Endocrine rhythms and sport: it is time to take time into account.

BACKGROUND: Studies of time-related biological phenomena have contributed to establishing a new scientific discipline, the chronobiology, which considers biological phenomena in relation to time. Sports activity profoundly affects the temporal organization of the organism and endocrine rhythms play a key role in the chronoorganization of individuals and are particularly important for correct physical activity. Correctly reading rhythmic hormonal variations of the human organism opens new horizons to sports medicine. OBJECTIVE: This review is aimed at clarifying the relationship between endocrine rhythms and sports activities on the basis of the latest data in the literature. METHOD: Data acquisition was obtained from three databases (PubMed, Scopus and SPORTDiscus), paying particular attention to reviews, meta-analysis, original and observational studies on this issue. RESULTS: After the description of the general characteristics and parameters of biological rhythms, the main endocrine rhythms will be described, highlighting in particular the interrelationships with sports activity and focusing on the factors which can affect negatively their characteristics and consequently the psychophysical performances of the athletes. CONCLUSION: Knowledge of this issue may allow establishing the best form of competitive or amateur activity, through the collaboration of an informed athlete and a sports physician attentive to biological rhythms. By taking into account that alteration of physiological rhythmic temporal organization can favour the onset of important diseases, including cancer, this will lead to the expected performances without impairing the correct chronoorganization of the athlete.

Characterization of pituitary cells targeted by antipituitary antibodies in patients with isolated autoimmune diseases without pituitary insufficiency may help to foresee the kind of future hypopituitarism.

PURPOSE: Detection of antipituitary antibodies (APA) at high levels and with a particular immunofluorescence pattern in patients with autoimmune polyendocrine syndromes may indicate a possible future autoimmune pituitary involvement. This longitudinal study was aimed at characterizing in patients with a single organ-specific autoimmune disease the pituitary cells targeted by APA at start, verifying whether this characterization allows to foresee the kind of possible subsequent hypopituitarism. METHODS: Thirty-six APA positive and 40 APA negative patients with isolated autoimmune diseases participated in the study. None of them had pituitary dysfunction at entry. Characterization by four-layer immunofluorescence of pituitary cells targeted by APA in APA positive patients at entry and study of pituitary function in all patients were performed every 6 months during a 5 year follow-up. RESULTS: Antipituitary antibodies immunostained selectively one type of pituitary-secreting cells in 21 patients (58.3 %, group 1), and several types of pituitary cells in the remaining 15 (41.7 %, group 2). All patients in group 1 showed subsequently a pituitary insufficiency, corresponding to the type of cells targeted by APA in 18 of them (85.7 %). Only 8 out of 15 patients in group 2 (53.3 %) showed a hypopituitarism, isolated in 7 and combined in the other one. None of APA negative patients showed hypopituitarism. CONCLUSIONS: The characterization of pituitary cells targeted by APA in patients with isolated autoimmune diseases, when the pituitary function is still normal, may help to foresee the kind of subsequent hypopituitarism, especially when APA immunostained selectively only one type of pituitary cells. A careful follow-up of pituitary function in these patients is advisable to allow an early diagnosis of hypopituitarism, even in subclinical phase and a consequent timely replacement therapy.

Prospective investigation of pituitary functions in patients with acute infectious meningitis: is acute meningitis induced pituitary dysfunction associated with autoimmunity?

