Mammosomatotroph adenoma cells secrete both growth hormone and prolactin

Pituitary adenoma cells from a mammosomatotroph adenoma obtained from a 21-year-old female presenting with acromegaly and amenorrhea were investigated by sandwich cell immunoblot assay, immunohistochemistry, and electron microscopy. The new, simple technique of sandwich cell immunoblot assay could detect two hormones secreted in the same one cell, and found that 89% of mammosomatotrophs secreted both growth hormone (GH) and prolactin (PRL). Immunohistochemistry showed that the tumor cells were positive for both GH and PRL. Electron microscopy showed cells contained granules ranging in size form 150 to 500 nm. This is the first demonstration of both GH and PRL in the same mammosomatotroph cell. Sandwich cell immunoblot assay can measure the amount of secreted hormone, allowing a new approach to the investigation of mammosomatotroph adenomas.     

The relationship between cell proliferation activity and secretory activity in pituitary adenoma--a review of 63 cases

Determination of the cell proliferation activity of neoplasm is useful in making a prognosis. Immunohistochemical detection using MIB-1 monoclonal antibody has recently allowed us to assess tumor cell proliferation easily, because it can be performed on paraffin-embedded specimens and the results have been demonstrated to be positively correlated with the results of PCNA staining. In this study, surgical specimens of 63 pituitary adenomas were examined by immunohistochemical staining with MIB-1 monoclonal antibody. Twenty-nine cases were non-functioning pituitary adenomas, 20 were prolactin (PRL)-producing pituitary adenomas, and 14 were growth hormone (GH)-producing pituitary adenomas. The MIB-1 positive rates of the pituitary adenomas ranged from 0% to 6.46%. In the non-functioning pituitary adenomas, the MIB-1 positive rates ranged from 0% to 4.55% (mean : 0.76%), in the PRL-producing pituitary adenomas the MIB-1 positive rates ranged 0% to 6.46% (mean : 0.91%), and in the GH-producing pituitary adenomas the MIB-1 positive rates ranged 0% to 1.28% (mean: 0.58%). There were no significant differences between these values according to the results of the Wilcoxon signed-rank test. Although the size of the non-functioning pituitary adenomas was not correlated with their MIB-1 positive rate, tumor size was closely correlated with the interval between the onset of the initial symptoms and the date of surgery. In the PRL-producing pituitary adenomas, the MIB-1 positive rate was not correlated with serum PRL levels as an index of secretory activity, but was correlated with the PRL staining positive rate. Preoperative bromocriptine therapy proved effective in reducing tumor size and serum PRL levels, but had no effect on the MIB-1 positive rate. In the GH-producing pituitary adenomas, the MIB-1 positive rate was not correlated with serum GH levels as an index of secretory activity, but was closely correlated with the GH staining positive rate. All three groups included both invasive and noninvasive tumor types, but there were no close statistical correlations between the three tumor types.     

Some changes of receptor and postreceptor signal transduction regulated by somatostatin in pituitary hGH-secreting adenomas

OBJECTIVE: To investigate the disturbance in the function of SRIF receptor, Gi protein and Ca2+ channel in hGH adenoma cells and to evaluate their significance in the pathogenesis of pituitary hGH adenomas. METHODS: All 25 patients with pituitary hGH adenoma who were involved in this study had typical acromegalic manifestation and high fasting serum hGH levels of > 5.0 micrograms/L which were not suppressed to < 3.0 micrograms/L by oral glucose tolerance test. The pituitary hGH adenoma tissue obtained from transphenoidal operation was digested by collagenase and the dispersed adenoma cells were cultured in the monolayer. The effects of octreotide (SMS), a long-acting agonist of somatostatin, on hGH secretion and intracellular cAMP level were observed and the influences of pertussis toxin (PT), an inhibitor of Gi protein, and Ca2+ ionophore A23187 or KCl on the inhibitory action of octreotide on hGH secretion were also investigated in the cultured pituitary hGH adenoma cells. RESULTS: A total of 16.0% (4/25) of cultured pituitary hGH adenomas did not respond to octreotide (100 nmol). The inhibitory effect of octreotide on hGH secretion was not blocked by PT (50 ng/ml) and A23187 (10 mumol) or KCl (22.5 nmol) in 31.6% (6/19) and 35% (7/20) of hGH adenomas, respectively. The effects of octreotide on hGH secretion and intracellular cAMP levels were studied in 10 cultured hGH adenomas. Octreotide suppressed both hGH secretion and cAMP levels in 5 cases; inhibited only hGH secretion or the cAMP level in 3 cases and 1 case respectively; and affected neither hGH secretion nor cAMP level in the last case. CONCLUSION: There were abnormalities in the SRIF receptor and/or postreceptor signal transduction in 16.0% of hGH adenomas which did not respond to octreotide. The defects in Gi and/or Ca2+ channels were found in 52.4% (11/21) of hGH adenomas which had responded to octreotide. These defects might induce diminution of the inhibitory action of SRIF on hGH secretion and might be the causes of hypersecretion in some pituitary hGH adenomas.     

