor-α sustain secretion of specific CXC chemokines in human thyrocytes: a first step toward a differentiation between autoimmune and tumor-related inflammation?

CONTEXT Chemokines are chemotactic cytokines responsible for the attraction and recruitment of different cell types during leukocyte infiltration, the histopathological hallmark of autoimmunity. Previous data demonstrate that thyrocytes secrete CXC chemokines, particularly CXCL8 and CXCL10. However, the physiopathological significance of such secretion and the effects of a combination of proinflammatory stimuli in terms of preferential CXCL8 and CXCL10 release remain unclear. OBJECTIVE The aim of this study was to investigate whether the secretion of chemokines by human thyrocytes is a generalized inflammatory response or whether it is dependent upon specific proinflammatory stimuli. METHODS CXCL8 and CXCL10 were measured in supernatants of human thyrocytes in primary cultures basally and after 24 h stimulation with interferon-γ (IFNγ) (1000 U/ml) and TNFα (10 ng/ml), alone or in combination. RESULTS CXCL8 but not CXCL10 was detected in basal conditions. The two chemokines showed differences in their response to proinflammatory cytokines. Indeed, significant secretion of CXCL10 was induced by IFNγ (P < 0.01) and not TNFα, whereas CXCL8 was secreted in response to TNFα (P < 0.01) being inhibited by IFNγ (P < 0.01). The combination of TNFα plus IFNγ synergistically increased the IFNγ-induced CXCL10 secretion (P < 0.01) and reversed the TNFα-induced CXCL8 secretion (P < 0.01). CONCLUSIONS These results confirm that human thyrocytes secrete CXC chemokines and demonstrate that the secretion of CXCL8 and CXCL10 is sustained by specific proinflammatory cytokines or their combination, which ultimately determines the nature of the infiltrating lymphocytes in human thyroid diseases. These results indirectly support a major role for CXCL10 in thyroid autoimmunity whereas CXCL8 might be involved in tumor-related inflammation.

Expanding the therapeutic spectrum of metformin: From diabetes to cancer

IntroductionMetformin, an oral hypoglycemic agent, was introduced in the clinical practice for the treatment of type 2 diabetes mellitus more than a half-century ago. Over the years, several studies demonstrated that diabetic patients treated with metformin have a lower incidence of cancer, raising the hypothesis that the spectrum of clinical applications of the drug could be expanded also to cancer therapy. Following these initial findings, a large number of studies were performed aimed at elucidating the effects of metformin on different types of tumor, at explaining its direct and indirect anti-cancer mechanisms and at identifying the molecular pathways targeted by the drug. Several clinical trials were also performed aimed at evaluating the potential anti-cancer effect of metformin among diabetic and non-diabetic patients affected by different types of cancer. While the results of several clinical studies are encouraging, a considerable number of other investigations do not support a role of metformin as an anti-cancer agent, and highlight variables possibly accounting for discrepancies.AimWe hereby review the results of in vitro and in vivo studies addressing the issue of the anti-cancer effects of metformin.ConclusionsIf in vitro data appear solid, the results provided by in vivo studies are somehow controversial. In this view, larger studies are needed to fully elucidate the role of metformin on cancer development and progression, as well as the specific clinical settings in which metformin could become an anti-cancer drug.

CXCL8 in thyroid disease: From basic notions to potential applications in clinical practice

CXCL8 was the first chemokine shown to be secreted by thyrocytes. Experimental data suggest that CXCL8 plays a role in thyroid homeostasis but its role in thyroid diseases remains poorly investigated. Clinical studies measuring the serum levels of CXCL8 in patients with autoimmune-thyroid-diseases reported conflicting results. Solid evidences support a role of CXCL8 as a tumor-promoting agent in several human cancers. Studies in thyroid cancer are still in their initial stage, but promising. Several evidences indicate that thyroid cancer may share with other human malignancies some of the effects of CXCL8 and highlight the possibility of using CXCL8 as a marker of aggressiveness. Basic and clinical evidences in favor or against a role for CXCL8 in thyroid diseases are discussed.

Severe disability in patients with relapsing-remitting multiple sclerosis is associated with profound changes in the regulation of leptin secretion.