Previous case reports and retrospective studies suggest that pituitary dysfunction may occur after acute bacterial or viral meningitis. In this prospective study we assessed the pituitary functions, lipid profile and anthropometric measures in adults with acute bacterial or viral meningitis. Moreover, in order to investigate whether autoimmune mechanisms could play a role in the pathogenesis of acute meningitis-induced hypopituitarism we also investigated the anti-pituitary antibodies (APA) and anti-hypothalamus antibodies (AHA) prospectively. Sixteen patients (10 males, 6 females; mean ± SD age 40.9 ± 15.9) with acute infectious meningitis were included and the patients were evaluated in the acute phase, and at 6 and 12 months after the acute meningitis. In the acute phase 18.7% of the patients had GH deficiency, 12.5% had ACTH and FSH/LH deficiencies. At 12 months after acute meningitis 6 of 14 patients (42.8%) had GH deficiency, 1 of 14 patients (7.1%) had ACTH and FSH/LH deficiencies. Two of 14 patients (14.3%) had combined hormone deficiencies and four patients (28.6%) had isolated hormone deficiencies at 12 months. Four of 9 (44.4%) hormone deficiencies at 6 months were recovered at 12 months, and 3 of 8 (37.5%) hormone deficiencies at 12 months were new-onset hormone deficiencies. At 12 months there were significant negative correlations between IGF-I level vs. LDL-C, and IGF-I level vs. total cholesterol. The frequency of AHA and APA positivity was substantially high, ranging from 35 to 50% of the patients throughout the 12 months period. However there were no significant correlations between AHA or APA positivity and hypopituitarism. The risk of hypopituitarism, GH deficiency in particular, is substantially high in the acute phase, after 6 and 12 months of the acute infectious meningitis. Moreover we found that 6th month after meningitis is too early to make a decision for pituitary dysfunction and these patients should be screened for at least 12 months. In addition, the occurrence of AHA and APA positivity due to acute infectious meningitis was demonstrated for the first time. Further longer-term prospective investigations need to be carried out on a larger cohort of patients to understand the role of autoimmunity in the pathogenesis of late hypopituitarism after acute infectious meningitis.

Differential expression of estrogen receptor α and ß transcripts in tissues and in primary culture cells from pubertal gynecomastia.

BACKGROUND: Pubertal gynecomastia is a common problem occurring in up to 65% of adolescent boys. Gynecomastia comes at a time when self-image awareness is at its greatest and psychologically could be a psychologically disabling condition. Surgery is considered the mainstay of treatment for severe or persistent cases. A medical management aimed at altering the effective androgen/estrogen ratio has been suggested with inconstant results. Some promising results have been obtained by using anti-estrogens. Surprisingly there are no data on the estrogen receptor (ER) α and ß RNA expression in gynecomastia. AIM: We studied ER RNA subtypes in pubertal gynecomastia. METHODS: ERα and ß RNA were determined by real time RT-PCR in 50 mammary samples from pubertal boys with idiopathic gynecomastia subjected to reductive mammoplasty. To study ERα and ß pattern of expression, epithelial and stromal primary cell cultures were set up from fresh tissues. RESULTS: These analyses indicated that in all stromal cells ERß was expressed at higher level than ERα and in epithelial cells both ERα and ERß were barely detectable. CONCLUSIONS: Our data suggest that also stromal cells are involved in the pathophysiology of pubertal gynecomastia. The high level of expression of ERß seen in pubertal gynecomastia adds new insight on validation of ERß as a target for candidate diseases and exploration of ERß as a marker for clinical decision-making and treatment in pubertal gynecomastia. This could drive to search for new and selective anti-estrogen drugs for medical treatment of pubertal gynecomastia with a particular attention to the ERß-selective ligand.

Raloxifene induces cell death and inhibits proliferation through multiple signaling pathways in prostate cancer cells expressing different levels of estrogen receptor α and ß.

