Alpha and beta subunits of glycoprotein hormones in argyrophil pituitary tumors with small granule cells

A group of 33 functionless pituitary tumors with small argyrophil groups (SAG) were collected from a series of 200 pituitary adenomas (16.5% of all adenomas). Histologically, the tumors showed an unusually high frequency of trabecular patterns, perivascular pseudo-rosettes, and oncocytoid transformation. Immunoreactivity for glycoprotein hormone alpha-chain was found in more or less numerous cells of 20 cases (64.5% of SAG tumors). Thirteen of these cases also showed specific beta-chain immunoreactivity, especially for follicle-stimulating hormone (FSH) beta-chain, which was present in 11 tumors. Various admixtures of immature, oncocytic, sparsely granulated, and densely granulated cells were observed ultrastructurally, with prevalence of the latter cell variants in tumors showing immunoreactive cells and prevalence of the former cell variants in tumors lacking immunoreactive cells. It is suggested that some relationship may exist between SAG cell (glycoprotein hormone precursor cells?) tumors--or at least part of them--and glycoprotein hormone cell lines. Anyway, whatever their origin and interpretation, SAG cell tumors seem to represent a distinct clinicopathologic entity.     

Simultaneous production of the alpha-subunit of glycoprotein hormones and other hormones in pituitary adenomas

The immunohistological classification of 97 pituitary adenomas revealed in 34 cases alpha-subunit (a-su) positive cells in the tumor tissue. In 15 cases a-su was the only hormone found, in 11 cases the beta-subunits of the glycoprotein hormones could also be detected (10 cases with LH/FSH and 1 case with TSH). In 8 cases a-su was found simultaneously together with other hormones of the pituitary (ACTH and a-su in 1 case, GH and a-su in 4 cases, prolactin and a-su in 2 cases, prolactin, GH and a-su in 1 case). A-su could be demonstrated to be partly simultaneously produced together with these hormones in identical cells and secretory granules. Next to prolactin, the a-su was the second most frequently occurring hormone that could be detected immunohistologically in our material.     

Production of the alpha-subunit of glycoprotein hormones by pituitary adenomas

Positive immunoreactions with alpha-subunit antibodies were present in 43% of biopsy samples obtained from 147 subsequently operated pituitary adenomas representing all major endocrine types (57 endocrine inactive adenomas, 43 prolactinomas, 30 adenomas causing acromegaly, and 17 adenomas causing Cushing's disease or Nelson's syndrome). Marked variations of the incidence, however, were found among the individual endocrine groups. Positive reactions were present in 63% of endocrine inactive adenomas, 57% of adenomas causing acromegaly, 35% of ACTH-secreting adenomas, and 9% of prolactinomas. A positive alpha-subunit reaction was accompanied in a minority of cases only with positive glycoprotein hormone-beta-subunit reactions. There were 21 pure alpha-subunit adenomas in the group of endocrine inactive adenomas.     

Expression of glycoprotein hormones and intracytoplasmic distribution of cytokeratin in growth hormone-producing pituitary adenomas.

Sixteen growth hormone (GH)-producing pituitary adenomas were studied for the expression of glycoprotein hormone subunits and cytokeratin by light microscopic immunohistochemistry. Cytokeratin immunoreactivity was demonstrated in all adenomas, but its intracytoplasmic distribution showed two distinct patterns; a prominent, dot-like pattern and a diffuse, perinuclear pattern. Seven adenomas (type 1) were exclusively composed of cells with cytokeratin in a dot-like pattern, whereas 9 adenomas (type 2) comprised of cells with cytokeratin of perinuclear distribution. The expression of alpha-subunit of glycoprotein hormone was significantly different between the two types of adenomas; 8 of 9 adenomas of type 2 contained many alpha-subunit immunoreactive cells but none of type 1 adenomas showed any immunoreactivity. Only a small number of adenoma cells were positive for beta-subunit of thyrotropin stimulating hormone in 3 adenomas of type 2. beta-subunits of follicle stimulation hormone and luteinizing hormone were negative in all adenomas. These findings suggest that the expression of glycoprotein hormone subunits in GH-producing adenomas may be closely linked to their types distinguishable by the cytokeratin distribution pattern.     

