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Biomedical subjects

M Scanarini

Publications and source records attributed to M Scanarini.

At least 19 recordsLinked to original sources

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.

Adenoma↗

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.

Adult↗

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.

Animals↗

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.

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.

Adenoma↗

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.

Acromegaly↗

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.

Base Sequence↗

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.

Autoantibodies↗

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.

Amino Acid Sequence↗

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.

Adenoma↗

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.

Cloning, Molecular↗

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.

Adult↗

Giant-cell granulomatous hypophysitis: a distinct clinicopathological entity.

Idiopathic giant-cell granulomatous hypophysitis is a chronic inflammatory disorder of the pituitary gland. It presents clinically as a sellar mass lesion with pituitary insufficiency and/or hyperprolactinemia, and is radiologically indistinguishable from a pituitary tumor. In most of the previously reported cases the documentation of the disorder has been limited to autopsy tissue. Only a few cases documented by biopsy have been recorded. Four cases are presented here with radiological, endocrinological, and surgical findings. The appropriately documented cases collected from the literature and the present series are reviewed and the incidence, patterns of clinical and radiological presentation, and operative management of this disorder are discussed. This entity should be considered when evaluating patients with a pituitary mass and evidence of hypopituitarism and hyperprolactinemia.

Adult↗

Corticotropin releasing hormone stimulation test: diagnostic aspects in Cushing's syndrome.

The effect of exogenous ovine Corticotropin-Releasing Hormone (oCRH) on plasma ACTH and cortisol levels was investigated in 10 normal volunteers and in 37 patients with Cushing's syndrome (26 with pituitary-dependent disease, 5 with an adrenal adenoma, 2 with an adrenal carcinoma and 4 with bilateral nodular hyperplasia). In all normal subjects and in patients with Cushing's disease, oCRH 100 micrograms as a bolus produced an increase in both plasma ACTH and cortisol. The peak of ACTH occurred after 15-30 min, while plasma cortisol showed highest levels between 30 and 60 min after oCRH administration. The hormonal response in Cushing's disease showed great variability with a clear hyperresponsiveness at least in 6 out of 26 patients with Cushing's disease. A slight and delayed response occurred in 3 cases of bilateral nodular adrenal hyperplasia, while a fourth case showed hyperresponsiveness similar to that found in pituitary-dependent Cushing's disease. No response was observed in patients with an adrenal tumor. Eleven patients with Cushing's disease were tested before and 1 month after pituitary microadenomectomy. After surgery basal cortisol levels were reduced in 10 and became unresponsive or less responsive to oCRH. ACTH patterns were variable with a normal response only in few cases. Although this test seems of limited value in the diagnosis of hypercortisolism, it is a useful tool to differentiate some types of Cushing's syndrome (adrenal tumor from pituitary-dependent Cushing's disease). Variable patterns of response in cases with bilateral nodular adrenal hyperplasia limit the usefulness of this test in recognizing this rare form of hypercortisolism.

Adolescent↗

Pituitary microadenomas: surgical results and morphological findings.

The results of transsphenoidal microsurgery in treating 22 patients with pituitary microadenomas are reported. Histological and ultrastructural comparative studies were performed in attempting to obtain a more accurate morphological classification of pituitary microadenomas.

Adenoma↗

The acromegaly--gigantism syndrome. Report of four cases treated surgically.

Four cases of growth-hormone-secreting pituitary adenoma, with associated aspects of acromegaly and gigantism, are reported in patients aged 12-26. All of the patients had macroadenomas and were treated surgically, three by the transsphenoidal approach and one with a transfrontal craniotomy. Histologic examination revealed eosinophilic adenomas in three of the cases and a mixed eosinophilic--chromophobe adenoma in one, all with cellular irregularities (mitosis and cellular and nuclear polymorphism), local invasivity, or both. Because surgical treatment did not produce complete normalization of growth hormone levels, radiotherapy followed the operations in all four cases. In our opinion, the treatment of acromegalic gigantism poses more therapeutic problems than that of simple acromegaly, with combined treatment (surgical, radiation, and medical) often being necessary.

Acromegaly↗