G. Mazziotti

Serum and follicular fluid cytokines in polycystic ovary syndrome during stimulated cycles

OBJECTIVE To investigate the serum and intrafollicular tumor necrosis factor–α and interleukin-6 concentrations in infertile women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF). METHODS Thirty-one patients with PCOS undergoing IVF were studied. Thirty-nine normally ovulating women matched for age and body mass index and undergoing IVF for male infertility were the control group. Serum tumor necrosis factor–α, interleukin-6, and estradiol levels were assayed before recombinant follicle-stimulating hormone stimulation under gonadotropin-releasing hormone analogue suppression and 34–36 hours after human chorionic gonadotropin (hCG) administration at the time of the oocyte retrieval. Cytokine and estradiol concentrations were also evaluated in the follicular fluids obtained at the time of oocyte retrieval. RESULTS The patients with PCOS had higher serum and follicular fluid tumor necrosis factor–α and interleukin-6 concentrations (P < .001) and lower follicular fluid estradiol levels (P < .05) than control women. In both groups, the serum tumor necrosis factor–α, interleukin-6, and estradiol values increased significantly after hCG stimulation. In both groups, the follicular fluid cytokine concentrations were higher than those found in the serum. In the PCOS women the follicular fluid tumor necrosis factor–α values were significantly and inversely correlated to the follicular fluid estradiol values (ρ = −0.79; P < .001); this correlation was not found in the control subjects. CONCLUSION In infertile women with PCOS, 1) serum and follicular fluid interleukin-6 and tumor necrosis factor–α values were higher than those found in control women, 2) the cytokine concentrations were higher in the follicular fluid than in the serum, and 3) the intrafollicular tumor necrosis factor–α concentrations were significantly and inversely correlated to the estradiol levels. These results suggest an involvement of the immune system in PCOS.

Occurrence of thyroid autoimmunity and dysfunction throughout a nine-month follow-up in patients undergoing interferon-β therapy for multiple sclerosis

Thyroid autoimmunity and dysfunction are a well known side effect of IFN α therapy for viral hepatitis and tumors, while the IFN β effects on the thyroid gland in neurological patients have not been studied. The aim of this longitudinal study was to look for the appearance of thyroid autoimmunity as well as for the occurrence of overt thyroid disease in the patients affected by multiple sclerosis (MS) treated with IFN β 1b. Eight patients (4 males, 4 females) undergoing r-IFN β 1b treatment (8 M.U. every other day for 9 months) for relapsing remitting multiple sclerosis entered the study. We have analyzed thyroid function parameters and auto antibody levels before and after 1, 2, 3, 6 and 9 months of therapy. None of them referred to familiar thyroid pathology or presented clinically overt thyroid disease except for one patient (case 4) who showed TPO-Ab pretreatment positivity and another (case 8) who was in therapy with Levothy-roxine 100 μg/die for multinodular goiter. The number of patients with appearance of thyroid antibodies has slowly increased, until the third month of therapy with 3 patients out of 7 positive for TPO-Ab. The only case of overt thyroid dysfunction reported by us appeared after nine months of therapy and consisted of a hypothyroidism. Our data suggest that short-term interferon β treatment is able to induce thyroid autoimmunity (42.8%) and dysfunction (12.5%).

Interferon-related thyroid autoimmunity and long-term clinical outcome of chronic hepatitis C.

BACKGROUND A high incidence of thyroid autoantibodies and/or disorders was observed in subjects with hepatitis C virus-related chronic hepatitis during interferon-alpha therapy. AIM To evaluate whether thyroid autoimmunity and dysfunction, induced by interferon-alpha therapy, could be viewed as predictors for treatment response and as valid prognostic markers of liver disease progression. PATIENTS A total of 136 subjects (96 males/40 females; median age 48 years; range 23-64) affected by biopsy-proven chronic hepatitis C (33.1% with compensated liver cirrhosis). METHODS All subjects were treated with interferon-alpha therapy at 6 MU 3 times weekly for 12 months and then followed up for an average period of 60 months (range 12-108). Routine laboratory tests, virological assessment, liver ultrasound, thyroid function tests (serum free-triiodothyronine, free-thyroxine, serum thyrotropin), and autoimmunity were performed for all subjects. RESULTS Percentage of thyroid autoimmunity and thyroid dysfunction in long-term responders was not significantly different compared to that in non-responders (47.0% and 11.8% vs 35.3% and 5.9%, respectively; non significant). The multivariate model demonstrated that the absence of cirrhosis was the only factor significantly related to successful response to therapy (odds ratio: 14.9; 95% confidence interval: 1.9-115.0 for chronic hepatitis C vs presence of cirrhosis). Moreover, the occurrence of thyroid autoimmunity during interferon therapy was similar both in patients with or without worsening of liver disease (33.3% and 39.8%, respectively; p = not significant). No subject with on-going liver disease developed thyroid dysfunction during treatment, as opposed to the 10/118 (8.4%) with a better course of liver disease; however, this difference was not statistically significant. The multivariate model showed that age was the only covariate significantly associated with unfavourable outcome of liver disease (odds ratio: 18.6; 95% confidence interval: 2.3-151.9, for those over 48 years vs younger patients). CONCLUSIONS There is no evidence that the immune mechanism involved in the pathogenesis of thyroid autoimmune phenomena is the same as that regulating the therapeutic clearance of HCV or modulating the unfavourable course of HCV-related chronic hepatitis. However, our study confirmed that liver disease seems to progress more slowly in younger subjects.

