Biological aspects of pituitary tumors induced by synthetic salmon calcitonin (TZ-CT) in Sprague-Dawley rats. Immunohistochemical and ultrastructural studies.

Rat pituitary tumors induced by synthetic salmon calcitonin (TZ-CT) were studied by the indirect peroxidase-labeled antibody method, together with ultrastructure and serum hormone measurement. Immunohistochemically, TZ-CT-induced pituitary tumors showed staining for only rLH alpha subunit, and were negative for other peptide hormones including GH, PRL, alpha MSH and ACTH, and the beta subunit of glycoprotein hormones. Electron microscopic examination showed that the majority of tumor cells possessed numerous small secretory granules, 100 to 200 nm in diameter. The serum PRL concentrations of rats with TZ-CT-induced pituitary tumors were markedly elevated, but not beyond 130 ng/ml. From our data, TZ-CT-induced pituitary tumors are considered to be endocrinologically inactive and to produce alpha subunit. Furthermore, these tumors are thought to be potentially useful models of alpha subunit-producing pituitary tumors in humans. This is the first report to document the tumorigenesis of alpha subunit-producing pituitary tumors in rats after long-term treatment with calcitonin.     

Immunoreactivity of normal and neoplastic human tissue mast cells

Immunoreactivity of human tissue mast cells (TMCs) was studied in one case of solitary mastocytoma of the skin, three cases of malignant mastocytosis, and in six lymph nodes with reactive intrasinusoidal increase of TMCs. Immunohistochemically, TMCs reacted positively to antisera against vimentin, common leukocyte antigen (CLA), lysozyme, alpha 1-antitrypsin (alpha 1-AT), and alpha 1-antichymotrypsin (alpha 1-ACT) and to a monoclonal antibody (KiB3) that detects preferentially B-lymphocytes. Additionally, strong positive reactions to polyclonal antisera against adrenocorticotropic hormone (ACTH) and human peptide histidine isoleucine (PHI) and weaker reactions to antisera against leu-enkephalin and met-enkephalin were observed; all other antisera tested yielded negative results. Positive stainings for vimentin, CLA, alpha 1-AT, alpha 1-ACT, and lysozyme further support the hypothesis that human TMCs may be related to the myeloid-monocytic system. The positive reactivity of TMCs to antisera against ACTH, PHI, leu-enkephalin, and met-enkephalin has not been reported previously. These findings suggest that TMCs are able to store and/or produce regulatory peptides in addition to many other well-known, granule-bound mediators.     

Gel chromatographic characterization of immunoreactive adrenocorticotropin in patients with ACTH hypersecretion

Summary We investigated the molecular size of circulating immunoreactive ACTH by gel chromatography in patients with ACTH hypersecretion due to various disorders of the hypothalamic-pituitary-adrenal axis. 4 patients with Addison's disease, 2 with Nelson's syndrome, 4 with Cushing's disease, 6 with the ectopic ACTH syndrome (2 bronchial carcinoma, 1 medullary carcinoma, 1 metastatic islett cell carcinoma, 1 benign bronchial carcinoid and 1 patient with occult ectopic Cushing's syndrome) and 1 patient with hypersecretion of ACTH from a clinically nonfunctioning pituitary adenoma were studied. Analysis of the molecular size of immunoreactive ACTH was performed by gel chromatography on a Sephadex G-75 column (superfine, 100×1.5 cm) equilibrated with 1% formic acid. 2 ml fractions were collected and evaporated to dryness. The ACTH content of the recovered samples was determined by RIA. In Addison's disease, Nelson's syndrome and Cushing's disease the plasma showed a single peak of ACTH immunoreactivity at the expected position of 1–39 ACTH. In the ectopic ACTH syndrome the plasma of 4 patients revealed at chromatography at least one other peak eluting between the void volume and 1–39 ACTH suggestive of a high molecular weight form of ACTH whereas plasma of 2 patients showed only a single ACTH peak at the position of labeled 1–39 ACTH. The patient with a clinically non-functioning pituitary adenoma revealed a gel filtration pattern similar to the patients with ectopic ACTH syndrom and secretion of high molecular weight ACTH. We conclude that secretion of high molecular weight forms of ACTH is not a unique feature of the ectopic ACTH syndrome. It may therefore not serve as a marker of the ectopic Cushing's syndrome in the differential diagnosis of the ACTH dependent Cushing's syndrome. Vice versa, lack of high molecular weight ACTH does not exclude an ectopic source of ACTH secretion as cause of Cushing's syndrome.Abbreviations ACTH adrenocorticotropin - MSH melanocyte stimulating hormone - I iodine - POMC proopiomelanocortin - RIA radioimmunoassay Supported by the Landesamt für Forschung NordrheinwestfalenDedicated to Professor Dr. W. Kaufmann on the occasion of his 65th birthday     