A chromogranin peptide is co-stored with insulin in the human pancreatic islet B-cell granules

The immunoreaction of a rabbit chromogranin A and B antiserum was studied in normal human pancreatic islets. By examination of consecutive light microscopical sections, it was revealed that, at high antiserum concentrations (1:2000 or less), the whole islet area was heavily labelled, although the peripheral glucagon (A)-cells were the most intense in their immunoreaction. At low antiserum concentrations (1:4000 or more) the A-cells still showed the same intense labelling reaction, but the central B-cells were weakly labelled. Electron microscopically, reactivity towards the chromogranin A and B antiserum and the monoclonal insulin antibodies was present in the same central electron-dense core of the B-cell secretory granules, as demonstrated after application of the immunogold technique at different antibody dilutions. In the A-cells, the chromogranin immunoreactivity was concentrated at the peripheral mantle of the secretory granules. The D-cell granules showed a weak immunolabelling. Examination of human islets with the monoclonal chromogranin A antibody LK2H10 revealed immunogold labelling only in the peripheral mantle of the A-cell granules, while the B-cell granules were unlabelled. The present results show that a chromogranin peptide is co-stored with insulin the in normal human B-cell secretory granules. Although the exact composition of this B-cell chromogranin is unknown, it is not identical to that of the chromogranin A present in the A-cell granules.     

Differential distribution of synaptotagmin I and rab3 in the anterior pituitary of four mammalian species

In the anterior pituitary of rat, gerbil, hamster and guinea pig, the presence and cellular distribution of the synaptic vesicle-associated proteins synaptotagmin I and rab3 were analyzed by immunoblotting and by immunocytochemical staining of serial semithin sections. Our results show that rab3 proteins are ubiquitously expressed in all endocrine cell types of both the anterior and intermediate lobe. In many cells, rab3 immunoreactivity was concentrated beneath the plasmalemma. This intracellular distribution coincided with the distribution of secretory granules, suggesting a possible association of rab3 proteins with the latter organelles. The staining patterns observed using two monoclonal rab3 antibodies with different isoform specificities are compatible with the recent suggestion that rab3B is the dominant rab3 isoform in anterior pituitary cells. However, we could demonstrate that also rab3A is present in endocrine adenohypophyseal cells, albeit at low levels. In contrast to rab3, synaptotagmin I immunoreactivity was only detected in a limited number of adenohypophyseal endocrine cells. Whereas the monoclonal synaptotagmin I antibody consistently failed to immunostain lactotrophs and endocrine cells of the intermediate lobe, other endocrine cell types displayed variable immunoreactivities towards this antibody. Although a low level of synaptotagmin I expression in the immunonegative cells cannot be excluded, the above observation may reflect a differential distribution of synaptotagmin isoforms in endocrine organs, as it has been described for the nervous system. Our study has established that endocrine cells of the anterior pituitary are endowed with proteins of the rab3 and synaptotagmin families which are generally thought to play important roles in the regulation of the trafficking and/or exocytosis of secretory organelles and, hence, probably fulfil similar functions in adenohypophyseal cells.     

Hepatitis C virus E2 protein purified from mammalian cells is frequently recognized by E2-specific antibodies in patient sera

The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDS-polyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzyme-linked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by beta-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s).     

The human external auditory canal, secretory system--an ultrastructural study

Human external auditory canal skin, with a special emphasis on the secretory system, was studied by light, transmission electron and scanning electron microscopy. Two types of secretory glands were observed: modified apocrine (ceruminous) and sebaceous. The sebaceous secretory cellere homogeneous; on the other hand, modified apocrine secretory cells contained heterogeneous secretory granules. They were ither dark granules or light granules. Evidence to support both the apocrine as well as the eccrine mode of secretion was noted in the modified apocrine gland. This finding is partly in agreement with early reports based on light microscopy with suggested only an apocrine mode and data based on transmission electron microscopy which showed only the eccrine mode. Significance of the secretion by the external ear canal and its role in a local immune defense system is discussed.     

