The glucose-dependent insulinotropic polypeptide receptor is overexpressed amongst GNAS1 mutation-negative somatotropinomas and drives growth hormone (GH)-promoter activity in GH3 cells.

Somatic mutations in the GNAS1 gene, encoding the α-subunit of the heterotrimeric stimulatory G protein (Gαs), occur in approximately 40% of growth hormone (GH)-secreting pituitary tumours. By altering the adenylate cyclase-cAMP-protein kinase A pathway, they unequivocally give somatotroph cells a growth advantage. Hence, the pathogenesis of somatotropinomas could be linked to anomalies in receptors coupled to the cAMP second-messenger cascade. Among them, the glucose-dependent insulinotropic polypeptide receptor (GIPR) is already known to play a primary role in the impaired cAMP-dependent cortisol secretion in patients affected by food-dependent Cushing's syndrome. In the present study, 43 somatotropinomas and 12 normal pituitary glands were investigated for GIPR expression by quantitative reverse transcriptase-polymerase chain reaction, western blotting and immunohistochemistry. Tumoural specimens were also evaluated for GNAS1 mutational status. The effect of GIPR overexpression on cAMP levels and GH transcription was evaluated in an in vitro model of somatotropinomas, the GH-secreting pituitary cell line GH3. GIPR was expressed at higher levels compared to normal pituitaries in 13 GNAS1 mutation-negative somatotropinomas. GIP stimulated adenylyl cyclase and GH-promoter activity in GIPR-transfected GH3 cells, confirming a correct coupling of GIPR to Gαs. In a proportion of acromegalic patients, GIPR overexpression appeared to be associated with a paradoxical increase in GH after an oral glucose tolerance test. Whether GIPR overexpression in acromegalic patients may be associated with this paradoxical response or more generally involved in the pathogenesis of acromegaly, as suggested by the mutually exclusive high GIPR levels and GNAS1 mutations, remains an open question.

Volumetric MRI analysis of hippocampal subregions in Cushing's disease: a model for glucocorticoid neural modulation.

Several preclinical studies have demonstrated neuronal effects of glucocorticoids on the hippocampus (HC), a limbic structure with anterior-posterior anatomical and functional segmentation. We propose a volumetric magnetic resonance imaging analysis of hippocampus head (HH), body (HB) and tail (HT) using Cushing's disease (CD) as model, to investigate whether there is a differential sensitivity to glucocorticoid neuronal damage in these segments. We found a significant difference in the HH bilaterally after 12 months from trans-sphenoidal surgical selective resection of the adrenocorticotropic hormone (ACTH)-secreting pituitary micro-adenomas. This pre-post surgery difference could contribute to better understand the pathopysiology of CD as an in vivo model for stress-related hypercortisolemic neuropsychiatric disorders.

Peroxisome proliferator-activated receptor gamma in the human pituitary gland: expression and splicing pattern in adenomas versus normal pituitary.

Pituitary adenomas are slow-growing tumours arising within the pituitary gland. If secreting, they give rise to well-known syndromes such as Cushing's disease or acromegaly; when hormonally inactive, they come to clinical attention often with local mass effects or pituitary deficiency. Peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor with a key role in fat and glucose metabolism, but also involved in several neoplasia, has recently been detected in pituitary adenomas. In the present study, we evaluated the occurrence and splicing profile of PPARgamma in 43 cases of pituitary adenoma of different subtypes and compared it to 12 normal pituitary glands. By real-time polymerase chain reaction, PPARgamma was expressed as much in adrenocorticotrophic hormone (ACTH)-secreting and ACTH-silent adenomas as in controls, with a moderate underexpression in somatotrophinomas and prolactinomas and overexpression in 54% of nonfunctioning pituitary adenomas (NFPA). There was no apparent qualitative change in the splicing profile of pathological pituitary glands, nor was the presence of specific isoforms with dominant negative effects against PPARgamma detected. Western blotting revealed similar expression levels in the different subgroups of pituitary adenomas and normal glands. Immunohistochemistry confirmed PPARgamma expression in approximately one-half of analysed samples. The intra- and intergroup differences observed in pituitary adenomas may represent new elements in the process of understanding the different clinical responses of Cushing's and Nelson patients to PPARgamma-ligand treatment. Moreover, the higher level of PPARgamma expression detected in the NFPA subgroup may suggest its possible role as a molecular target in these pituitary adenomas, paving the way for investigations on the effectiveness of treatment with thiazolidinediones in such patients.