The effectiveness of vasopressin as an ACTH secretagogue in cattle differs with temperament

By using the temperament selection criterion of exit velocity (EV), cattle typically exhibiting hypercortisolism and a blunted response to exogenous corticotrophin-releasing hormone (CRH) can be identified via individual behavioral responses to handling. To further characterize hypothalamic-pituitary-adrenal (HPA) axis dysfunction associated with bovine temperament, the present study compared pituitary and adrenal activity, following stimulation with exogenous vasopressin (VP), in steers with an excitable or calm temperament. Serial blood samples were collected via indwelling jugular cannula for 6 h preceding and 6 h following administration of a VP bolus. Plasma concentrations of adrenocorticotropic hormone (ACTH) and cortisol were quantified by RIA to determine pituitary and adrenal responsiveness within temperament groups. Cortisol concentrations in excitable steers during the pre-challenge period revealed an increased initial adrenal reactivity to interactions with humans. Subsequent acclimation to the experimental surroundings yielded greater baseline cortisol concentrations in the cattle with an excitable temperament. Pituitary stimulation with VP resulted in a greater ACTH output from the excitable compared to the calm animals. The data presented herein provide additional evidence that HPA axis function in cattle of an excitable temperament may be akin to a state of chronic stress. The bovine temperament model may be of further use to both decipher mechanisms associated with HPA dysfunction and to elucidate physiological phenotypes or pathologies that have parallels in other species.     

Comparison of the antibodies elicited by the individual structural polypeptides of foot-and mouth disease and polio viruses.

Antibody produced against preparations of VP1, one of the four structural polypeptides of foot-and-mouth disease virus, neutralized the virus and reacted with both full and empty particles in radioimmunoassays (RIA). Antiserum against VP2 reacted with artificial empty particles of the virus but not with full particles. In contrast, none of the individual polypeptides of poliovirus produced antisera which neutralized the virus nor reacted with it in RIA. However, antisera produced with VP1 and VP2 reacted with artificial empty particles in RIA.     

Quantitation of antibodies to IgM, IgD and beta2-microglobulin in antisera to chronic lymphatic leukemia lymphocytes.

Rabbit antisera were prepared to chronic lymphatic leukemia (CLL) lymphocytes and were tested for their reaction with radioiodinated, solubilized, cell surface proteins of normal and CLL lymphocytes. A pooled, hyperimmune antiserum precipitated at least 16 polypeptides from both normal and CLL lymphocytes as shown by sodium-dodecyl sulfate polyacrylamide gel electrophoresis. These polypeptides varied in molecular weight from 11,000 to 180,000. None was prominent or unique to the CLL lymphocytes, although four peptide bands in the CLL cells usually showed more radioactivity than their counterparts in normal cells. Precipitation with antisera of known specificity showed that the cell surface proteins from CLL and normal lymphocytes included HLA antigens and beta2-microglobulin. IgM and IgD were found in preparations of normal cells and in cells of 3 of 5 CLL patients. Of cells from the other 2 patients, one showed only IgD and the other no Ig. An antigen-binding capacity test was employed to quantitate the antibody content of a pooled anti-CLL lymphocyte serum. The antiserum reacted with all Ig classes; however, after absorption with light chains and F(ab')2 fragments, the serum retained activity only for IgM and IgD. The absorbed antiserum bound 45 microgram IgM, 1.5 microgram IgD and 4.5 microgram beta2-microglobulin per milliliter. These data indicate that rabbit anti-CLL lymphocyte sera fail to detect a qualitatively unique tumor-specific polypeptide on the surfaces of CLL cells. However, such antisera contain antibodies to many cell surface proteins including IgM, IgD, HLA antigens and beta2-microglobulin.     

ACTH response induced in capsaicin-desensitized rats by intravenous injection of interleukin-1 or prostaglandin E.