Respiratory health of children at schools near a fertilizer plant

Two antibodies, one monoclonal and one polyclonal, were produced and used to identify Taenia solium eggs, using the enzyme-linked immuno-electrotransfer blot technique (EITB). Different life-stages of Taenia solium and T. saginata, including eggs from gravid proglottids recovered, post-treatment, from patients infected with the tapeworms, and eggs of Diphyllobothrium pacificum and Hymenolepis nana from other patients were tested with these antibodies. The monoclonal antibody only recognized the eggs and immature proglottids of T. solium. The polyclonal antibody, however, not only reacted with the eggs, cysticerci and immature proglottids of T. solium but also with the eggs and immature proglottids of T. saginata. The sensitivity and specificity of the EITB were both 100% using the monoclonal antibody but only 78% and 60%, respectively, using the polyclonal antibody. Diagnostic bands for T. solium eggs corresponded to proteins of 22.5 kDa using the polyclonal antibody and 22.5-37 kDa using the monoclonal antibody. Species-specific fluorescence was obtained with an anti-T. solium monoclonal antibody which bound to egg-derived oncospheres of T. solium but not to those of T. saginata.     

Characterization of two ELISAs for NGAL, a newly described lipocalin in human neutrophils

NGAL is a newly described member of the lipocalin protein family, secreted from specific granules of human neutrophils upon activation of the cells. Its ability to bind the bacterial chemotactic formylpeptide FMLP indicates, that NGAL may have modulatory effects on the immune response. We here describe monoclonal and polyclonal antibodies against NGAL, which can be used for Western blotting and immunohistochemistry, and furthermore describe two ELISAs using either exclusively the polyclonal anti-NGAL antibodies or the polyclonal and monoclonal antibodies in combination. The assays are equally specific, reproducible, accurate, and sensitive, with a detection limit of 32 ng/l. The antibodies and assays will be valuable tools in the future investigation of NGAL expression in inflammatory and malignant disorders and in the elucidation of the function of NGAL as a modulator of the inflammatory response.     

Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography

A subfamily of small GTP-binding proteins, rab, has been shown to be involved in regulation of vesicular traffic in eukaryotic cells. The goal of this study was to identify the rab proteins associated with atrial secretory granules. A [32P]GTP-overlay assay showed the presence of multiple small GTP-binding proteins on the atrial granules. By biochemical analysis, we have demonstrated that one of the small GTP-binding proteins associated with the atrial granules is a rab12 protein (rab12p), one of the rab proteins that are most closely related to a Sec4 protein of yeast. Association of rab12p with the atrial granules was confirmed by immunogold electron microscopy. Immunoprecipitation followed by immunoblot analysis with anti-rab12 antibody showed that in addition to atria, rab12p was expressed in multiple other organs and cell lines. These results suggest that rab12p may function in vesicular traffic in multiple diverse types of cells.     

Mechanism-based design of prolactin receptor antagonists

The mechanism of action of two forms of the prolactin (PRL) receptor was studied using analogs of human growth hormone (hGH). At low concentrations (approximately 1 pM), hGH binds and stimulates proliferation of Nb2 cells containing the 391-residue PRL receptor as well as murine lymphoid FDC-P1 cells transfected with the 591-residue hPRL receptor. However, at high concentrations (approximately 70 microM) hGH inhibits proliferation of both these cell lines. Such a "bell-shaped" hormone response curve was observed for hGH stimulation of the hGH receptor (Fuh, G., Cunningham, B.C., Fukunaga, R., Nagata, S., Goeddel, D. V., and Wells, J.A. (1992) Science 256, 1677-1680) and is consistent with the sequential formation of an active hormone-(receptor)2 complex in which hGH binds through a first site (Site 1) to a first receptor and then through a second site (Site 2) to a second receptor. By analogy to hGH activation of the hGH receptor, we find that hGH variants that are mutated in Site 1 or Site 2 are greatly reduced as agonists. Similarly, only Site 2 mutants are potent antagonists of either hGH or hPRL stimulated cell proliferation. These and other data support the notion that hGH and hPRL activate the PRL receptor by sequential dimerization and provide a rational basis for the design of potent antagonists to the prolactin receptor.     