Raloxifene (RAL), a selective estrogen receptor (ER) modulator (SERM) seems to induce apoptosis in both androgen-dependent and -independent prostate cell (PC) lines via activation of ERß and an antagonistic effect on ERα. In this study, we evaluated the effects of RAL on epithelial PC growth using the two following in vitro models: the androgen-dependent cell line EPN which expressed both ERs; and a stabilized epithelial cell line derived from a prostate cancer specimen (CPEC), which expressed low levels of ERß and lacked ERα. In EPN cells, there was an increase in the pre-G1 apoptotic peak and a reduction in the S phase of the cell cycle with G0/G1 arrest after E2 or RAL treatment; bcl-2 mRNA and Bcl-2 protein levels were significantly reduced, while activated caspase-3 and Par-4 levels increased significantly after either E2 or RAL treatment; in addition, c-myc transcript was inhibited after 10(-6) M RAL treatment. A dose-dependent increase of metallothionein II gene RNA level was also induced by RAL in EPN. In CPEC, there was only a weak apoptotic peak associated with caspase-3 activation and Par-4 increase after either E2 or RAL treatment; while c-myc transcript level increased. RAL induced a rapid but transient phosphorylation of ERK 1/2 in EPN cells but generated a sustained effect in CPEC. These findings suggest that RAL effects on PC growth control in vitro are cell-specific, depending on ERß or ERß/ERα relative expression levels. Moreover, this study demonstrated that RAL affected both transcriptional regulation and non-genomic signals, which resulted in the modulation of multiple signaling pathways of apoptosis and of cell cycle progression.

Simultaneous evaluation of the circulating levels of both Th1 and Th2 chemokines in patients with autoimmune Addison's disease.

BACKGROUND: Chemokines play a key role in the recruitment of the immune cells into the autoimmune process. Thus, the simultaneous evaluation of circulating levels of Th1-related chemokines, such as CX chemokine ligand 10 (CXCL10) and macrophage inflammatory proteins 1α (CCL3/MIP-1α), and Th2-related chemokines, such as macrophage inflammatory proteins 1 ß (CCL4/MIP-1ß) could be useful in the approach to some autoimmune diseases, including autoimmune Addison's disease (AAD). AIM: To evaluate plasmatic levels of MIP-1α, MIP-1ß, CXCL10 and adrenocortical antibodies in patients with AAD under treatment with corticosteroids. PATIENTS AND METHODS: Twelve women and 5 men (group 1) were divided in 2 subgroups: 9 subjects with isolated AAD (group 1a) and 8 with AAD associated with chronic autoimmune thyroiditis (group 1b). MIP-1α, MIP- 1ß and CXCL10 were evaluated in the serum of all patients and in 20 healthy controls, using a system for microarray suspension. RESULTS: The levels of MIP-1α, MIP-1ß and CXCL10 resulted significantly increased vs controls (p<0.001). An inverse significant correlation between the serum levels of MIP- 1ß and the duration of the disease was observed. CONCLUSION: High levels of MIP-1α and MIP-1ß associated with increased levels of CXCL10 in AAD seem to indicate a role of these chemokines in the autoimmune pathology of adrenal gland through the recruitment in loco of Th1 and Th2 cells. The simultaneous measurement of Th1-related chemokines (CXCL10 and MIP-1α) and of Th2-related chemokine MIP-1ß in the serum of patients with AAD would sustain a novel preliminary hypothesis on the immune microenvironment of chronic autoimmune inflammation within adrenal glands.

Efficacy of recombinant human follicle stimulating hormone at low doses in inducing spermatogenesis and fertility in hypogonadotropic hypogonadism.

BACKGROUND: Recombinant-FSH (rFSH) added to hCG at dose of 450 IU weekly is effective in inducing spermatogenesis in patients with hypogonadotropic hypogonadism (HH), but there are no data on the use of lower doses. AIM: This observational retrospective study evaluated whether 150-225 IU of rFSH weekly were able to induce spermatogenesis in HH men who failed to start it with hCG alone. SUBJECTS AND METHODS: Thirty-four patients with pre-pubertal onset HH (20-44 yr old) without adverse fertility factors were considered for this study. After hCG pre-treatment they received also either rFSH (Group 1) or highly purified urinary FSH (hpFSH) (Group 2) 75 IU sc 2 or 3 times weekly. Semen analysis was performed every 3 months during pre-treatment and the 1st yr of combined therapy. Patients were also invited to refer pregnancies in their partners during the subsequent 12 months. RESULTS: Total sperm count/ejaculate did not show significant difference between 2 groups, while a significantly higher forward motility was observed in Group 1 (p<0.05). The median times to achieve sperm output thresholds (first sperm appearance, sperm concentration >1.5 or >5 mil/ml) were significantly lower in Group 1 (p<0.04, 0.03, and 0.001, respectively). A tendency to a shorter time to pregnancy was shown in partners of Group 1. CONCLUSIONS: Our data indicate that lower rFSH week dose than that so far used was able to induce potentially fertilizing sperm output in HH men previously treated with hCG. The rFSH effects are comparable to those of hpFSH but with a trend to a faster outcome achievement.