1. We investigated whether afferent nerves are involved in the development of adrenocorticotrophic hormone (ACTH) responses induced either by systemic administration of interleukin-1 beta (IL-1 beta) and prostaglandin E2, or by psychological stress. The capsaicin desensitization method was used to impair afferent C fibres and we compared the ACTH responses between capsaicin desensitized and vehicle pretreated control rats. 2. The present results showed that the capsaicin desensitized rats had significantly smaller increases in plasma ACTH than the control rats in response to intravenous injection of IL-1 beta or prostaglandin E2. 3. There were no significant differences between the capsaicin desensitized and control rats in the ACTH responses induced by cage switch stress. 4. The capsaicin desensitized rats responded to intravenous injection of corticotrophin releasing factor (CRF) with a greater increase in the plasma level of ACTH than the control rats, indicating that capsaicin pretreatment resulted in augmentation of pituitary gland sensitivity to CRF. 5. These results suggest that afferent neurons play an important role in the ACTH responses induced by systemic injection of IL-1 beta or prostaglandin E2.     

Calcitonin determination by a fast and highly sensitive enzyme amplified immunoassay

A colorimetric enzyme amplification system was used to develop an immunoassay for human calcitonin (hCT) with a sensitivity of 6 pmol/l, and intra- and inter-assay CVs of 12% and 11.8% respectively for the low pool, and 10% and 11.2% for the high pool. The mean recovery of added synthetic hCT (58.5 pmol) from the plasma of 10 patients was 110% (64.4 pmol). The correlation coefficient between radioimmunoassay (RIA) and amplified enzymo-immunoassay was found to be 0.96 (p 0.001). The assay was successfully applied to the measurement of elevated calcitonin levels in plasma from patients with medullary carcinoma of the thyroid (MCT). AEIA offered a reliable and sensitive alternative to RIA for calcitonin determination with the added advantage of convenience as the label employed was much more stable.     

Potentiation of the hCRF-induced release of ACTH in man by an opioid antagonist

Summary Administration of synthetic human corticotropin-releasing factor (hCRF; 2 µg/kg body weight) to six normal male subjects produced a significant rise in plasma ACTH, followed by an increase in circulating cortisol. Simultaneous treatment with the opioid antagonist naloxone (1.6 mg i.v. bolus, followed by an infusion at a rate of 1.2 mg/h) significantly potentiated the hCRF-induced rise in ACTH and enhanced the cortisol response to hCRF. It is suggested that naloxone acts by antagonizing an inhibitory ultra-short-loop feedback effect of coreleased -endorphin on pituitary corticotrophs, thereby amplifying the net effect of hCRF, i.e., the release of ACTH.ACTH adrenocorticotropic hormone - CRF corticotropin-releasing factor - hCRF human CRF - oCRF ovine CRF - EKG electrocardiogram - POMC proopiomelanocortin - RIA radioimmunoassay - S.E.M. standard error of the mean This study was supported by the Deutsche Forschungsgemeinschaft (Go 299/3-2; Mu 585/2-2)     

Abnormal pituitary function during melancholia: reduced alpha-melanocyte-stimulating hormone secretion and increased intact ACTH non-suppression.

In order to investigate pituitary alpha-melanocyte-stimulating hormone (alpha-MSH), intact (1-39 structure) adrenocorticotropic hormone (ACTH), and adrenal cortisol secretion, we measured 8 a.m. plasma levels of those hormones before and after administration of 1 mg dexamethasone in 39 depressed inpatients and 10 healthy controls. We found a significantly lower baseline alpha-MSH secretion in melancholic patients as opposed to healthy controls. There were no significant relations between alpha-MSH secretion on the one hand and ACTH or cortisol secretion on the other. Dexamethasone did not affect the 8 a.m. alpha-MSH circulating levels. The post-dexamethasone intact ACTH and cortisol values were significantly higher in melancholics as compared with healthy, minor and simple major depressed subjects. ACTH non-suppression was defined as post-dexamethasone intact ACTH greater than or equal to 12 pg/ml. ACTH non-suppression was found to be more sensitive (70%) and specific (100%) for melancholia than cortisol non-suppression. By means of pathway analysis we have established that cortisol non-suppression during a severe depression is completely determined by an augmented ACTH escape from suppression by dexamethasone. It is concluded that the assay of post-dexamethasone intact ACTH could, in the future, replace post-dexamethasone cortisol determination.     

Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.     

