Effect of systemic steroid therapy in Graves' orbitopathy on regulatory T cells and Th17/Treg ratio.

PURPOSE: Glucocorticoids are a mainstay treatment for Graves' orbitopathy, yet their exact mechanisms of action remain unclear. We aimed to determine whether the therapeutic effects of systemic steroid therapy in Graves' orbitopathy are mediated by changes in regulatory T lymphocytes (Tregs) and T helper 17 lymphocytes (Th17). METHODS: We assessed Treg and Th17 levels in the peripheral blood of 32 patients with active, moderate-to-severe Graves' orbitopathy who received 12 weekly pulses of methylprednisolone, and determined their association with disease severity, disease activity, and treatment outcomes. The acute orbitopathy phase was confirmed based on clinical evaluation and magnetic resonance imaging, and assessed using the clinical activity score (CAS). The severity of the disease was classified according to ETA/EUGOGO guidelines, and quantified based on the total eye score. Treatment response was determined based on specific criteria (e.g., changes in CAS score, diplopia grade, visual acuity, etc.). Treg and Th17 cells were identified using flow cytometry. RESULTS: Methylprednisolone treatment improved the activity of the disease and altered the Th17/Treg balance (i.e., the percentage of Tregs decreased while the number of Th17 cells remained unchanged). There was no association between the Treg/Th17 ratio and the activity and severity of the disease or the treatment response. CONCLUSIONS: Therapeutic effects of steroid therapy in Graves' orbitopathy are not mediated by Treg and Th17 alterations in the peripheral blood. The decrease in peripheral Treg percentage is likely a consequence of the non-specific effects of steroids and does not impact clinical outcome.

CARDIOVASCULAR COMPLICATIONS OF ACROMEGALY.

Acromegaly is associated with increased mortality and decreased life expectancy. Cardiovascular disease is the principal cause of premature mortality in patients with acromegaly, accounting for about 60% of deaths. GH and/or IGF-I exert direct cardiac effects: enhance cardiac contractility, stimulate cardiomyocyte growth, influence calcium influx in cardiomyocytes. Cardiac remodelling is influenced by hypertension and insulin resistance. Among cardiovascular risk factors arterial hypertension, reported in 35% of patients with acromegaly, ranks among most important negative prognostic factors for mortality. Hypertension plays significant role in the development of cardiac hypertrophy, especially in older acromegalic patients and diastolic blood pressure is best predictive factor for cardiac hypertrophy. Therefore, early and aggressive hypertension treatment is essential for prognosis in acromegaly. Other important risk factors are: valvular defects, arrhythmias, endothelial dysfunction, heart failure, lipid abnormalities and coronary artery disease. Numerous studies suggest that patients with acromegaly are under threat of arrhythmias, especially those with structural heart abnormalities. Congestive heart failure as end-stage acromegalic cardiomyopathy occurs usually in older patients, with long-term uncontrolled disease and other cardiovascular and metabolic complications. Relation between acromegaly and coronary artery disease is controversial as it seems to be connected rather with classical cardiovascular risk factors than GH and IGF-1 overexpresion.

Growth Hormone Treatment Increases Plasma Irisin Concentration in Patients with Turner Syndrome.