Antipituitary antibodies in dutch patients with idiopathic hypopituitarism.

BACKGROUND: Despite extensive research, in the majority of patients with isolated growth hormone deficiency (IGHD) and multiple pituitary hormone deficiency (MPHD), the cause of their clinical picture remains unknown. Recent articles suggest that some cases of idiopathic growth hormone deficiency might be explained by a silent form of autoimmune hypophysitis based on the presence of antipituitary antibodies (APA) at high titers (>1:8). METHODS: We collected clinical data and serum from 71 patients participating in the Dutch HYPOPIT study. APA screening in 40 IGHD patients and 31 MPHD patients was performed by an indirect immunofluorescence method. APA, when present, were related to clinical and morphological pituitary findings. RESULTS: APA were present at high titers in 7 of 31 MPHD patients (23%) and 1 of 40 IGHD patients (2.5%). Among APA-positive MPHD patients, apart from growth hormone deficiency, all patients of pubertal age had gonadotroph defi- ciency, all had thyroid hormone deficiency and 50% had ACTH deficiency. CONCLUSION: The high frequency of APA in our idiopathic MPHD population indicates that, in 23% of the patients diagnosed with idiopathic MPHD, the hormone deficiencies might actually be caused by a silent form of autoimmune hypophysitis. Screening for APA should therefore be considered in all patients with 'idiopathic' MPHD.

Epidemiology, diagnosis, and treatment of male hypogonadotropic hypogonadism.

Hypogonadotropic hypogonadism (HH), or secondary hypogonadism, is a clinical condition due to an impairment of the pituitary function, characterized by low testosterone plasma levels associated with normal or low FSH and LH plasma levels. An impairment of gonadotropin secretion and, therefore, a reduced efficiency of spermatogenesis was reported to be frequently associated to conditions different from the classical causes of secondary hypogonadism. These conditions (metabolic, endocrine and eating disorders, physical exercise etc.) have been associated with a non-classical form of HH that could be called "functional" HH (FHH). FHH differs from the classical one by the evidence that gonadotropin levels are in the low-normal range, but are inadequate for the testosterone levels, that often are also in the low-normal range. This commentary aims at reviewing knowledge on the forms of male HH in order to indicate and discuss clinical context, diagnostic and therapeutic approach in the less known non-classical form, i.e. FHH.

Effect of long-term cabergoline therapy on the immunological pattern and pituitary function of patients with idiopathic hyperprolactinaemia positive for antipituitary antibodies.

OBJECTIVE: The occurrence of antipituitary antibodies (APA) in patients with idiopathic hyperprolactinaemia (IH) and the effects of dopamine agonists on these antibodies and long-term pituitary function outcome have been so far not evaluated. This longitudinal study was aimed at investigating, in patients with IH the occurrence of APA and the effect of cabergoline on the pituitary function and behaviour of APA. DESIGN: Sixty-six patients with IH were studied. APA (by indirect immunofluorescence) and pituitary function were investigated every year for 3 years. RESULTS: Seventeen patients resulted APA positive (Group 1) and 49 APA negative (Group 2). Eight patients of Group 1 (Group 1a) and 24 of Group 2 (Group 2a) were asymptomatic and then not treated; instead, nine patients in Group 1 (Group 1b) and 25 in Group 2 (Group 2b), showing symptoms of hyperprolactinaemia, were treated with cabergoline for 2 years. Among the untreated patients, during the follow-up, those with APA positive (Group 1a) showed an increase of APA titres and PRL levels with partial pituitary impairment in some of them; instead those with APA negative (Group 2a) persisted negative with normal pituitary function despite persistent hyperprolactinaemia. Among the treated patients, those with APA positive (Group 1b) showed normalization of PRL levels, APA disappearance and recovery of pituitary function (when initially impaired) during cabergoline treatment, persisting also at last observation (off-therapy). Instead all patients of Group 2b persisted with APA negative during the follow-up with normalization of PRL levels and stable normal pituitary function during cabergoline therapy but showing a further increase of PRL at the last observation. CONCLUSIONS: The presence of APA in some patients with IH suggests a possible occurrence of autoimmune hypophysitis at potential/subclinical stage; an early and prolonged cabergoline therapy could interrupt the progression to an overt clinical stage of the disease. However, the small amount of patients investigated suggests caution against generalization of our assumption and prompts to further controlled studies on a more numerous population to verify these conclusions.