Lack of association between changes in plasma leptin concentration and in food intake during the menstrual cycle

Changes in plasma leptin concentration and food intake occur during the menstrual cycle; because leptin regulates food intake, one could hypothesize that changes in plasma leptin concentration and in food intake are associated throughout the menstrual cycle. However, no data have ever been provided to support such a relationship. The aim of our study was to investigate, during the different phases of the menstrual cycle, (a) the changes in plasma leptin concentration and, if such changes were demonstrated, (b) the potential relationship between the changes in plasma leptin concentration and food intake.

Long-term treatment with interferon-β therapy for multiple sclerosis and occurrence of Graves’ disease

Interferon (IFN)-β has become a widespread therapy for multiple sclerosis. As already reported for IFN-α, thyroid autoimmunity and dysfunctions have been observed also in course of IFN-β therapy. Nevertheless, very few cases of Graves’ disease, occurred in such condition, have been reported in literature. We here describe the case of a 40-year-old female affected by multiple sclerosis, who received IFN-β-1b, 8 million IU sc every other day for her condition. After 22 months of cytokine administration, she developed a severe Graves’ disease with persistently positive TRAb which suggested the withdrawal of the treatment. Our patient had performed a complete thyroid evaluation with normal findings, before and during the first 6 months of therapy. This case suggests that patients undergoing long-term IFN-β therapy should be monitored for thyroid hormones and antibodies throughout the treatment as thyroidal side effect can be a late event.

Insulin resistance and advancing age: What role for dehydroepiandrosterone sulfate?

The relationship between insulin resistance and aging is still debated. This study aims to investigate the role that age-related differences in plasma dehydroepiandrosterone sulfate (DHEAS) concentration may have on insulin action. For this reason, 75 subjects (42 men and 33 women) with a wide age range (21 to 106 years) were studied. In all subjects, plasma DHEAS and total testosterone concentrations were measured and a euglycemic clamp was used, but substrate oxidation was not determined in centenarians (n = 15). Plasma DHEAS correlated with age (r = -.77, P < .001) and whole-body glucose disposal (WBGD) (r = .57, P < .001). After controlling for age, sex, body fat, and waist to hip ratio (WHR), the association between plasma DHEAS and WBGD was still observed (r = .31, P < .005). Comparing subjects at the third tertile versus those at the first and second tertiles of plasma age-adjusted DHEAS concentration, the former group showed a weaker association between WBGD and age (r = -.38, P < .05) than the latter group (r = -.43, P < .002). The difference between the two regression lines was also significant (P < .03). After controlling for sex, body fat, and WHR, the association between plasma DHEAS and WBGD was dependent on the age of the subjects, being strong in adults (n = 30, age < 50 years, r = .69, P < .001), weak in old subjects (n = 30, age 51 to 99 years, r = .23, P < .05), and absent in centenarians (r = -.05, P < .88). With the subjects divided by sex throughout the different age groups, the univariate association between plasma DHEAS and WBGD was present in females (r = .43, P < .01) but not in males (r = .17, P < .32). Plasma total testosterone and insulin-like growth factor-1 (IGF-1) concentrations declined with advancing age and were significantly correlated with DHEAS and WBGD. In a multivariate analysis with WBGD as the dependent variable, a model including age, sex, body fat, WHR, DHEAS, total testosterone, and IGF-1 explained 66% of WBGD variability, with DHEAS significantly and independently associated with WBGD (P < .004). In conclusion, the negative relationship between advancing age and insulin action seems related to plasma DHEAS concentration. Differences in plasma total testosterone and IGF-1 concentrations may provide a further contribution to the relationship between DHEAS and WBGD.

Is the IFN-alpha-related thyroid autoimmunity an immunologically heterogeneous disease?