The usefulness of combined biochemical tests in the diagnosis of Cushing's disease with negative pituitary magnetic resonance imaging.

OBJECTIVE: The etiological diagnosis of ACTH-dependent Cushing's syndrome is often a problem. In fact, no endocrine or radiological examination can conclusively distinguish the ectopic from the pituitary source of disease. The aim of our study was to evaluate the role of stimulation and suppression endocrine tests in the diagnostic and therapeutic approach of patients with Cushing's disease (CD) and negative pituitary magnetic resonance imaging (MRI), considering their post-surgical outcome in comparison with patients with CD and positive MRI. PATIENTS AND METHODS: We retrospectively analyzed 31 patients (25 women and 6 men, median age 40 +/- 15 years) with a confirmed diagnosis of CD who underwent transsphenoidal pituitary surgery by the same neurosurgeon between 2001 and 2005. Preoperative endocrine assessment included corticotropin-releasing hormone (CRH), desmopressin (dDAVP), and overnight 8 mg dexamethasone suppression tests (8-DST) in all patients. Fifteen patients had a normal pituitary MRI and sixteen had a clearly evident pituitary microadenoma. Bilateral inferior petrosal sinus sampling (BIPSS) was performed in patients with discordant biochemical results or with signs and symptoms highly suggestive of an ectopic source of ACTH. Post-surgical median follow-up was 38.4 +/- 22.0 months. RESULTS: Among patients with negative MRI, 60% had concordant positive endocrine tests and underwent neurosurgery without other examinations. BIPSS was performed in three other patients prompted by discordant endocrine tests (negative dDAVP) and in two patients with clinical suspicion of ectopic disease. Among patients with positive MRI, 87% underwent neurosurgery without BIPSS that was performed in two patients because of negative concomitant response to dDAVP and CRH tests. A pituitary adenoma, confirmed by pathological examination, was found in 40 and 81% of patients with negative and positive MRI respectively (P<0.05), corticotroph hyperplasia resulted more frequent in the group with negative MRI. Remission rate was not different between patients with negative and positive MRI (73 and 75% respectively; P=0.61) and between patients with negative MRI who did not undergo BIPSS and patients with positive MRI (P=0.56). The recurrence rate was also similar between groups (P=0.64), but higher, although not statistically different (P=0.07) in patients with corticotroph hyperplasia at histology. CONCLUSIONS: An accurate evaluation of presurgical endocrine tests results enabled us to reduce the number of BIPSS in patients with a negative MRI without any fallout on their post-surgical outcome. In the hands of an expert pituitary surgeon, the outcome after surgeryand the subsequent recurrence rate are much the same in patients with negative or positive MRI.

Effect of short-term therapy with recombinant human growth hormone (GH) on metabolic parameters and preclinical atherosclerotic markers in hypopituitary patients with growth hormone deficiency.