Seminal anti-Müllerian hormone level is a marker of spermatogenic response during long-term gonadotropin therapy in male hypogonadotropic hypogonadism.

BACKGROUND: In adult men, anti-Müllerian hormone (AMH) levels are higher in semen than in serum, but the significance and control of its seminal secretion are still unknown. This study evaluated seminal and serum AMH levels during long-term gonadotropin therapy in men with hypogonadotropic hypogonadism (HH). METHODS: A total of 20 men with never treated prepubertal-onset HH received i.m. hCG to normalize testosterone (T) and induce puberty. Afterwards, 11 of them, requiring fertility, were treated with HCG plus recombinant FSH (rFSH) (75 IU) twice a week, whereas 9 continued to receive hCG alone for 12 months. Before and during therapy, serum AMH, inhibin B and T levels were assessed. Semen samples were also collected during therapy for sperm count and seminal AMH assay. RESULTS: HCG alone decreased basal high serum AMH and stimulated T and inhibin B levels. rFSH plus hCG increased seminal AMH levels, which were consequently significantly higher than with hCG alone, and positively correlated to sperm densities and testicular volumes at 3 and 12 months (P < 0.001). CONCLUSIONS: Our data demonstrate that rFSH, added to hCG, stimulates seminal AMH and spermatogenesis in HH. Thus, seminal AMH levels are under T and FSH control and are closely related to progression of spermatogenesis. Our results also suggest that an early seminal AMH increase may be a marker of good future response to gonadotropin therapy in HH.

Autoimmunity as a possible cause of growth hormone deficiency.

A possible autoimmune aggression to pituitary somatotrophs has been suggested by the occurrence of antipituitary antibodies (APA) directed against GH-secreting cells in some cases of GH deficiency (GHD) both in adults and in children and in some patients with autoimmune poliendocrine syndrome. We also detected APA in some patients with idiopathic short stature (ISS) and suggested that the presence of these antibodies could identify those of them prone to develop GHD. In fact, patients with ISS, resulted positive for APA at the first observation, during a longitudinal follow-up showed an impaired GH response to the stimuli in subsequent years suggestive of acquired GHD. Also in such patients we demonstrated that the target of APA were the somatotrophs and that an autoimmune attack to these cells may be the underlying cause of hormonal impairment in several children with GHD positive for APA. In this connection we suggested that in these patients an early iso-hormonal therapy with recombinant GH may be useful to interrupt or delay the progression towards a clinical GHD.

Natural and synthetic retinoids in prostate cancer.

Prostate cancer (PCa) is the most common cancer for men in Europe, North America, and some parts of Africa. Initially, growth of prostate cancer is usually androgen-dependent, but often it becomes androgen-independent after androgen-deprivation therapy. Managing hormone-refractory prostate carcinoma remains a difficult challenge for clinicians. Retinoids, vitamin A and its synthetic analogs are one of the most studied class of chemopreventive drugs for PCa. Retinoids play a key role in several vital functions as vision and development, and also exert anti-proliferative actions. Anti-proliferative effects of retinoids rely on the regulation of many biological processes, including differentiation, cell proliferation, and apoptosis. Retinoid actions are mediated by two classes of nuclear proteins called retinoic acid (RARalpha,beta and gamma and retinoic alpha,beta and gamma receptors, which are ligand-regulated transcription factors. Effects of both all-trans-retinoic acid (RA), the natural active derivative of vitamin A, and its synthetic derivatives, on prostate gland or prostate cell lines implicate retinoids in the regulation of prostate growth and suppression of PCa development. Deficient retinoid availability and action at the cellular level because of either decreased content or altered metabolism in PCa cells can play a key role in abnormal cellular differentiation pathways, and the loss of anti-proliferative effects. Here we review the in vitro and in vivo effects of retinoids in PCa.