In vivo characterization of sustained-release formulations of human growth hormone

Long-acting formulations of recombinant human growth hormone (rhGH) were prepared by stabilizing and encapsulating the protein into three different injectable, biodegradable microsphere formulations composed of polymers of lactic and glycolic acid. The formulations were compared in juvenile rhesus monkeys by measuring the serum levels of rhGH and two proteins induced by hGH, insulin-like growth factor-I and IGF binding protein-3 (IGFBP-3) after single s.c. administration. All three formulations, which differed principally in the composition of the polymer, provided sustained elevated levels of all three proteins for several weeks, and the rate of release of rhGH differed among the formulations consistent with the molecular weight of the polymer used. All three formulations induced a higher level of insulin-like growth factor-I and insulin-like growth factor binding protein than was induced by daily injections of the same amount of rhGH in solution. After three monthly injections of one of the formulations, both the rhGH and IGF-I levels remained elevated for nearly 90 days. Immunogenicity of the rhGH released from this formulation, as assessed by the incidence of seroconversion to hGH and the titer of anti-hGH antibody in both the rhesus monkeys and transgenic mice expressing rhGH, was no greater than that of the unencapsulated protein. In addition, the microsphere injection sites appeared normal by macroscopic evaluation between 1 to 2 mo after microsphere administration and by microscopic evaluation between 2 to 3 mo. These results show that serum levels of a therapeutic protein can be sustained for an extended period when encapsulated into different formulations of injectable, biodegradable microspheres.     

p53 Protein accumulates in Cushings adenomas and invasive non-functional adenomas

The p53 protein, a negative regulator of cell growth, plays an important role in the pathogenesis of many human tumours following gene mutation and/or deletion. We screened a large number of sporadic pituitary tumours for p53 protein accumulation suggestive of gene mutation. Samples were divided into benign adenomas (n = 95) and invasive tumours with local or distant invasion (n = 26). All main tumour classes were represented. Putative p53 mutations were detected by immunohistochemistry on paraffin-embedded sections using polyclonal CM-1 and monoclonal DO-7 and PAb1801 antibodies. Results were compared to normal post-mortem pituitary tissue controls (n = 17). p53 protein accumulation was detected in invasive tumours (16%), but only in corticotrophinomas (2/4) and non-functional tumours (4/15). In non-invasive adenomas, protein accumulation was observed only in ACTH-secreting tumours where 50% were positive (16/32). No protein accumulation was identified in any control tissue. These results indicate that p53 protein accumulation may play a role in the development of Cushings adenomas and in the progression of non-functional tumours to the invasive state.     

Localization of human intestinal defensin 5 in Paneth cell granules

Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells but recently also found to be produced by epithelial cells. We biosynthesized recombinant human intestinal defensin 5 (rHD-5) using the baculovirus-insect cell expression system. Since insect cells process defensin incompletely and secrete the precursor proHD-5, we substituted a methionine for an alanine at a likely processing site to allow selective chemical cleavage with cyanogen bromide, and rHD-5 was used to elicit polyclonal antibodies. By the immunoperoxidase-staining technique, the antibodies selectively stained Paneth cells of the normal adult small intestine. Immunogold electron microscopy further localized HD-5 to the Paneth cell secretory granules. Since some defensins exert activity cytotoxic to mammalian cells, we assayed the effect of rHD-5 on the human intestinal cell lines Caco2 and Int407. proHD-5 did not exert cytotoxic activity, and rHD-5 showed only minimal activity against Int407 and was inert against Caco2. Since Paneth cells release their granules adjacent to the mitotic cells of the intestinal crypts, HD could protect this cell population against invasion and parasitization by microbes.     

The homeobox-containing gene Wariai regulates anterior-posterior patterning and cell-type homeostasis in Dictyostelium

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.     

Modulations of human growth hormone receptor level on the cell surface

Using a monoclonal antibody (GHRP2-88) raised against the extracellular portion of human growth hormone receptor (hGHR), the mechanisms on modulations of cellular levels of hGHR were investigated in human IM-9 cells. Upon stimulation with human growth hormone (hGH), hGHRs on the cell surface are down-regulated through internalization and degradation of hGHR. For hGHR internalization, hGH-mediated dimerization of hGHRs, but not staurosporine-sensitive phosphorylation is required. For hGHR degradation, however, staurosporine-sensitive phosphorylation is necessary. In the absence of hGH, hGHRs on the cell surface are cleaved to release human growth hormone-binding proteins (hGH-BPs), probably by a metalloprotease. In the presence of hGH, the hGH-BP release was rather decreased based on the reduction in cell surface hGHRs. Thus, the cell surface level of hGHR may be regulated post-translationally by the two mechanisms depending on the external hGH levels.     