OBJECTIVE: This study examines the effects of growth hormone replacement on body composition, insulin sensitivity, lipid profile, endothelial dysfunction and carotid intima media thickness in patients with adult-onset growth-hormone (GH) deficiency. METHODS: Twelve patients with severe GH deficiency received GH replacement for one year. In all patients, the following parameters were evaluated before and after six and twelve months of therapy: fasting glucose, insulin levels and lipid profile, bone mineral density and body composition. Carotid intima media thickness and brachial flow-mediated dilatation were also evaluated by arterial ultrasonography at basal condition and after one year of therapy. RESULTS: No significant changes were seen in body weight and blood pressure, total fat and lean mass, or bone mineral density after six months of GH replacement. There was an increase in triglycerides (p = 0.05), while total and HDL cholesterol, blood glucose, insulin levels did not change significantly. After twelve months, an increase in lean mass and a decrease in fat mass (p < 0.01 vs. baseline), a decrease in insulin resistance (p < 0.01 vs. six months; p = 0.01 vs. baseline) and a decrease in triglycerides (p < 0.01) were observed. Intima media thickness was greater in GH deficiency than in controls (p = 0.01) before therapy, and was unchanged after twelve months of therapy, whereas the flow-mediated dilatation tended to improve (p = 0.05). CONCLUSIONS: GH replacement is able to reverse typical metabolic and body composition alterations in patients with adult GH deficiency after twelve months, but it is unable to revert the vascular alteration completely. Flow-mediated dilatation seems to be a more precocious marker of the remission of arterial damage.

Dopamine receptor subtype 2 and somatostatin receptor subtype 5 expression influences somatostatin analogs effects on human somatotroph pituitary adenomas in vitro.

Dopamine (DA) and somatostatin (SRIF) receptor agonists inhibit growth hormone (GH) secretion by pituitary adenomas. We investigated DA subtype 2 receptor (DR2) and SRIF receptor (sst) subtypes 2 and 5 expression in 25 GH-secreting pituitary adenomas and tested in primary culture the effects on GH and prolactin (PRL) secretion of sst agonists selectively interacting with sst2 (BIM-23120), sst5 (BIM-23206), and sst2 and sst5 (BIM-23244). All adenomas expressed sst2; eight adenomas expressed both sst5 and DR2, eight sst5 but not DR2, and eight DR2 but not sst5. One tissue lacked expression of DR2 and sst5. GH secretion was inhibited by BIM-23120 in all samples, while it was reduced by BIM-23206 only in adenomas not expressing DR2. BIM-23120's inhibitory effects correlated with sst2 and DR2 expression, whereas DR2 expression correlated inversely with BIM-23206 inhibitory effects on GH secretion. In seven mixed GH-/PRL-secreting pituitary adenomas, PRL secretion was inhibited in sst5-expressing tumors by BIM-23206, but not by BIM-23120. BIM-23244 reduced PRL secretion only in adenomas expressing sst2, sst5 and DR2. sst5 and DR2 expression correlated directly with BIM23206 inhibitory effects on PRL secretion. Our results suggest that adenomas expressing DR2 are less likely to respond to clinically available SRIF analogs in terms of GH secretion inhibition. Therefore, drugs interacting also with DR2 might better control secretion of pituitary adenomas.

Cerebral aneurysm and acromegaly: a case report.

Few cases are reported concerning the association between cerebral aneurysms and acromegaly, and this is the first case report documenting an increase in diameter of a cerebral aneurysm in persistent acromegaly. Persistently elevated GH plasma levels might promote an increase in diameter of cerebral aneurysms. An accurate follow-up in acromegalic patients is important, especially concerning the cerebrovascular system. Establishing the effectiveness and usefulness of this strategy will require future prospective studies.

Folliculo-stellate cells of human pituitary adenomas: immunohistochemical study of the monocyte/macrophage phenotype expression.