Objectives: Experimental evidences indicate that leptin is involved in the neuroinflammatory process sustaining multiple sclerosis (MS). However, the relationship between leptin and body fat, as assessed by body mass index (BMI), in MS was not previously evaluated. It was the aim of this study to compare serum leptin levels between patients with MS and healthy controls and to evaluate the possible relationship between circulating leptin levels and disease severity. Patients and Methods: Eighty-four MS patients and 57 sex-matched healthy volunteers were enrolled. Serum leptin levels were measured in all patients and controls. MS patients were stratified in 3 groups according to their degree of disability as assessed by the Expanded Disability Status Scale (EDSS). Patients were classified as having low (33 patients with an EDSS score <1.5), intermediate (28 patients with an EDSS score from 2 to 3) and high disability (23 patients with an EDSS score ≥3.5). Results: No significant differences in serum leptin levels and BMI were observed between patients and controls. In patients with MS, serum leptin levels were significantly correlated with BMI in those patients with low (R2 = 0.363; p < 0.001) and intermediate disability (R2 = 0.408; p < 0.001), but not in patients with a higher disability score (R2 = 0.064; p = 0.256). Conclusion: BMI, the major determinant of leptin level in physiological conditions, has a minor role in determining the serum levels of leptin in MS patients with a high EDSS score. Future longitudinal studies will be required in order to provide further insights into the regulation of leptin secretion in patients with MS.

Metformin reverts the secretion of CXCL8 induced by TNF-α in primary cultures of human thyroid cells: An additional indirect anti-tumor effect of the drug

CONTEXT Metformin displays both direct and indirect anti-tumor effects. CXCL8 is a crucial downstream mediator of Nuclear-Factor-κB signaling related to the growth and progression of thyroid cancers. Targeting CXCL8 results in prolonged survival and reduced metastatic spread in in-vivo animal models of thyroid tumors. OBJECTIVE This study aimed to evaluate whether metformin inhibits the secretion of CXCL8 induced by Tumor-Necrosis-Factor-α (TNF-α) in primary cultures of normal and tumor human thyroid cells as well as in thyroid cancer cell lines. METHODS Normal human thyrocytes, papillary thyroid cancer cells, and thyroid cancer cell lines (TPC-1 and BCPAP) were stimulated with TNF-α (10 ng/mL) alone or in combination with metformin (0.01, 0.1, 1, 2.5, 5, and 10mM). CXCL8 levels were measured in the cell supernatants after 24 hours. RESULTS Metformin significantly and dose-dependently inhibited the TNF-α-induced CXCL8 secretion in both normal thyrocytes (ANOVA: F = 42.04; P < .0001) and papillary thyroid cancer cells (ANOVA: F = 21.691; P < .0001) but not in TPC-1 and BCPAP cell lines. CONCLUSION Metformin inhibits the TNF-α-induced CXCL8 secretion in primary cultures of normal thyroid cells and differentiated thyroid cancer cells at least of the most frequent poorly aggressive phenotype. The recruitment of neutrophils within the thyroid gland is a crucial metastasis-promoting factor, and it depends on the amount of CXCL8 produced by both tumor cells and by the more abundant normal thyroid cells exposed to TNF-α. Thus, the here-reported inhibiting effect of metformin on TNF-α-induced CXCL8 secretion could be considered as a further indirect anticancer property of the drug.

Interferon-β but not Glatiramer acetate stimulates CXCL10 secretion in primary cultures of thyrocytes: A clue for understanding the different risks of thyroid dysfunctions in patients with multiple sclerosis treated with either of the two drugs

Autoimmune thyroid disease (AITD) has been reported in patients with multiple sclerosis (MS) receiving interferon-beta (IFN-β), but not in those receiving Glatiramer acetate (GA). CXCL10 is a chemokine playing a pathogenetic role in AITD and MS. Our aim was to evaluate the effects on CXCL10 secretion of IFN-β and GA, alone and in combination with TNF-α, in primary cultures of thyrocytes (PCT). Significant and dose-dependent secretions of CXCL10 were induced by IFN-β but not GA. TNF-α synergistically increased IFN-β induced CXCL10 secretion. These results may provide an explanation for the occurrence of AITD during IFN-β, but not during GA, treatment for MS.

