Malignant gastric carcinoid causing ectopic ACTH syndrome: discrepancy of plasma ACTH levels measured by different immunoradiometric assays

Discrepancy of plasma ACTH levels measured by different immunoradiometric assays (IRMA) in a case with malignant gastric carcinoid causing ectopic ACTH syndrome was examined by gel chromatography and immunohistochemical analysis. A 49-year-old male was found to have a large gastric tumor, with muscle wasting, hypertension, diabetes and hypokalemia caused by hypercortisolemia. His plasma ACTH levels, although initially elevated, were found to be almost in normal ranges. The discrepancy of plasma ACTH levels was proven to be due to different IRMA kits used; the initial assay was performed by a kit that could recognize high-molecular weight (HMW) form as well as ACTH(1-39), but the later assay by another kit that could recognize only ACTH(1-39). Pathological examination of the gastric tumor was consistent with the diagnosis of malignant carcinoid. Immunohistochemical study revealed that immunoreactivity of proopiomelanocortin (POMC) was positive within the tumor cells, whereas those of ACTH and prohormone convertase 1/3 were negative. Molecular sieving analysis of patient's plasma by gel chromatography coupled with ACTH radioimmunoassay which could recognize HMW form and ACTH(1-39) and two different IRMAs revealed that the predominant form of ACTH was HMW form with a minor peak of ACTH(1-39). This is a rare case of ectopic ACTH syndrome caused by malignant gastric carcinoid with preferential production of HMW form of ACTH, possibly due to unprocessed POMC.     

Demonstration of nuclear 3,5,3'-triiodothyronine receptor proteins in gonadotrophs and corticotrophs in rat anterior pituitary by double immunohistochemical staining.

Previously we raised an antiserum (4BII) against nuclear T3 receptor (NT3R), which recognizes c-erb A/NT3R alpha 1 and beta, but not alpha 2. An immunohistochemical study using 4BII revealed that among the tissues examined, the cerebral cortex, anterior pituitary, and thyroid gland exhibited strong immunostaining in the nuclei. In the present study we examined the localization of NT3R proteins in individual cell types of rat anterior pituitary, using the technique of double immunostaining with 4BII and antibodies against pituitary hormones. The cryostat sections and paraffin sections of rat pituitary were incubated with 4BII at 4 C overnight. The NT3R proteins were visualized as brown in the nuclei by avidin-biotin peroxidase staining. The control sections incubated with an antiserum which had previously absorbed with c-erb A peptide or an inactive antiserum showed very poor nuclear staining under the same condition. After being washed with 0.1 M glycine-HCl buffer (pH 2.2) for 2 h to remove 4BII, the sections were incubated with antiserum against individual anterior pituitary hormones at 4 C overnight. Using the method of alkali phosphatase/naphthol/Fast blue staining, the peptide hormones in the cytosol were stained blue. Double staining with 4BII and anti-FSH beta or anti-LH beta anti-serum clearly demonstrated that gonadotrophs contained the NT3R proteins. Similarly, the NT3R proteins in corticotrophs were demonstrated by the immunostaining with 4BII and anti-ACTH antiserum. As expected, the NT3R proteins were present in somatotrophs, thyrotrophs, and lactotrophs. On the other hand, folliculo-stellate cells, which are nonhormone secreting cells and are identified by their S-100 protein immunoreactivity, were stained by 4BII only faintly. The present study demonstrated the existence of NT3R proteins not only in somatotrophs, thyrotrophs and lactotrophs but also gonadotrophs and corticotrophs, suggesting some action of thyroid hormone through the receptor in these cells.     