Shared receptor components but distinct complexes for alpha and beta interferons

Protease protection assays of apolipoprotein B100 (apoB) in digitonin-permeabilized HepG2 cells indicated that multiple domains of apoB are exposed to the cytosol through an extensive portion of the secretory pathway. The intracellular orientation of apoB in the secretory pathway was confirmed by immunocytochemistry using antibodies recognizing specific domains of apoB in streptolysin-O (STP-O)- and saponin-permeabilized HepG2 cells. Lumenal epitopes on marker proteins in secretory pathway compartments (p63, p53, and galactosyltransferase) were not stained by antibodies in STP-O-treated cells, but were brightly stained in saponin-treated cells, confirming that internal membranes were not perforated in STP-O-treated cells. An anti-apoB peptide antibody (B4) recognizing amino acids 3221-3240 caused intense staining in close proximity to the nuclear membrane, and less intensely throughout the secretory pathway in STP-O-permeabilized cells. Staining with this antibody was similar in STP-O- and saponin-treated cells, indicating that this epitope in apoB is exposed to the cytosol at the site of apoB synthesis and throughout most of the remaining secretory pathway. Similar results indicating a cytosolic orientation were obtained with monoclonal antibody CC3.4, which recognizes amino acids 690-797 (79-91 kD) in apoB. Two polyclonal antibodies made to human LDL and two monoclonal antibodies recognizing amino acids 1878-2148 (D7.2) and 3214-3506 (B1B6) in apoB did not produce a strong reticular signal for apoB in STP-O-treated cells. The anti-LDL and B1B6 antibodies produced almost identical punctate patterns in STP-O-treated cells that overlapped with LAMP-1, a membrane marker for lysosomes. These observations suggest that the B1B6 epitope of apoB is exposed on the surface of the lysosome. The results identify two specific regions in apoB that are exposed to the cytosol in the secretory pathway.     

Significant correlations between tissue factor and thrombin markers in trauma and septic patients with disseminated intravascular coagulation

The authors report on the morphological features of a pituitary adenoma that produced growth hormone (GH) and adrenocorticotropic hormone (ACTH). This hormone combination produced by a single adenoma is extremely rare; a review of the available literature showed that only one previous case has been published. The tumor, which was removed from a 62-year-old man with acromegaly, was studied by histological and immunocytochemical analyses, transmission electron microscopy, immunoelectron microscopy, and in situ hybridization. When the authors used light microscopy, the tumor appeared to be a bimorphous mixed pituitary adenoma composed of two separate cell types: one cell population synthesized GH and the other ACTH. The cytogenesis of pituitary adenomas that produce more than one hormone is obscure. It may be that two separate cells--one somatotroph and one corticotroph--transformed into neoplastic cells, or that the adenoma arose in a common stem cell that differentiated into two separate cell types. In this case immunoelectron microscopy conclusively demonstrated ACTH in the secretory granules of several somatotrophs. This was associated with a change in the morphological characteristics of secretory granules. Thus it is possible that the tumor was originally a somatotropic adenoma that began to produce ACTH as a result of mutations that occurred during tumor progression.     

Suppression of immunological response against a transgene product delivered from microencapsulated cells

A potential obstacle to successful gene therapy for some patients is the in vivo production of neutralizing antibodies against the recombinant therapeutic product delivered. To mimic this clinical situation, we implanted microencapsulated recombinant cells producing human growth hormone into C57B1/6 mice to provoke antihuman growth hormone antibody production. We then investigated the efficacy of different immunosuppressive treatments to inhibit the development of neutralizing antibodies. The experimental mice were treated with either an immunosuppressive drug (FK506 or cyclophosphamide), a cytokine (interferon-gamma [IFN-gamma] or interleukin-12 [IL-12], or a monoclonal antibody (anti-CD4, anti-gp39, or CTLA4-Ig). Serum human growth hormone and mouse anti-human growth hormone antibody levels were measured by enzyme-linked immunosorbent assay (ELISA) for 4 weeks. There were three patterns of response noted among the seven treatment groups. First, the mice receiving IFN-gamma, IL-12, anti-gp39, or CTLA4-Ig were similar to the untreated controls-no suppression of anti-hGH antibodies and no improvement in delivery of hGH. Next, the mice receiving FK506 or cyclosphosphamide showed > or = 90% suppression of antibodies but also no improvement in product delivery. Last, the mice receiving anti-CD4 showed almost complete antibody suppression over 1 month postimplantation. Furthermore, only anti-CD4 permitted a sustained level of human growth hormone delivery to day 28, in contrast to the controls whose human growth hormone delivery was undetectable by day 14 postimplantation. Hence, the use of anti-CD4 inhibited formation of neutralizing antibodies against a recombinant gene product delivered in vivo, and allowed prolonged delivery of a foreign protein. Its role as adjunct treatment for appropriate patients receiving gene therapy should be examined further.