Serum negative autoimmune thyroiditis displays a milder clinical picture compared with classic Hashimoto's thyroiditis

BACKGROUND Despite high sensitivity of current assays for autoantibodies to thyroperoxidase (TPO) and to thyroglobulin (Tg), some hypothyroid patients still present with negative tests for circulating anti-thyroid Abs. These patients usually referred to as having seronegative autoimmune thyroiditis (seronegative CAT) have not been characterized, and definite proof that their clinical phenotype is similar to that of patients with classic chronic autoimmune thyroiditis (CAT) is lacking. OBJECTIVE To compare the clinical phenotype of seronegative CAT (SN-CAT) and CAT as diagnosed according to a raised serum level of TSH with negative and positive tests for anti-thyroid Abs respectively. METHODS A case-control retrospective study enrolling 55 patients with SN-CAT and 110 patients with CAT was performed. Serum free triiodothyronine (FT3), free thyroxine (FT4), TSH, Tg Abs, and TPO Abs were measured in all patients. RESULTS Patients with SN-CAT displayed significantly lower mean levels of TSH (6.6±3.4 vs 10.2±9.8 μU/ml; P=0.009), higher mean FT4 levels (1.1±0.2 vs 0.9±0.2 ng/dl; P=0.0002), and similar FT3 levels when compared with CAT patients. Mean thyroid volume was significantly greater in patients with CAT when compared with SN-CAT patients (11.2±6.5 vs 8.1±3.7 ml; P=0.001). Logistic regression demonstrated that FT4 (0.123 (0.019-0.775); (P=0.026)) and thyroid volume (1.243 (1.108-1.394); (P=0.0002)) were significantly and independently related to the diagnosis (CAT/SN-CAT). Patients with SN-CAT had a similar prevalence of thyroid nodules and female gender but a lower prevalence of overt hypothyroidism (5.4 vs 20.9%; P=0.012) as opposed to patients with CAT. CONCLUSIONS These results suggest an autoimmune etiology of SN-CAT, which, however, seems to have a milder clinical course when compared with CAT.

High circulating levels of CCL2 in patients with Klinefelter's syndrome

We would like to thank Dhindsa and colleagues for their interest and comments on our recent review article published in Clinical Endocrinology entitled ‘The Role of Obesity and Type 2 Diabetes Mellitus in the development of Male Obesity-associated Secondary Hypogonadism’. We would also like to thank the Editor for the opportunity to respond. We would like to confirm that we agree with the useful comments raised and provide further comment below. In our published review, we commented that the mechanisms implicated in the pathogenesis of secondary hypogonadism in men and its association with obesity and type 2 diabetes mellitus are multiple, complex and incompletely understood. We also stated that one of the pathogenic mechanisms implicated in the development of male obesity-associated secondary hypogonadism is increased aromatase activity within adipocytes. This results in increased peripheral conversion of testosterone into oestradiol and subsequent negative feedback on secretion of luteinizing hormone secretion from the pituitary. The suppressive effect of such a mechanism on the male hypothalamo–pituitary–gonadal axis results in a reduction in plasma testosterone levels and secondary hypogonadism. Consistent with this hypothesis, it has been reported in the literature that obese men show increased levels of oestrogens and decreased levels of bioavailable androgens within the serum. It has also been noted that use of aromatase inhibitors in men with obesity-related hypogonadism may normalize serum testosterone, again consistent with an important pathogenic role for aromatization in this condition. However, we acknowledge that there is controversy in the literature regarding the role of aromatization in the pathogenesis of male obesity-associated secondary hypogonadism. There is some inconsistency in the literature regarding relationships between serum levels of testosterone and oestradiol and the severity of obesity in this condition. We acknowledge that in some studies on obese men, serum levels of free oestradiol directly correlate with free testosterone. One explanation for this direct correlation is that with increasing obesity in men, as serum levels of testosterone fall (following suppression of the male gonadal axis), serum oestradiol levels would also be expected to fall eventually due to lack of substrate (testosterone) for the aromatase enzyme. This hypothesis has been supported by the European Male Ageing Study. We feel that it is important to emphasize that there are many potential mechanisms that underlie the complex pathogenesis of male obesity-associated secondary hypogonadism other than enhanced aromatase activity, as outlined in our published review article. These include the increasingly important roles of leptin, inflammatory mediators (TNF-a, IL-1b, CRP), the role of sleep disruption and the serotoninergic system and endogenous kisspeptin. We also outline the effects of insulin resistance at various levels including lipases, suppression of hepatic SHBG synthesis and the suppression of the hypothalamo–pituitary unit as potentially important pathogenic mechanisms. There may also be other, as yet unknown mechanisms at play. Clearly, there is a need for further focused studies in this field to develop a clear understanding of pathogenesis and to inform future novel treatment strategies.