Binding of bovine and porcine pituitary glycoprotein hormone alpha-subunit to TSH antibody in serum of patients with Graves' disease.

The characteristics of autoantibodies reactive with bovine (b) TSH were examined in the sera of six patients with Graves' disease selected on the basis of highly negative values in the TSH receptor assay. Test sera were incubated with other 125I-labeled pituitary glycoprotein hormones and their isolated subunits (alpha and beta) [human (h) TSH, bTSH, porcine (p) TSH, pFSH, bFSH, bLH and equine (e) chorionic gonadotropin (CG)] (purity was confirmed by gel-filtration on Sephadex G-100 and SDS-PAGE), and the antibody bound fraction was precipitated by the addition of anti-human gamma-globulin (goat). Almost all sera showed detectable binding to bTSH, pTSH, pFSH, pTSH-alpha, bFSH-alpha, bLH-alpha, but not to hTSH, hTSH-alpha, hTSH-beta, hFSH, hLH, hCG, pTSH-beta, bLH-beta, eCG-alpha. Exceptions were very low binding to bLH-beta by one serum and to pTSH-beta, by two sera. The level of binding (B/T%) of the patients' sera to pTSH-alpha, bFSH-alpha and bLH-alpha was 3.0-27.7%, 2.6-45.3% and 2.2-39.0%, respectively; that of sera from normal healthy adults was 1.9 +/- 0.3%, 0.8 +/- 0.2% and 0.9 +/- 0.2% (mean +/- SD), respectively. These results indicate that the TSH antibodies recognize mainly an epitope in the alpha subunit of bovine and porcine pituitary glycoprotein hormones (TSH, FSH, LH).     

Demonstration of pituitary tissue with 6 cells immunoreactive to pituitary hormones in a sacrococcygeal teratoma

A sacrococcygeal teratoma, containing mature-appearing anterior pituitary tissue, was first reported with the result of an immunohistochemical analysis for pituitary hormones. All kinds of adenohypophyseal endocrine cells were demonstrated in the anterior pituitary tissue in this teratoma. This study revealed that the anterior pituitary tissue being contained together with nerve tissues in a sacrococcygeal mature teratoma has the capacity to produce at least six anterior pituitary hormones.     

Sex steroid hormones in serum and tissue of benign and malignant breast tumor patients

The ability of breast tumors to synthesize sex steroid hormones is well recognized and their local production is thought to play a role in breast cancer development and growth. The aim of this study was to estimate local intra-tumoral and circulating levels of Estrone (E1), Estrone Sulfate (E1S), Estradiol (E2), Estriol (E3), and Testosterone (T) in 33 pre- and postmenopausal women with primary breast cancer in comparison to 12 pre- and postmenopausal women with benign breast tumors. The mean levels of the studied sex hormones were higher in serum and tumor tissue of breast cancer women than those with benign breast tumors apart from Testosterone which showed a significant decrease in pre- and postmenopausal women with breast cancer (P<0.001for follicular phase, P<0.05 for luteal phase, and P<0.005 for postmenopausal). The levels of the five hormones were significantly higher intra-tumoral than in serum of both benign and malignant breast tumor women with E1S as the predominant estrogen. There was only a positive significant correlation between serum and tumor tissue levels of E1 (rs=0.52, P<0.05 for follicular; rs=0.63, P<0.05 for luteal and rs=0.58, P<0.05 for postmenopausal) and a significant correlation between serum and tumor tissue of T (rs=0.64, P<0.05 for follicular; rs=-0.51, P<0.05 for luteal and rs=-0.81, P<0.04 for postmenopausal).     

Immunoelectron microscopic studies of GH and alpha subunit in GH secreting pituitary adenomas

9 of 15 cases of GH secreting adenomas showed the localization of GH and a subunit in the adenoma cells. GH and a subunit were frequently colocalized in the same adenomas. Immunoelectron microscopically, GH and a subunit were localized in secretory granules which were packed in the cytoplasm. Immunoelectron microscopic double staining (preembedding method and post-embedding method) showed colocalization of GH and a subunit in the same secretory granules. These data suggested corelease of GH and a subunit by the same secretory granules.     