Irisin (Ir) deficiency may be a contributing factor in metabolic disease. This study aimed to investigate the effect of supraphysiological doses of recombinant human growth hormone (rhGH) on Ir plasma concentration in relation to metabolic disorders, including obesity and other components of metabolic syndrome. We studied 36 girls with Turner syndrome (mean age 8.2 years) treated with rhGH (0.05 mg/kg/day). Anthropometric data and fasting blood levels [e. g., Ir, insulin, glucose, glycated hemoglobin (HbA1c), IGF-1, IGFBP-3, cholesterol, insulin resistance (HOMA-IR), and ß-cell function (HOMA-ß)] were analyzed prior to and following rhGH therapy [mean (SD) follow-up of 1.47 (0.89) years]. Insulin sensitivity (Matsuda index) was calculated before and after the glucose load. Following rhGH therapy, an increase in IGF-1 [mean (SD) of 119.40 (62.47) ng/ml to 439.08 (209.91) ng/ml, p=0.000], Ir [2.10 (1.03) µg/ml to 2.48 (0.78) µg/ml, p=0.036], HOMA-IR [median (IQR) of 0.64 (0.45-1.30) to 0.92 (0.67-2.36), p=0.0206], and HOMA-ß values [45.00 (27.69-72.00) to 81.53 (51.43-132.00), p=0.0447] were observed. Multiple regression analysis yielded no associations between Ir and metabolic and hormonal parameters before rhGH treatment; however, on rhGH, the model (R2=0.56, adjusted R2=0.45) showed positive associations between Ir and IGF-1 standard deviation score and HbA1c, and negative associations between Ir and fasting blood glucose, HDL-cholesterol, and triglycerides. Despite manifestation of insulin resistance, rhGH application had a positive effect on Ir regulation, and restored physiological conditions of lipid and glucose metabolism.

Acromegaly due to GHRH-secreting large bronchial carcinoid. Complete recovery following tumor surgery.

A case of acromegaly, secondary to GHRH secretion by a large bronchial carcinoid is reported. A 61-year-old woman presented with typical symptoms and signs of acromegaly for at least 10 years. She suffered from recurrent pneumonias, but repeated chest X-ray examinations failed to demonstrate the bronchial tumor. The diagnosis was confirmed by elevated GH, IGF-1 and GHRH secretion. We have shown an enlarged pituitary gland without focal lesions together with a cerebral meningioma on MRI and the presence of a bronchial carcinoid tumor. The latter was confirmed by histology carried out after bronchoscopy and tumor excision. We observed partial suppression of GH secretion following short-term oral bromocriptine administration in this patient. Surgical removal of the carcinoid tumor resulted in a complete clinical, hormonal and radiological cure of acromegaly. This case of acromegaly due to ectopic GHRH secretion by bronchial carcinoid differs from others described in the literature by an atypical large tumor size, the suppression of elevated GH secretion by oral bromocriptine and a concomitant meningioma.

Intratumoral levels of estrogens in breast cancer.

Breast cancer tissue is an endocrine organ and particularly the estrogen biosynthetic properties of this tissue have been well studied. The concentration of estradiol in breast cancer tissue from postmenopausal patients is considerably higher than that in the circulation and appears to depend largely on local production. Androgenic precursor steroids are abundantly present, but estrogen storage pools like fatty acid derivatives appear to be less important than initially thought. New, potent and highly specific aromatase inhibitors effectively inhibit peripheral conversion of androgens to estrogens (Cancer Res. 53: 4563, 1993) as well as intratumour aromatase, median aromatase activity being 89% lower in the tissue from patients pretreated with aromatase inhibitor 7 days prior to surgery (P < 0.001). Also the intratissue concentrations of estrogens were decreased (64% and 80% reduction, respectively for estrone and estradiol; P = 0.001 and <0.05; Cancer Res. 57: 2109, 1997). These results illustrate that intratissue estrogen biosynthesis is effectively inhibited by the new generation of aromatase inhibitors. The pathophysiological consequences of this finding are currently under study.

Concentration of sex steroids in adipose tissue after menopause.

Adipose tissue is a site of uptake, storage, action, and metabolism of sex steroids. After menopause aromatization of androgens to estrogens in adipose tissue is one of the most important sources of estrogen in the circulation and for peripheral tissues. The aim of this study was to estimate local sex steroid concentrations in breast and abdominal subcutaneous (s.c.) adipose tissue, to compare them with plasma concentrations and to investigate possible correlations with body mass index (BMI). The patients were postmenopausal women undergoing surgery for non-oncological reasons (Group A; n = 35) and breast cancer patients (group B; n = 19). The concentrations of estrone, 17 beta-estradiol, estrone sulfate, 17 beta-estradiol sulfate, androstenedione, androstenediol (androst-5-ene-3 beta, 17 beta-diol), testosterone and dehydroepiandrosterone were measured. The method was based on frozen tissue homogenization, extraction with ethanol: acetone, delipidation, extraction of estrogens with ether, and of androgens with iso-octane in toluene, followed by RIA. The mean levels of steroids were higher in fat than in plasma, apart from testosterone. Levels of sulfates of estrogens and androstenediol were higher in breast than abdominal adipose tissue, and levels of estradiol lower. Positive correlations were found between BMI and tissue and plasma concentration of both estrone and androstenedione.