Blindness impairs plasma growth hormone response to L-dopa but not to arginine.

To investigate how blindness influences GH secretion, we studied the GH response to L-dopa and arginine in 8 blind adult males and 10 normal age-matched control males. Arginine and L-dopa tests were performed in random order at least 1 week apart at 0800 h, and plasma GH was measured by RIA. The blind subjects showed GH responses to arginine similar to those in normal subjects [peak, 22.1 +/- 1.3 vs. 20.5 +/- 1.3 micrograms/L (+/- SE)], but their GH response to L-dopa was significantly reduced [peak, 5.4 +/- 1.3 vs. 20.3 +/- 2.4 micrograms/L (+/- SE); P less than 0.01]. Because L-dopa is believed to release GH by stimulating endogenous GHRH, whereas arginine may act by suppressing endogenous somatostatin secretion, we propose that blindness may impair GH release by inhibiting GHRH secretion.

L-Dopa stimulated growth hormone release in the blind.

Following L-Dopa administration (single oral dose of 500 mg), plasma Growth hormone (GH) levels significantly rose in 7 out of 8 normal subjects (aged between 64-83) and in 4 out of 8 blind subjects (aged between 60-88). The mean peak +/- SE in the 8 normal subjects was 24.9 +/- 4.9 ng/ml and in the 8 blind subjects was 11.5 +/- 2.8 ng/ml. These findings represent an additional anomaly in pituitary hormone release in the blind and may indicate that dopamine receptors in the hypothalamus are less sensitive to L-Dopa in blind people than in normal subjects.

Different expression patterns of somatostatin receptor subtypes in cultured epithelial cells from human normal prostate and prostate cancer.

The transcripts of five SRIH receptor subtypes (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) were investigated by RT-PCR in epithelial cells (EC) and stromal cells (SC) from primary cultures of five normal human prostates and six prostate cancers. Primary cultures of prostate EC were established in serum-free keratynocyte medium with 5% FCS, epidermal growth factor, and bovine pituitary extract; SC were cultured in MEM with 10% FCS. Total RNA was extracted from EC and SC using a modified guanidine thiocyanate method. RT-PCR was performed after deoxyribonuclease treatment, using SSTR1-, SSTR2-, SSTR3-, SSTR4-, and SSTR5-specific-primers and adding glyceraldehyde-3-phosphate dehydrogenase-specific primers as internal control. A PCR product of the expected size of 334 bp, corresponding to SSTR1, was expressed only in EC from prostate cancer, whereas the expected 461-bp product of SSTR2 was found only in EC from normal prostate. SSTR3 messenger RNA was undetectable in normal and cancer EC, whereas SSTR4 and SSTR5 were present in both cell types. SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 messenger RNAs were not expressed in SC from both normal and cancer prostates. The RT-PCR method clearly demonstrated SSTRs' expression in the human prostate EC in vitro with differences between normal and tumoral samples. Our results may explain the ineffectiveness of some SSTR2 selective SRIH analogues in the treatment of prostate cancer and suggest that the absence of SSTR2 could represent a growth advantage in prostate cancer.

Loss of estrogen receptor beta expression in malignant human prostate cells in primary cultures and in prostate cancer tissues.