Immunohistochemical demonstration of alpha1-antichymotrypsin and alpha1 -antitrypsin in the normal pituitary gland and its tumors

The immunohistochemical distribution of the protease inhibitors alpha₁-antichymotrypsin (alpha₁-ACT) and alpha₁-antitrypsin (alpha₁-AT) has been documented in the normal human pituitary gland and in a series of pituitary tumors. In normal gland, alpha₁-ACT was localized mainly in the dendritic folliculostellate cells, identified by immunopositivity for S 100 protein. A minority of endocrine cells also stained in 3 of 10 autopsy glands. Folliculostellate cells were identified in 11 of 28 tumors, and again, the distribution of alpha₁-ACT positivity corresponded to these cells. In 4 cases, there was staining of a small minority of tumor cells. Alpha₁-AT was localized to colloid in the microfollicles of the anterior lobe. In I normal gland, there was granular staining of endocrine cells. Alpha₁-AT was present in 5 tumors, in microfollicles and in scattered endocrine cells in 2 adenomas. These data would support a physiological role for alpha₁-ACT and alpha₁-AT in the pituitary gland. Their differing distribution might reflect different functions.     

Coding sequence mutations in the alpha subunit of propionyl-CoA carboxylase in patients with propionic acidemia.

Propionic acidemia is a rare autosomal recessive disorder of intermediary metabolism. It is caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric protein composed of two subunits, alpha and beta. PCC requires ATP and biotin as cofactors for the reaction, the latter enzymatically added onto the alpha subunit. We investigated coding sequence mutations in the alpha subunit of PCC by analyzing fibroblast RNA from propionic acidemia patients deficient in alpha subunit function by single-strand conformation polymorphism and direct sequencing. Five missense mutations and one short in-frame deletion were found among different patients. Four mutations were located in the putative biotin carboxylase domain, whereas the two others were within the 67-amino-acid C-terminal domain previously shown to be required to obtain biotinylation of the alpha subunit. We analyzed fibroblast extracts for the presence of a biotinylated alpha subunit by Western blot analysis using streptavidin coupled to alkaline phosphatase. Four of five cell lines failed to show a biotinylated alpha subunit, regardless of the position of the mutations within the coding sequence. Two mutations located in the biotinylation domain were expressed in an Escherichia coli-based system and shown to abolish biotinylation of the domain. The results suggest that most mutations have a severe impact on the stability or the functionality of the alpha subunit.     

Hydrophobic interaction chromatography of human serum alpha1-antitrypsin and alpha1-acid glycoprotein

The relative hydrophobicity of human serum alpha1-antitrypsin (AAT) and alpha1-acid glycoprotein (AGP) in comparison to various reference proteins was determined by hydrophobic interaction chromatography (HIC). Apolar character of glycoproteins was generated using three different cosmotropic salts, ammonium sulfate, sodium sulfate, sodium citrate and isocratic, or reversed linear gradient elution techniques. Human serum AAT and AGP showed different apolar properties on Fractogel EMD phenyl and propyl columns modulated either by the type and concentration of cosmotropic salts, or by the pH of the mobile phase. According to its higher carbohydrate content AGP proved to be more polar than AAT. Human serum AAT and AGP were pre-separated by Cibacron Blue F3G-A dye ligand affinity chromatography and based on their different hydrophobicity were fractionated and purified by HIC.     

Immunoexpression of inhibin alpha subunit, inhibin/activin betaA subunit and CD99 in ovarian tumors