Bombesin plasma levels in patients with benign and malignant breast disease.

Some reports suggest a role for bombesin-like peptides in the pathology of breast tumors. Bombesin and related gastrin-releasing peptides have been shown to influence the inositol phospholipid signalling pathway and stimulate growth of some cells, including some human breast cancer cell lines. We measured the plasma concentration of bombesin in 23 breast cancer patients, 32 patients suffering from benign breast disease and in 21 healthy controls. The bombesin concentration in plasma taken from the thoracodorsal vein, in the vicinity of the tumor in breast cancer patients was higher than that in the peripheral circulation (mean +/- SEM, 91.3 +/- 54.3 vs. 40.9 +/- 27.4 pg/ml; p < 0.05). Bombesin concentrations in the cubital vein in breast cancer patients and in those with benign breast disease (61.7 +/- 49.3 pg/ml) was significantly higher than that in the control group (23.7 +/- 5.06 pg/ml; p < 0.05). Our findings suggest storage or synthesis of bombesin-like peptides within the affected breast and may confirm the role of these peptides in the growth of breast tumors.

Local aromatase activity in human breast tissues.

The presence of oestradiol in malignant breast cells is considered to be an important factor in the promotion of growth of the tumor. Therefore the regulation of the local high intra-tissue oestradiol concentrations, regardless of plasma concentrations, has been investigated. Experimental evidence suggests that in situ biosynthesis of oestrogens is at least partly responsible for the local accumulation of these steroids. In this paper we report further data on measurements in fatty and tumor tissues of local aromatase activities and of concentrations of substrates and products of this enzyme. Data are given on localization of aromatase and on steroid concentrations in tumors and in adipose tissues dissected from different quadrants of breasts with malignant tumors. In adipose tissues small variations in steroid concentrations in fatty tissues were found. No tumor-directed gradients in the adipose tissue-concentrations of the androgens dehydro-epiandrosterone, 5-androstene-3 beta, 17 beta-diol, 4-androstene-3,17-dione and testosterone and of the oestrogens oestradiol, oestrone and their sulfates could be detected. Furthermore no consistent pattern could be recognized in the aromatase activities in the fatty tissues dissected from tumor-bearing and non-affected quadrants of the same breast. No correlations between aromatase activity measured in vitro and product concentrations in vivo were found. Therefore the mechanisms for regulation of the local oestradiol levels in breast tissues remain unknown.

Tissue androgens and the endocrine autonomy of breast cancer.

To evaluate whether a tumour-directed gradient in androgen levels in fatty tissue can account for the maintenance of intra-tissue oestradiol levels, androstenedione (Adione), dehydroepiandrosterone (DHEA), testosterone (Testo) and androstenediol (Adiol) were assayed in breast tumour tissues and in fatty tissue taken at different distances from the tumour. The concentration of Adione was significantly lower in tumour tissue (5.6 +/- 1.5 pmol/g tissue; mean +/- SEM; n = 14) than in the adjacent fatty tissue (20.4 +/- 2.2; P less than 0.005). Testo, by contrast, occurred in equal concentrations in tumour (0.80 +/- 0.11) and in adjacent fatty tissue (0.70 +/- 0.07). Adione levels tended to be lower after the menopause only in fatty tissue, not in the tumour tissue; for Testo no differences were observed between samples from pre- and postmenopausal patients. Tumour DHEA levels (57 +/- 12 pmol/g tissue) were lower than those in fatty tissue (117 +/- 17; P less than 0.02). As with Adione, fatty tissue DHEA concentrations tended to be higher in pre- than in postmenopausal patients. Adiol showed a similar pattern as Testo. For none of the aromatase substrates nor their precursors a tumour-directed gradient was observed. The concentration of Adione in breast cancer tissue is much lower than the reported Km of the aromatase system for Adione. We have concluded, therefore, that the maintenance of oestradiol concentrations in tumour tissues is not substrate-driven.