The aim of this study was to investigate the expression of estrogen receptor (ER) beta and alpha genes in normal (N) and malignant (C) primary cultures of human prostate epithelial cells (PEC) and fibroblasts (PFC) and in the prostate tissue donors. Both ERbeta and ERalpha messenger ribonucleic acids were found by RT-PCR analysis in six NPECs and normal prostate tissues and in only one of six CPECs and in the respective cancer tissue donor. The other five CPECs and related cancer tissue donors and all normal and cancer PFCs expressed ERalpha messenger ribonucleic acid alone. Immunoblot analysis, using a polyclonal anti-ERbeta (C-terminal) antibody, demonstrated ERbeta protein in all NPEC lysates and in one of the six CPECS: ERalpha protein was expressed in both NPECs and CPECs when a polyclonal antibody directed against the ERalpha N-terminal domain was used. In contrast, ERalpha protein was not detected in two of the six CPEC lysates when ERalpha C-terminal monoclonal antibodies were used. Using a set of primers designed to amplify the region from exons 6-8, RT-PCR analysis demonstrated the absence of the expected transcript in these cells. The present study shows that the ERbeta gene is expressed together with ERalpha in normal prostates and NPECs, whereas it is barely detectable in prostate cancer and CPECS: Moreover, in some CPECs, the ERalpha gene may be transcribed in a changed protein, resulting from the expression of a deletion variant. Together, these data suggest that prostate malignancy is associated with a potential disorder of ER-mediated pathways.

Remission of subclinical adrenocortical failure in subjects with adrenal autoantibodies.

Idiopathic Addison's disease is a chronic organ-specific autoimmune disorder with a long subclinical period characterized only by the presence of adrenal autoantibodies (AA) with or without adrenal function failure. The aim of this longitudinal study was to evaluate the behavior of AA using, an indirect fluorescence method, and adrenal function in 20 AA-positive and 50 AA-negative patients screened by an investigation of a large population of organ-specific autoimmune disease patients without clinical Addison's disease. As controls, 100 normal age-matched subjects were tested only once. In the 20 AA-positive and 50 negative patients, AA and adrenal functional tests were evaluated every 4 months for 5 yr. The AA-positive patients were grouped into 5 adrenal functional stages, specifically: stage 0, normal adrenal function; stage 1, high PRA and low (or normal) aldosterone levels alone; stage 2, along with impaired cortisol response to ACTH, stage 3, along with increased ACTH levels; and stage 4, clinically overt Addison's disease. On the basis of the behavior of AA, the 20 positive patients were grouped as follows: group A, 11 patients with AA titer of 1:8 or higher at the first observation and persistently AA positive in subsequent observations, with titers ranging from 1:8 to 1:64; group B, 6 patients with initial AA titers of 1:8 or lower and AA disappearance in subsequent observations; and group C, 3 patients with AA titer of 1:32 or higher, undergoing corticosteroid therapy after the start of the study and showing AA disappearance in subsequent observations. With respect to adrenal function in group A, 2 patients initially in stage 1 and 1 patient initially in stage 2 did not progress to the upper stages, whereas 5 patients initially in stage 0 and 3 initially in stage 1 progressed subsequently to the upper stages, in 2 cases reaching overt clinical Addison's disease (stage 4). On the other hand, all of the patients of group B showed both a spontaneous disappearance of AA and recovery of adrenal function during the study span. Also, the 3 patients of group C showed disappearance of AA after corticosteroid therapy with recovery of adrenal function. None of the 50 patients who were initially AA negative became AA positive subsequently or showed impairment of adrenal function. We reached the following conclusions. 1) AA, even if present initially in some subjects without clinical Addison's disease, can subsequently disappear. 2) Restoration of adrenal function after disappearance of AA indicates that a spontaneous remission of subclinical adrenocortical failure can occur.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel case of multiple endocrine neoplasia type 2A associated with two de novo mutations of the RET protooncogene.