Leptin and the Pituitary

In 1994, Zhang et al. of Rockefeller University in New York reported the first successful complementary DNA (cDNA) cloning of leptin by the positional cloning method. Leptin was identified as the gene of ob/ob mouse in genetic obesity syndromes. It has very strong food intake control, and body weight and energy expenditure. The name "leptin" derived from the Greek word leptos, meaning "thin." We hereby review major advances leading to our current finding of leptin, leptin receptor and its structure, the outline of homozygote, and also influence of leptin in the pituitary. (The structure of leptin) The mouse obese gene has been localized to chromosome 6. With human leptin gene on chromosome 7q31.3, its DNA has more than 15000 base pairs and consists of three exons and two introns. For bioactivation of leptin the importance of disulfide-binding site is suggested. Human leptin which replaced the 128-th arginine with glutamine has the function of an aldosteron antagonist, which is reported to have the function of athrocytosis inhibition. The resemblance of leptin precursor of human, mouse and rat is very high, i.e., mouse and rat homology is 96% and mouse and human homology is 83%. (The structure of leptin receptor) The mutant gene, which is the cause of obesity, was shown on map on diabetic mouse (db/db) chromosome 4, and it was proven to be the same as the leptin receptor gene cloned by Tartaglia et all. Further studies have found the Zucker fatty rat (fa/fa) to be incorporated into a linkage map of rat chromosome 5, whose region of rat is the equivalent to the region of conserved synteny of the db/db mouse gene. The leptin receptor is glycoprotein consisting of a single transmembrane-spanning component. The primary structure of leptin receptor belongs to the cytokine-class1 family, the single membrane-spanning receptor, and is highly related to the gp130 signal-transducing component of the interleukin-6 (IL-6) receptor, the granulocyte colony-stimulating factor (G-CSF) receptor, and the leukemia inhibitory factor (LIF) receptor. The leptin receptor is known to have at least six existing isoforms (Ob-Ra, b, c, d, e, f) from the difference in splicing. (Homozygote Mutation of Leptin and Leptin Receptor :Hormone Secretion Disorders) The point mutation of ob/ob mouse and the splicing mutation of db/db mouse show remarkable obesity and hyperphagia. These obesity models show a reproduction disorder with both the male and the female, and they develop with homozygote. The cause is thought to be the gonadotropin secretory abnormality in pituitary. Three family lines report the cases of this deficiency, and it is considered that the secretory abnormality in pituitary develops into hypogonadotropic. These patients show low value in plasma FSHbeta (follicle stimulating hormone-beta and LHbeta (luteinizing hormone-beta which are produced from pituitary, and the plasma GnRH (gonadotropin releasing hormone) level is also low. Furthermore, the leptin receptor deficient family line was reported in 1998, in which case only the homozygote developed. The plasma leptin concentration of normal human is about 8.0 ng/ml, and this case with leptin receptor deficiency has high value of 500-700 ng/ml, which is the equivalent to the db/db mouse. (Role of Leptin in Hypothalamus-Pituitary-Periphery Function) The role of leptin which regulates pituitary hormones suggests the promotion the GHRH (growth hormone releasing hormone) secretion in hypothalamus-pituitary axis, with the possibility of the rise in secretion of GH (growth hormone) in pituitary, i.e. effects of icv (intracerebroventricular) infusion of leptin has spontaneously stimulated GHRH, which promotes GH secretion in the normal rats. On the other hand, topical treatment of GH3 (derived from a rat pituitary GH-secreting cell line) with leptin directly inhibits cell proliferation. The obesity model animals (ob/ob, db/db, fa/fa) have equally plump body compared to the normal models, which shows signs of sufficient growth. (Localization and Functional Relevance of Leptin and Leptin Receptor in Rodents Pituitary) Aside from being the food intake inhibitor and the energy control factor, leptin takes part in controlling the pituitary hormones. Promoting the secretion of GH, PRL (prolactin), TSHbeta (thyroid stimulating hormone-beta, FSHbeta/LHbeta, and inhibiting the secretion of ACTH (adrenocorticotropic hormone) are the major changes of pituitary hormones which are brought on by leptin. The expressive localization is specific, and immunohistochemistry (IHC) method recognized leptin in granular state in FSHbeta, LHbeta and TSHbeta positive cells. In our biochemical examination, the bulk of the expression of leptin is recognized in fraction of the secretory granule. In particular, FSHbeta cells had the highest percentage rate of colocalized leptin in rat pituitary. On the other hand, leptin receptor has been reported to be found only in normal rat pituitary, human pituitary adenoma, and respective cell lines in pituitaries by the RT-PCR method until now, but we disclosed for the first time the localization of leptin receptor on the plasma membrane of GH-secreting cells with the IHC method that has not been cleared so far. These findings show that leptin and leptin receptor have been expressed in different cells, and that the rat pituitary glands entertain paracrine mechanism between leptin (FSHbeta/LHbeta cells) and leptin receptor (GH cells). The function of paracrine in this pituitary suggests a new point of view in hypothalamus-pituitary axis, and it shall be concerned with many aspects such as hormone secretions and proliferation/inhibition. (Human Pituitary Adenoma) Preliminary report of leptin and leptin-receptor relationship with pituitary adenoma that has secretion abnormality has been filed, and its manifestation is being observed by the RT-PCR. Leptin and leptin receptor are expressed in most adenoma, and it is thought to function by autocrine and paracrine pathway in the adenomas. Leptin has been located in ACTH-secreting adenoma most frequently, especially in ACTH carcinoma. The leptin receptor is detected in all adenomas with high percentage rate, with both long and short forms, and then many cases of nonfunctioning pituitary adenomas, compared with other adenomas, have been reported to be positive with both long and short forms of leptin receptor as detected by RT-PCR. The HP75 cell line is derived from the nonfunctioning pituitary adenoma, which produces FSHbeta and LHbeta. The expression of leptin receptor in nonfunctioning pituitary adenoma, and the suppression of HP75 multiplication may lead to the possible hypothesis of leptin becoming one factor for the treatment of pituitary adenoma, especially in gonadotropin adenomas.     