Type I and type II interferons inhibit both basal and tumor necrosis factor-α-induced CXCL8 secretion in primary cultures of human thyrocytes.

Interferons (IFNs) and tumor necrosis factor-α (TNF-α) cooperate in activating several inflammation-related genes, which sustain chronic inflammation in autoimmune thyroid disease (AITD). Much is known about the positive signaling of IFNs to activate gene expression in AITD, while the mechanisms by which IFNs negatively regulate genes remain less studied. While IFNs inhibit CXCL8 secretion in several human cell types, their effects on thyroid cells were not evaluated. Our aim was to study the interplay between TNF-α and type I or type II IFNs on CXCL8 secretion by human thyroid cells. CXCL8 was measured in supernatants of primary cultures of thyroid cells basally and after a 24-h incubation with TNF-α. CXCL8 was detected in thyroid cell supernatants in basal conditions (96.2±23.5 pg/mL) being significantly increased (784.7±217.3 pg/mL; P<0.0001 vs. basal) by TNF-α. Twenty-four hour incubation with IFN-γ or IFN-β or IFN-α dose dependently and significantly inhibited both basal and TNF-α-induced CXCL8 secretion. The degree of the inhibitory effect was IFN-γ>IFN-β>IFN-α. This study demonstrates that type I and type II IFNs downregulate both basal and TNF-α-induced CXCL8 secretion by human thyrocytes, IFN-γ being the most powerful inhibitor. Future studies aimed at a better comprehension of the interplay between CXCL8 and thyroid diseases appear worthwhile.

Obesity Does Not Modify the Risk of Differentiated Thyroid Cancer in a Cytological Series of Thyroid Nodules

Background: A possible impact of obesity on the risk of thyroid cancer has been postulated in some studies, but it remains controversial. Objective: To investigate the association between obesity and differentiated thyroid carcinoma in a population of unselected patients subjected to fine-needle aspiration cytology (FNAC) for thyroid nodules. Methods: We retrospectively evaluated the results of FNAC of thyroid nodules in 4,849 patients (3,809 females and 1,040 males; mean age 55.9 ± 14.1 years). Patients were stratified according to their body mass index (BMI). There were 1,876 (38.7%) normal-weight patients (BMI 18-24.9), 1,758 (36.2%) overweight (BMI 25-29.9), 662 (13.7%) grade 1 obese (BMI 30-34.9), 310 (6.4%) grade 2 obese (BMI 35-39.9) and 243 (5.0%) grade 3 obese (BMI >40). Results: The prevalence of suspicious or malignant nodules (Thy4/Thy5) did not differ across the 5 BMI groups, i.e. it was 6.8% in normal-weight patients, 6.3% in overweight patients, 6.3% in grade 1 obese patients, 4.0% in grade 2 obese patients and 4.2% in grade 3 obese patients (p = 0.29). The prevalence of Thy4/Thy5 nodules did not differ when males and females were evaluated separately (p = 0.22 and p = 0.12, respectively). A significant, lower rate of Thy4/5 cytology was observed in female patients with grade 2-3 obesity (odds ratio 0.51; 95% confidence interval 0.284-0.920; p = 0.009). Conclusions: The results of this study, in a retrospective series of patients with thyroid nodules, do not confirm previous findings reporting an association between obesity and differentiated thyroid carcinoma. Thus, obese patients with nodular thyroid disease should be managed the same as normal-weight patients.