Immune response of the guinea pig to bovine parathyroid hormone antigen: influence of the booster dose on antisera titer and sensitivity

Thirty guinea pigs were immunized with 40 microgram of bovine parathyroid hormone, bPTH(1-84), and divided into 6 groups with a similar primary response. Each group was boosted twice with a different dose of antigen (0.04 to 40 microgram). The titer (30% binding of tracer) and affinity (% displacement with 320 pg of bPTH(1-84) were studied. The primary response was maximal at 61 days; titers were 1 x 10(-4) or less and affinity was low. The response was maximal 25 days after each booster. After the first, the highest titers, 5.2 +/- 2.6 x 10(-4) (mean +/- S.D.) were seen in the 40 microgram group and a positive correlation was observed between the dose of antigen and the titer (r = .7386, p less than 0.001) for doses greater than 0.6 microgram. This remained true after the second booster although titers were lower in all groups. Affinity was greater after the first booster (35.85 +/- 20.38%, n = 30) than after the second (23.24 +/- 20.73, n = 30, p less than 0.0025), but was similar in all groups. Selected antisera reacted with antigenic determinants in regions 1-34, 53-84 and 35-53 of the bPTH molecule. Cross-reactivity with human PTH(1-84) was maximal in the 53-84 region. In conclusion, antibodies against all regions of bPTH can be raised in the guinea pig. Once a primary response is elicited, best titers are achieved by boosting with high doses of antigen without any detrimental effect on antisera affinity.     

Biological Aspects of Pituitary Tumors Induced by Synthetic Salmon Calcitonin (TZ–CT) in Sprague-Dawley Rats

Rat pituitary tumors induced by synthetic salmon calcitonin (TZ–CT) were studied by the indirect peroxidase-labeled antibody method, together with ultrastructure and serum hormone measurement. Immunohistochemically, TZ–CT-induced pituitary tumors showed staining for only rLHα subunit, and were negative for other peptide hormones including GH, PRL, αMSH and ACTH, and the α subunit of glycoprotein hormones. Electron microscopic examination showed that the majority of tumor cells possessed numerous small secretory granules, 100 to 200 nm in diameter. The serum PRL concentrations of rats with TZ–CT induced pituitary tumors were markedly elevated, but not beyond 130 ng/ml. From our data, TZ–CT-induced pituitary tumors are considered to be endocrinologically inactive and to produce α subunit. Furthermore, these tumors are thought to be potentially useful models of α subunit producing pituitary tumors in humans. This is the first report to document the tumorigenesis of α subunit producing pituitary tumors in rats after long-term treatment with calcitonin.     

Null cell adenomas of the pituitary gland. An immunohistochemical study

Fourteen null cell adenomas of the pituitary gland were examined immunohistochemically with antisera against three general neuroendocrine markers and 22 hormones. All cases showed positive immunostaining for neuron-specific enolase, ten cases for synaptophysin, and six cases expressed chromogranin immunoreactivity. Hormone immunoreactivity was detected in a few cells in ten of the 14 cases studied and the number of hormones demonstrated in each case was one or two. Thyroid-stimulating hormone was detected in five of the 14 cases, gastrin in four, beta-endorphin in two, calcitonin gene related peptide in one, prolactin in one, and follicle-stimulating hormone in one.     

MEDULLARY CARCINOMA OF THE THYROID WITH ECTOPIC ACTH SYNDROME

An autopsied patient with medullary carcinoma of the thyroid and ectopic ACTH syndrome is reported. A microadenoma of pancreatic islet coexisted in this case, which is assumed to be of D cell origin. Immunohistochemical study revealed decreased number of pituitary ACTH cells. Some of them showed Crooke's degeneration. Hormone assay study of tumor tissue and plasma disclosed abnormal ACTH, β-MSH as well as calcitonin. Somatostatin and Substance P were also demonstrated in tissue. Histologically the tumor showed solid alveolar pattern with a minor part consisting of small cell variant and this histologic variation is discussed.     

Pituitary adenomas. An immunohistochemical study of hormone production and chromogranin localization.