Folliculo-stellate cells (FS) represent a small percentage of anterior pituitary elements of still undetermined embryological origin. They are sparse among endocrine pituitary cells and are characterized by the lack of secretory granules and by the presence of few branching processes inserted between hormone-secreting cells. Although FS cell role is still under discussion, recent reports showed that they produce monocyte-derived cytokines able to influence the hormone production and modulate the immunoendocrine connections. In this study we applied three monocyte-macrophage markers (HAM56, KP1, HLA-DR) to 15 pituitary adenomas in order to ascertain whether FS cells belong to the macrophage lineage. In this case FS cells could be considered the resident macrophages of the pituitary. FS cells were identified according to the reactivity to S-100, GFAP and vimentin. We confirm that S-100 represents the most useful marker for these cells that were detected scattered between tumor cells in more than half of the adenomas. GFAP stained only a percentage of FS cells, while vimentin recognized in addition to stellate cells endothelia, perivascular and infiltrating macrophages. We were unable to detect the expression of the macrophage markers on S-100 and GFAP reactive cells. Indeed, HAM56, KP1 and HLA-DR-positive cells were mostly round, small size and located in the perivascular and septal positions where FS cells were never detected. Lack of expression of monocyte-macrophage lineage markers by FS cells in pituitary adenomas suggests their preferential neuroectodermal origin. However, further studies on normal human pituitary will be needed before ruling out a possible role for FS cells as resident pituitary macrophages.

GSTM1 and CYP2D6 genotype frequencies in patients with pituitary tumours: effects on P53, ras and gsp.

We describe studies to determine if susceptibility to pituitary tumours is associated with the putatively high risk GSTM1 null and CYP2D6 EM genotypes. Frequency distributions of these genotypes were similar in cases and controls though the frequency of CYP2D6 PM and GSTM1 B tended to be lower (P = 0.072 and P = 0.095 respectively) in the tumour group. Immunopositivity for p53 was found in 18/97 tumours. In these samples GSTM1 null (39%) and CYP2D6 EM (56%) frequencies were not different to those in controls. The frequencies of CYP2D6 PM and GSTM1 B in the p53 immunonegative tumours tended to be lower (P = 0.055 and P = 0.1185 respectively) than in controls. Mutations in gsp and ras were studied using the polymerase chain reaction and allele specific oligonucleotide analysis. Eight of 19 somatotrophinomas demonstrated mutations in gsp; frequencies of GSTM1 null and CYP02D6 EM were similar to controls. No ras mutations were identified in 55-tumour studies. The data indicate the GSTM1 null and CYP2D6 EM genotypes are not associated with altered expression of p53 or, mutation of gsp and ras in these adenomas and, suggest the CYP2D6 PM genotype is associated with a reduced risk of pituitary adenomas and, that GSTM1*B confers greater protection than GSTM1*A.

Patterns of ACTH response to oCRH in Cushing's disease: correlation with histological/immunocytochemical findings.

The most common lesion in Cushing's disease is an anterior pituitary adenoma. However, normal or hyperplastic corticotropic pituitary tissue has also been found in some cases. In an attempt to distinguish the patterns of ACTH response to oCRH in different forms of anterior pituitary hypersecretion, 17 patients with pituitary adenoma and 17 without pathological evidence of adenoma were studied. These patients underwent transsphenoidal pituitary surgery by the same surgeon and were retrospectively evaluated. The diagnosis of pituitary lesions was confirmed by microscopical and immunohistochemical studies. Patients without pituitary adenoma showed a higher and more prolonged mean plasma ACTH response than that observed in patients with pituitary tumors. In patients with pituitary adenoma, the peak ACTH response was observed within 30 min after oCRH administration, and was followed by a gradual decrease to basal levels in the following 30 min. In those cases in whom no pituitary adenoma was found, oCRH injection produced a marked increase in plasma ACTH levels during the first 60 min with a slower decline at the subsequent time points. The mean response curves of the two groups, analyzed by Beherens-Fischer nonparametric ANOVA, showed significant differences, either when they were compared globally (p < 0.01), or at single time points. Differences in ACTH response to oCRH stimulation support the hypothesis of different pathogenetic mechanisms leading to ACTH hypersecretion in Cushing's disease with and without pituitary adenoma.

Molecular genetic studies of sporadic pituitary tumors.