We report a novel case of multiple endocrine neoplasia type 2A (MEN 2A) associated with two mutations of the protooncogene RET. One affects codon 634 and causes a cysteine to arginine substitution; the second at codon 640 causes an alanine to glycine substitution in the transmembrane region. The two mutations were present on the same RET allele and were detected in germline and tumor DNA. Both mutations were de novo, i.e. they were not found in the DNA of the parents or relatives. Immunohistochemical and RT-PCR analysis showed that the pheochromocytoma expressed calcitonin as well as both RET alleles. A cell line established from the tumor and propagated in culture sustained the expression of RET and calcitonin, as did the original pheochromocytoma. Because the patient presented with medullary thyroid carcinoma and pheochromocytoma without parathyroid gland involvement, we speculate that this clinical picture could be correlated with the two RET mutations and to the unusual calcitonin production. This is the first report of a MEN 2A case due to two mutations of the RET gene and associated with a calcitonin-producing pheochromocytoma.

Longitudinal study of vasopressin-cell antibodies and of hypothalamic-pituitary region on magnetic resonance imaging in patients with autoimmune and idiopathic complete central diabetes insipidus.

Diagnosis of autoimmune central diabetes insipidus (CDI) is based on the presence of autoantibodies to AVP-secreting cells (AVPcAb) or the coexistence of other autoimmune polyendocrine syndromes; moreover, it can be also suggested by the presence of lymphocytic infundibulo-neurohypophysitis, evidenced by biopsy of pituitary stalk and/or by pituitary stalk thickening on magnetic resonance imaging (MRI). However, so far, in clinical CDI patients with lymphocytic infundibulo-neurohypophysitis, AVPcAb have not been investigated and in those with or without autoimmune polyendocrine syndromes (APS), longitudinal studies on the behavior of AVPcAb alone, or of both AVPcAb and hypothalamic pituitary imaging on MRI are lacking. Aim of this work was to investigate in these patients the occurrence of AVPcAb (by indirect immunofluorescence) and of pituitary stalk thickening (by MRI) and their longitudinal changes during a follow-up period. We studied 22 patients, aged 29-53, with APS and complete CDI, grouped as follows: 10 with recent onset (< or =1.5 yr) of CDI (group 1a) and 12 with CDI of long-term duration (> or = 7 yr) (group 1b); moreover, a group of 13 patients with apparent idiopathic CDI of recent onset (<1.5 yr) were studied. They were divided, on the basis of the detection of AVPcAb as follows: 5 AVPcAb positive patients (aged 19-26) classified as isolated autoimmune CDI (group 2) and 8 AVPcAb negative patients (aged 21-26), classified as true idiopathic CDI (group 3). All patients were evaluated yearly, along 5 yr, for AVPcAb and for hypothalamic-pituitary region imaging. At study entry, 8/10 (80%) of patients in group 1a and 7/12 (58.3%) in group 1b were positive for AVPcAb and persisted positive subsequently, during all the follow-up period, even if at lower titers. All patients in group 2 were positive and all those in group 3 were negative for AVPcAb and persisted positive and negative, respectively, for all the follow-up study. Among the AVPcAb-positive patients, only 5 in group 1a and 2 in group 2 showed also pituitary stalk thickening at the first observations, which however spontaneously disappeared subsequently indicating a possible lymphocytic infundibulo-neurohypophysitis. All patients in the studied groups showed loss of the hyperintense signal of the neurohypophysis on MRI at entry and during all the follow-up period. Results of this longitudinal study suggest: 1) AVPcAb, frequently present at high titers in recent phases of CDI, persist subsequently, even if at lower titers, several years after the onset of disease. 2) The occurrence of a lymphocytic infundibulo-neurohypophysitis suggested by the pituitary stalk thickening on MRI only in patients positive for AVPcAb confirms a further autoimmune variant of CDI also in these cases. 3) The longitudinal behavior of patients in group 3 suggests that the absence of AVPcAb at the onset of clinical idiopathic CDI is able to exclude a subsequent appearance of these antibodies and consequently an autoimmune involvement in CDI of these patients. Instead the finding of AVPcAb in several patients with only CDI, thought at first clinical observation as idiopathic, indicates that the prevalence of autoimmune CDI must be considered much higher than that so far reported.