OBJECTIVE: Anti-inhibin alpha and inhibin/activin betaA subunit and anti-CD99 monoclonal antibodies (mAbs) have recently been demonstrated to be able to label ovarian granulosa cells; thus, they may be of value in the diagnosis of granulosa cell tumors. The present study aimed to determine what combination of these mAbs may be useful for the differential diagnosis of sex cord-stromal tumors of ovary. DESIGN: Immunohistochemical analyses with anti-inhibin alpha and inhibin/activin betaA subunit antibody and anti-CD99 mAb were performed on 42 ovarian tumors, including sex cord-stromal tumors (29), ovarian epithelial cancers (10), and Krukenberg tumors (3). RESULTS: All sex cord-stromal tumors were positive for inhibin alpha subunit, and 17 cases (58.6%) of sex cord-stromal tumors were immunoreactive for inhibin/activin betaA subunit. Epithelial tumors and Krukenberg tumors were all negative for inhibin/activin betaA subunit except mucinous carcinoma, which showed strong cytoplasmic immunoreactivity. All sex cord-stromal tumors except one granulosa cell tumor showed membranous staining for CD99. A case of serous carcinoma and a case of mucinous carcinoma were positive for CD99, and the remaining epithelial tumors and Krukenberg tumor were all negative for CD99. CONCLUSIONS: The results of immunohistochemical analysis, together with literature review, suggest that inhibin alpha subunit may be a useful diagnostic marker for sex cord-stromal tumor of the ovary. In addition, anti-CD99 antibody may be useful for the differential diagnosis between ovarian tumors. Inhibin/activin betaA subunit has a limited usefulness in the differential diagnosis of ovarian tumor because of its wider immunoreactivity for both sex cord-stromal tumors and mucinous carcinomas. The differential diagnosis of sex cord-stromal tumors of the ovary would be better made with a combined use of both anti-inhibin alpha subunit and anti-CD99 mAbs.     

Localization of tissue antigens with the fluorescent antibody technique: application to human anterior pituitary hormones

Antisera have been prepared to adrenocorticotrophic hormone, thyrotrophic hormone and growth hormone extracted from human pituitaries and to gonadotrophin, and thyrotrophic hormone extracted from human urine.Precipitin, absorption-precipitin and gel-diffusion tests have been performed with these antisera.No conclusive evidence was obtained of the presence of a hormone-specific antibody in any of the antisera.Cross-reactions occurred indicating the presence of an antigen common to those extracts prepared from the pituitary and a further antigen, or antigens, common to those extracts prepared from the urine.Despite the absence of hormone-specific antibodies fluorescein-labelled samples of the antisera gave staining in the cytoplasm of cells in the anterior pituitary and did not stain other tissues.The implication of these results in the interpretation of tissue localization studies with labelled antisera is discussed.     

Development of a rapid, immunochromatographic strip test for serum asialo alpha1-acid glycoprotein in patients with hepatic disease

Serum asialoglycoprotein (desialylated glycoproteins) concentrations have been reported to be elevated in patients with hepatic disease as compared with that of normal subjects. We recently developed a solid-phase sandwich assay for asialo alpha1-acid glycoprotein (AsAGP) as a representative of the serum asialoglycoproteins and evaluated the utility of this AsAGP as a diagnostic marker for liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC). In this study, we developed a rapid, one-step immunochromatographic strip capable of specifically detecting AsAGP in serum specimens. We have produced a monoclonal antibody (mAb) to AGP, and based on ELISA and Western blot analysis, we have selected four hybridoma clones which generated mAbs to recognize AsAGP. In the immunochromatographic strip test, one mAb was used for conjugation with colloidal gold microparticles. Ricinus communis agglutinin (RCA) was immobilized onto a nitrocellulose membrane strip to form a result line in the path of chromatographic migration. Likewise, a control line was created above the result line by the immobilization of anti-mouse IgG. A serum specimen was then applied to the sample pad. The AsAGP in the sample specifically bound to the microparticles via mAb (As16.89) and co-migrated upward until the AsAGP was sandwiched with the immobilized lectin (RCA), revealing a visible result line. The colloidal gold microparticles without bound AsAGP continued to migrate, forming a visible control line. Thus, an AsAGP-positive specimen (>1.5 microg/mL) yielded a result line and a control line, whereas an AsAGP-negative specimen (<1.5 microg/mL) produced only a single control line. The entire test procedure was completed in less than 5 min. In order to examine the reliability of the testing procedures, we carried out the immunochromatographic strip test with 102 serum samples and compared the results of these tests with those obtained by ELISA. The two methods showed excellent correlation, with 83-100% above/below the cut-off value (1.5 microg/mL). Therefore, we concluded that the results of the immunochromatographic test are in excellent accordance with those of the sandwich ELISA.     

Protocol for the examination of specimens from patients with primary pituitary tumors

In an effort to improve the diagnosis of pituitary tumors, we propose a synoptic approach to pituitary pathology reporting that will provide clear information to endocrinologists, neurosurgeons, neuropathologists, and surgical pathologists to advance the diagnosis and classification of pituitary adenomas.