Tumors from 42 surgically resected pituitaries and from 13 autopsy cases were studied immunohistochemically with polyclonal antisera to 7 anterior pituitary hormones and with a newly developed monoclonal antibody directed against human chromogranin for evaluation of the distribution of chromogranin in normal and neoplastic pituitaries. In addition, a prospective study was done for assessment of the prevalence, morphology, and endocrine cell types of pituitary tumors in 100 autopsy subjects. When these 55 pituitary adenomas were examined with monoclonal antibody (LK2H10) directed against human chromogranin, selective staining of normal adenohypophyseal cell types and pituitary tumors was observed. Most null-cell adenomas (12/14) were positive for chromogranin, whereas all prolactin (PRL)-producing adenomas (19/19) were negative. Growth hormone (GH) adenomas were focally positive (9/9). All oncocytomas (2/2), 1 thyrotropin (TSH) adenoma, and a follicle-stimulating hormone/luteinizing hormone adenoma were positive for chromogranin. One or more adenomas were present in 14% of the autopsy cases. The tumors occurred most frequently in patients in the fifth through the seventh decades of life. Immunohistochemical staining of 13 adenomas revealed 1 TSH, 1 ACTH, and 4 PRL-producing tumors, whereas 7 other tumors, which were null-cell or undifferentiated adenomas, failed to stain for any of the seven principle pituitary hormones. These results indicate that antibody LK2H10 to human chromogranin is useful in the immunohistochemical characterization of pituitary adenomas. Incidental pituitary microadenomas from autopsy-derived pituitaries most commonly produce PRL, or they belong to the null-cell or undifferentiated tumor group.     

ACTH secretion in adrenocortical disorders

Secretion of cortisol is under stimulatory regulation by ACTH, and cortisol secreted, in turn, inhibits ACTH secretion by the pituitary. Therefore, measurement of plasma ACTH is indispensable in the diagnosis of the adrenocortical diseases. The adrenal cortex is included in the hypothalamo-adenohypophysial-adrenocortical system, and the pathogenesis of these disorders must be evaluated. An interaction between the hypothalamo-adenohypophysial-adrenal system and the immune system have been suggested. We studied the effect of interleukin-1 (IL-1) on ACTH secretion by cultured rat pituitary cells in vitro. Our results suggest that IL-1 stimulates the ACTH secretion by enhancing its synthesis by the pituitary, rather than ACTH release.     

Cytodifferentiation and immunocharacteristics of adenohypophysial cells in the toad, Bufo melanostictus

Summary Employing the unlabelled antibody enzyme technique cytodifferentiation, immunocharacteristics and topographical distribution of melanotropic (MSH), adrenocorticotropic (ACTH), thyrotropic (TSH), prolactin (PRL), gonadotropic (GTH) and growth hormone (GH) secreting cells in the embryonic/larval as well as adult pituitary gland of the common Indian toad, Bufo melanostictus, have been studied by using antisera raised in rabbit against mammalian hypophysial hormones. Immunoreactive MSH and ACTH cells appear first in the dorsocaudal and rostral regions of the pituitary anlage (PA) at stage 21 (Gosner's classification) of the embryonic development. This is followed by the differentiation of TSH and PRL cells at stage 22 in the midventral and central regions of the PA respectively. Finally, at stage 23 the GTH cells appear in the fostral and the GH cells in the caudal regions of the PA. With the progress of the development, cells showing immunoreactivity to various antisera gradually increase in number, size, granular content and finally occupy the characteristic adult disposition. The MSH cells comprise the pars intermedia. In the pars distalis (PD) the ACTH cells are localized in the rostroventral region, TSH cells in the central region and the GH cells in the dorsocaudal region. However, GTH and PRL cells are distributed throughout the PD.Dedicated to Late Dr. Maurice P. Dubois     

Antisera to human cytomegalovirus produced in hamsters: reactivity in radioimmunoassay and other antibody assay systems.

Hamsters immunized with human cytomegalovirus (CMV) concentrated and purified by polyethylene glycol precipitation and density gradient centrifugation produced antisera with high titers of specific viral antibody, and which showed no significant reactivity with human host cell components. The antisera had high titers of CMV antibody in complement fixation, indirect fluorescent-antibody (FA), and neutralization tests, but titers obtained by indirect radioimmunoassay (RIA) were markedly higher. The antisera were used to follow the development of CMV antigen in infected host cells by indirect RIA and indirect FA staining. Virus-specific antigen was first detectable by RIA at 8 h after infection, and by FA staining at 16 h; cells contained optimal amounts of antigen for RIA and FA assays at 72 to 100 h postinfection. Immune globulins from the antisera were labeled with 125I for use in direct RIA. The labeled globulins gave highly specific reactions with CMV-infected cells, including those infected with low-passage isolates, and showed no reactivity with cells infected with other human herpesviruses or certain other human viruses.