Immunohistological evidence for renin in human endocrine tissues

The peroxidase-labeled antibody method and the avidin-biotin-complex method with antiserum to purified human kidney renin were used to identify renin in human endocrine tissues. Renin immunoreactivity was found in some large cells of the anterior pituitary, the zona glomerulosa and the zona reticularis of the adrenal, the Leydig cells of the testis, and the follicular epithelial cells of the thyroid and prostate glands. The specificity of the immunohistochemical reaction was confirmed by immunoabsorption tests. The specific localization of immunoreactive renin in each tissue suggests a possible role of renin in the function of these tissues.     

Immunocytochemical detection of Chlamydia trachomatis on cytological specimens

Chlamydia trachomatis can cause many kinds of diseases in humans, both men and women. Its diagnosis depends on the observation of intracytoplasmic inclusion bodies on smears. To observe intracytoplasmic inclusion bodies we applied the Papanicolaou and immunocytochemical techniques. Elementary and reticulate bodies were seen more easily using the immunocytochemical technique in 3 hours on gynecological and urethral specimens. The immunocytochemical technique is rapid, accurate and specific with unstained gynecological and urethral specimens as well as destained Papanicolaou smears. It appears that fixation is the most important factor in immunocytochemical observations of Chlamydia. Absolute methanol or ethanol was the fixation agent of choice based on our experience. After proper fixation, the immunocytochemical technique can be applied for routine examinations to detect Chlamydia trachomatis.     

Prohormone convertases (PC1/3 and PC2) in rat and human pancreas and islet cell tumors: Subcellular immunohistochemical analysis

Prohormons convertase 1/3 (PC1/3; also termed PC1 or PC3) and PC2 are enzymes that activate prohormones by cleaving the pairs of basic amlno acids. This mechanlsm was inltlally Interred lrom the series of several endocrine and neuroendocrine precursor protoh, inciudlng proinsulin and prolusion. To determine the cellular and sub cellular distribution of PC1/3 and PC2 in the rat snd human pancreas, Immunohlstochemistry was performed using polyclonal antlers against mouse PC1/3 (ST-28) and mouse PC2 (ST-29). These studles showed light and dsctron mlcroacoplc co-locailzation of Insulln, PC1/3 and PC2, and the coexistence of glucagons and PC2 In the pancreatic islets. This tendency of colocalizstion was also depicted In one case of human insulin and three cam of human glucagonomas, as well as In rat Insullnomas. in two cases of human Insullnomas, Incomplete processing of proinsulin was suggested by the absence of PC2. At the sub cellular level in the rat pancreatic lslet, the colocalizstion of PC1/3 and insulin, and that of PC2 and glucagons, were observed in the same secretor granules by immunoelectron, microscopy and Image analysis. These studles suggest that PC1/3 and PC2 can functlon with the specifictties In the processing of proinsulin and proglucagon Into their active forms, respectively, in the normal and neoplastic pancreatic islets.