Dear Sir, Over the last years, many studies have aimed to investigate the relationship between the appearance of autoimmune phenomena in the course of interferon-alpha (IFN-a) treatment and the therapeutic response of the underlying viral or neoplastic disease [1–5]. We read with great interest the paper by Dalgrad et al. which demonstrated that, in patients treated with IFN-a plus ribavirin for HCV-related chronic hepatitis, the response to the antiviral treatment was not correlated to the appearance of thyroid dysfunction [5]. This result was in accordance with previous studies [1, 2, 4], but in disagreement with others demonstrating that the IFN-induced thyroid disease occurred more frequently in patients with a sustained response to the cytokine treatment [3]. The authors considered the different duration of the cytokine treatment as a possible factor explaining this discrepancy. We think that immunological and genetic aspects should also be considered in this context. The type 1 immune response plays a critical role in the resolution of viral infection, as well as in the development of many autoimmune diseases [6–8]. In thyroid autoimmune disease, however, the T helper (Th)1 and Th2 responses coexist in the same patients, although with different reciprocal intensity in relationship with the clinical expression of the disease process [9]. The type 1 response seems to be dominant in the course of hypothyroidism occurring in patients with Hashimoto’s thyroiditis and in the course of silent thyroiditis, whereas the type 2 response has been correlated mainly with the appearance of hyperthyroidism in the cases affected by Graves’ disease [10–13]. In particular, the thyroid-destructive process in Hashimoto’s thyroiditis seems to be induced mainly by cell-mediated immunity, as demonstrated by the increase of serum Th1 cytokines in this condition [13]. However, Graves’ thyrotoxicosis is induced by the thyroidstimulating autoantibodies to thyrotropin receptor, the production of which is likely to depend on Th2 cell function [12]. The same mechanisms should be considered for the IFN-related thyroid autoimmunity. In this view, the patients who develop Graves’type thyrotoxicosis in the course of treatment with IFN-a should be considered immunologically different to those showing hypothyroidism or transient thyrotoxicosis by a destructive process in the thyroid gland [14, 15]. The fact that in the various studies the different clinical pictures of the IFN-related thyroid autoimmunity were not considered separately could explain the controversial data of literature about the correlation between the response to the antiviral treatment and the appearance of thyroid disease. Unfortunately, such differentiation is not always possible because different expressions of the thyroid disease can occur in the same patient, suggesting a possible evolution of the immunological process throughout the IFN treatment [16]. However, a probable correlation between the remission of chronic hepatitis and the appearance of thyroid disease should be considered only for those patients with a Th1-mediated destructive process in the thyroid gland, mainly when the antiviral treatment is performed with IFN-a plus ribavirin [17]. Nevertheless, it does not mean that the two events are constantly correlated. In fact, most of the patients with sustained response to antiviral treatment show neither thyroid autoimmunity nor thyroid dysfunction [17]. This apparent paradox is easily explained by the fact that whilst the response to the antiviral treatment is related to both host and viral factors [18, 19], the genetic background seems to play a predominant role in determining the occurrence of the IFN-related thyroid autoimmunity [20]. In this view, the IFN-a plus ribavirin treatment can trigger a Th1-mediated cytotoxic process in the thyroid gland Journal of Internal Medicine 2002; 252: 377–378

An Improved Polymerase Chain Reaction (PCR) Protocol for Unambigous Detection of Growth Hormone Gene Deletions

hGH-1 gene deletions are detected by simultaneous PCR amplification along the two homologous DNA sequences flanking the hGH-1 gene on both sides and are differentiated by SmaI restriction enzyme digestion. We have observed that among the SmaI digested PCR products from normal homozygous subjects, from those heterozygous for the 7.6 kb deletion and from those heterozygous for a 6.7 kb deletion, along with the expected fragments there is an unexpected 1470 bp fragment. This fragment arises from the co-amplification of a third homologous sequence located downstream from the hGH-1 gene and it confuses differentiation between normal homozygous and heterozygous for 7.6 kb subjects from the 6.7 kb heterozygous subjects. To overcome this problem we have improved PCR conditions using a different reverse primer. These changes avoid the interaction of the primers with the third homologous sequence located downstream from the hGH-1 gene and prevent the appearance of this additional band that complicates the interpretation of the results. We conclude that the new reverse primer sequence avoids the amplification of the downstream hGH-1 gene sequence and the production of the 1474 bp band after SmaI endonuclease enzyme digestion and makes it possible to differentiate homozygous normal subjects and those who are heterozygous for a 7.6 kb deletion from those who are heterozygous for a 6.7 kb deletion.

Diabetes insipidus and increased serum levels of leptin and lactate-dehydrogenase (LDH) in an adolescent boy with a primary intracranial germinoma. Case Report and an endocrinological revaluation of literature

A 16-year-old boy presented with a four-month history of polyuria-polydipsia and a diplopia which had reverted after treatment. The neuroimaging studies performed had been strongly suggestive of an optic nerve glioma, while en-docrinological investigation (β-hCG 420 IU/L) has lead to the correct diagnosis later confirmed at the immunohystochemical analysis performed at biopsy. The high serum level of hCG was unaffected by bromocriptine nor octreotide, while the PRL level (80.0 μg/L) was reduced only by bromocriptine. Among the several tumor markers which may be secreted by such lesions, ours is the first reported case of an elevation of serum LDH for a primary intracranial germinoma. Moreover, the elevated value of serum leptin reported by us might be due to the insensitivity of the hypothalamic structures to endogenous leptin.