Estrogens in plasma and fatty tissue from breast cancer patients and women undergoing surgery for non-oncological reasons.

Breast cancer tissue is able to maintain the tissue estradiol level in spite of the massive decrease in plasma estradiol associated with menopause, whereas fatty tissue from breasts with malignancies more closely reflects the changes in plasma. In the present study estrone and estradiol levels in fatty tissues from different origins were compared to evaluate the capacity of distant fatty tissues to act as estrogen reservoirs. Abdominal fat was obtained from 25 premenopausal and 20 postmenopausal women who underwent surgery for non-oncological reasons. Estrone and estradiol levels in these tissues were compared to those in breast fatty tissue from breast cancer patients. Plasma estrogen levels were not different in the two groups. In both groups, median plasma estradiol levels dropped sharply with menopause (from 363 to 40 pmol/l in breast cancer patients; from 280 to 45 pmol/l in the non-oncological patients; p less than 0.002), whereas a significant decrease in plasma estrone was observed only in the breast cancer patients (from 238 to 140 pmol/l; p less than 0.02). In premenopausal women, median estrone and estradiol levels in breast fatty tissue (1135 and 375 fmol/g, respectively) and abdominal tissue (1390 and 470 fmol/g, respectively) were not different. In postmenopausal women, however, significantly higher estrone levels (663 vs. 508 fmol/g; p less than 0.01) and estradiol levels (245 vs. 187 fmol/g; p less than 0.02) were found in abdominal fatty tissue. In view of the absolute estrogen levels in breast and abdominal fatty tissue and in plasma, we conclude, however, that it is unlikely that remote fat contributes substantially to the maintenance of estrogen levels in breast cancer tissue.

On the significance of in situ production of oestrogens in human breast cancer tissue.

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.

Endogenous steroid hormones and local aromatase activity in the breast.

To test the hypothesis of an increased activity of the enzyme aromatase in adipose tissue from affected when compared with non-affected quadrants of patients with breast cancer, the aromatase activity has been measured in tumour and fatty tissues dissected at specific sites from the breasts of 16 patients. Activity was measured after extensive purification of the product formed. Results, expressed in fmol/g of tissue, did not show a higher activity in the affected vs the non-affected quadrants. In the tumours, higher activities were found when expressed per g of tissue. Per mg of DNA, an indicator of the number of cells, tumour enzymatic activity was lower than in fatty tissues. The relations between the products of aromatase, oestrone and oestradiol in the various tissues point to the importance of additional enzymatic processes, especially of the reductive 17 beta-oestradiol dehydrogenase, in the accumulation of high quantities of oestradiol in the malignant tissue.

A quantitative structure-activity relationship (QSAR) analysis of the effects of aromatic substitution on the anticonvulsant activity and toxicity of arylsuccinimides.

Anticonvulsant activity and toxicity of 20 arylsuccinimides were quantitatively correlated with the hydrophobic, electronic and steric parameters of the substituents in the benzene ring and at the nitrogen atom. The activity was highest when the benzene ring substituents X were characterized by hydrophobic fragmental constants fx lying in the 1.0-1.7 range, though toxicity increased with fx.

Potential acetylcholinesterase reactivators: pyridine and alpha-oxooxime derivatives.

The synthesis and pharmacological screening in vitro and in vivo of pyridine-2-carbaldoxime derivatives I and alpha-oxooximes II are described. Four compounds elicited reactivating effect on phosphorylated acetylcholinesterase comparable with that of pralidoxime used as reference substance. Among the compounds tested, interesting structures are those of oximes bearing a thioether substituent [RA 49 (Table 1) and RA 59 (Table 2)] chloro derivative of MINA [RA 55 (Table 2)] and dipyridyl glyoxime methiodide RA 56 (Table 1).