Tumor formation may result from the activation of dominant oncogenes or by inactivation of recessive, tumor suppressor genes. The role of such mutations in the development of pituitary tumors has been studied. Tumors from 88 patients, representing the 4 major classes of adenoma, were investigated. In DNA extracted from matched leukocyte and tumor samples, allelic deletions were sought with 15 probes identifying restriction fragment length polymorphisms on chromosomes 1, 5, 10, 11, 13, 17, 20, and 22. Evidence of amplification or rearrangement of 10 recognized cellular oncogenes (N-ras, mycL1, mycN, myc, H-ras, bcl1, H-stf1, sea, kraS2, and fos) was sought in tumor DNA. Activating dominant mutations of Gs alpha were detected using the polymerase chain reaction to amplify exons 7-10 and hybridizing the product to normal and mutant allele-specific oligonucleotides. Allelic deletions on chromosome 11 were identified in 16 tumors (18%) representing all 4 major subtypes. Deletions on other autosomes were observed in less than 6% of tumors. Three adenomas had deletions on multiple autosomes, 2 of these were aggressive and recurrent. Mutations of Gs alpha were confirmed to be specific to somatotrophinomas, being identified in 36% of such tumors in this series. No evidence of amplification or rearrangement of other recognized cellular oncogenes was found. Inactivation of a recessive oncogene on chromosome 11 is an important and possibly early event in the development of the four major types of pituitary adenoma, whereas activating mutations of Gs alpha are confirmed to be specific to somatotropinomas. Two aggressive tumors were found to have multiple autosomal losses, suggesting a multistep progression in the development of tumors of this phenotype.

Eukaryotic expression of recombinant human centromere autoantigen and its use in a novel ELISA for diagnosis of CREST syndrome.

The cDNA coding for the human full-length centromere protein B (CENP-B) was isolated from a liver cDNA library by oligonucleotide screening and extended at the 5' and 3' ends by linker addition. The cDNA was inserted into a modified baculovirus transfer vector which mediated high-level expression of recombinant human CENP-B with a histidine-hexapeptide as affinity ligand at its N-terminus in infected Spodoptera frugiperda (Sf9) insect cells. Based on the histidine-hexapeptide moiety, the recombinant CENP-B was purified to homogeneity by single-step affinity chromatography using metal chelating matrix. An ELISA established with the eukaryotically expressed and purified recombinant human full-length CENP-B demonstrated its excellent specificity, sensitivity and reproducibility for the measurement of autoantibodies directed to the human CENP-B (ACA-B) representing a diagnostic marker for CREST syndrome, an autoimmune rheumatic disease. In this study, all pathological sera from patients (n = 80) with serologically and clinically diagnosed CREST were positively assayed for ACA-B, whereas 399 sera obtained from blood donors and 82 out of 84 sera from patients with autoimmune rheumatic disorders which were unrelated to CREST were negative in the ELISA.

Association of somatotrophinomas with loss of alleles on chromosome 11 and with gsp mutations.

The molecular pathology of somatotrophinomas has been investigated by a combined search for dominant mutations of the gene encoding the Gs alpha protein and for recessive mutations involving chromosome 11q13, which contains the gene causing multiple endocrine neoplasia type 1 (MEN1). Somatotrophinomas and peripheral leukocytes were obtained from thirteen patients with acromegaly; one patient also suffered from MEN1. Five DNA probes identifying restriction fragment length polymorphisms from 11q revealed allele loss in pituitary tumors from five (four non-MEN1 and one MEN1) patients. Deletion mapping revealed that the region of allele loss common to the somatotrophinomas involved 11q13. An analysis for similar allelic deletions at 12 other loci from chromosomes 1-5, 7-9, 12-14, and 17 did not reveal generalized allele loss in the somatotrophinomas. These results, which represent the first report of chromosome 11 allele loss occurring in non-MEN1 somatotrophinomas, indicate that a recessive oncogene on 11q13 is specifically involved in the monoclonal development of somatotrophinomas. In addition Gs alpha mutations were detected in two non-MEN1 somatotrophinomas, one of which also revealed allele loss of chromosome 11. Thus, our results reveal that the development of somatotrophinomas is associated with alterations in both dominant and recessive oncogenes and further characterization of these genetic abnormalities will help to elucidate the multistep etiology and progression of somatotrophinomas.

Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques.

Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.

Production of recombinant rat interleukin-6 in Escherichia coli using a novel highly efficient expression vector pGEX-3T.

Interleukin-6 (IL-6) is one of the most important mediators of the acute phase reaction in liver. For the production of recombinant rat IL-6 in Escherichia coli, a previously isolated cDNA coding for the rat IL-6 was cloned into the modified novel expression vector pGEX-3T. The IL-6 cDNA was highly expressed as a fusion protein with the glutathione S-transferase (GST) at its C-terminus and rat IL-6 at its N-terminus. The GST-IL-6 fusion protein was controlled by a tac-promoter and could be induced very efficiently by isopropyl-beta-D-thiogalactopyranoside. The synthesized GST-IL-6 fusion protein was insoluble and precipitated intracellularly in E. coli. Using an advanced technique, the insoluble protein was solubilized and purified to homogeneity by affinity chromatography using immobilized glutathione in a one-step procedure.

Purification of recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen using the expression system pH6EX3 followed by metal chelating affinity chromatography.

A novel plasmid expression vector (pH6EX3) that directs the synthesis of a fusion protein with a histidine hexapeptide at its N-terminus and a foreign protein at its C-terminus was constructed. The fusion gene is controlled by a strong tac promoter, leading to high-level expression of recombinant protein in several bacterial strains; the protein is deposited mainly as an insoluble mass in inclusion bodies. The fusion protein can be purified from the insoluble cell fraction by one-step affinity chromatography based on the selective interaction between the histidine hexapeptide and a metal chelating matrix charged with Ni2+ ions. The principle of this new system was tested by expressing and purifying antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein autoantigen. With the use of column chromatography and pH gradient elution, about 25 micrograms recombinant protein/ml of bacterial culture was obtained.

Cloning and expression of antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen in Escherichia coli.

Autoantibodies directed against the 68-kDa (U1) ribonucleoprotein antigen are mainly found in sera of patients with mixed connective tissue disease. The corresponding cDNA was fragmented into four regions coding for the major antigenic epitopes A', B', C' and D'. All the epitopes were subcloned and expressed as fusion proteins with the glutathione S-transferase in Escherichia coli using the novel expression system pGEX that allows very high yields of recombinant proteins after a single-step purification. The sera of patients with the autoimmune disease were analyzed for the expressed recombinant proteins by an immunoblotting technique. All positive sera showed a patient-specific behavior and could be divided into four groups regarding recognition of the four antigenic epitopes of the 68-kDa (U1) ribonucleoprotein antigen. The epitope B' was reactive to all patient sera positively tested and classified as the marker antigenic epitope for the mixed connective tissue disease.

Primary intrasellar coccidioidomycosis simulating a pituitary adenoma.

The case of a 68-year-old woman who had relatively acute, unilateral ophthalmoplegia is reported. Radiological studies indicated a mass lesion involving the pituitary gland and left cavernous sinus. Pathological tissue obtained by the transsphenoidal approach revealed the presence of a Coccidioides granuloma. This pathological entity should be considered when evaluating patients with a pituitary mass and ophthalmoplegia.

Measurement of melatonin in blood by radioimmunoassay. Analytical considerations and clinical usefulness.

Melatonin assay has proved to be clinically useful for the diagnosis and investigation of several diseases (e.g., Cushing's syndrome, depressive disorders). We have evaluated the analytical performance of a radioimmunoassay for the determination of melatonin in plasma. The interference of some anticoagulants has been investigated, as well as the preliminary extraction of the hormone using a liquid-liquid and a solid-phase extraction method. The variation coefficients of the assay, within and between runs, were between 3.8% and 9.2% and between 4.1% and 10.5%, respectively. The well-documented circadian rhythm of melatonin secretion was confirmed in our healthy subjects. Preliminary results appear to confirm the significance of melatonin measurement in neurosurgical patients.