Detection of inhibin α and βA subunits (inhibin A, B, activin A,AB) in the human pituitary gland and in pituitary adenomas by immunohistochemistry and nonradioisotopicin situ hybridization

Inhibin and activin are gonadal hormones produced in human ovaries. They are known to act on anterior pituitary cells to regulate the synthesis and secretion of follicle-stimulating hormone (FSH). The purpose of the present study was to determine the localization of inhibin and activin subunits α and βA as endocrine markers in the human normal pituitary gland and pituitary adenomas, using immunohistochemistry andin situ hybridization (ISH) methods. Pituitary tissues from surgical and autopsy materials were fixed in 10% formalin and embedded in paraffin. Five normal pituitary glands and 79 pituitary adenomas were immunostained with the avidin-biotin peroxidase complex (ABC) method using polyclonal antibodies against inhibin and activin subunits α and βA. The other antibodies against anterior pituitary hormones used in this study were as follows: antigrowth hormone (anti-GH), antiprolactin (anti-PRL), antiadrenocorticotropic hormone (anti-ACTH), anti-FSHβ, antilutenizing hormone (anti-LH) β, antithyroid-stimulating hormone (anti-TSH) β, and antiglycoprotein α-subunit (anti-α-SU). We analyzed gene expressions of subunits α and βA by nonradioisotopic ISH in pituitary adenomas. In the normal human pituitary glands, inhibin and activin subunits α and βA immunoreactivities were found diffusely in the cytoplasm of anterior pituitary cells. The percentage of subunit α-immunopositive cells was 40% of the anterior pituitary cells. Subunit βA immunoreactivities were observed in about 15% of the anterior pituitary cells. By the double-staining method, subunit α immunoreactivity was detected in all types of anterior pituitary cells, and it was colocalized most frequently with GH and α-SU-positive cells. Subunit βA immunoreactivity was colocalized predominantly with PRL, FSH-β, LH-β, and α-SU. Among the 79 adenomas, 75 cases (94.9%) were positive for subunit α, and 50 cases (63.3%) were positive for subunit βA. Subunit βA was positive in tumor cells with the following incidences: GH adenomas, 3 of 14 (21.4%); PRL adenomas, 5 of 8 (62.5%); ACTH adenomas, 6 of 6 (100%); TSH adenomas, 7 of 7 (100%); nonfunctioning adenomas, 29 of 44 (65.9%), including gonadotropin-positive, 16 of 22 (80.0%). The ISH signals for subunits α and βA were strongly expressed in gonadotropin-positive adenomas among the nonfunctioning adenomas. The mRNA signals were low and infrequent in the GH-producing adenomas. Inhibin and activin subunit α localization did not demonstrate cell-type specificity in pituitary adenomas. In contrast, subunit βA demonstrated predominant positivity in the functioning pituitary adenomas (ACTH- and TSH-secreting) and nonfunctioning adenomas (including gonadotropin-positive adenomas). The present results suggest that the functional role of inhibin and activin in the differentiation of cells in normal human pituitary glands and adenomas is present in subunit βA.     

A Serum-Free System for Primary Cultures of Human Pituitary Adenomas

We report the successful use of a serum-free culture system for primary cultures of human pituitary adenomas. The system utilizes histiotypic suspension culture with low protein-binding membrane inserts that enable cells to retain their three-dimensional tissue configuration, closely mimicking the growth pattern in vivo. A serum-free defined medium was developed with CMRL-1969 (Connaught, Willowdale, Ontario, Canada) supplemented with 0.375% albumin bovine Fraction V, 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL sodium selenite, 30 μg/mL putrescine, 6.85 × 10⁻¹¹ M hydrocortisone, and 3.7 × 10⁻¹¹ M tri-iodothyronine (T₃). We analyzed eight surgically resected human pituitary adenomas. Basal pituitary hormone secretion measured by radioimmunoassay of pituitary hormones was compared with hormone hypersecretion in vivo and with control cells of the same tumors cultured in CMRL-1969 with 10% fetal calf serum. The light microscopic, immunocytochemical, and ultrastructural morphology of cells cultured in this serum-free histiotypic system was compared with cells cultured in serum-supplemented media and with cells cultured on collagen-coated plastic; all cultured cells were compared with the morphology of surgically resected tissues of the same specimens. Basal pituitary hormone secretion during 24-hour incubations correlated with the clinical patterns of hormone excess; the data were similar in serum-enriched and serum-free cultures, however, hormone secretion decreased less rapidly in the serum-free cultures. Cells maintained in the histiotypic culture system closely resembled the corresponding surgically resected tumor using the morphologic parameters and were better preserved than those plated in collagen-coated plastic wells. This comparative study indicates that this serum-free histiotypic culture system provides an ideal method of examining pituitary adenomas in vitro without altering the profile of hormone secretion and cell morphology documented in vivo. This system can be used to examine the production and effects of a wide range of hormones and growth factors that have been implicated as causative agents in pituitary tumorigenesis.     

Pituitary Adenoma with Neuronal Choristoma (PANCH): Composite Lesion or Lineage Infidelity?

Fifteen cases of the rare association of pituitary adenoma and neuronal choristoma (PANCH) were investigated by histology, immunohistochemistry, and electron microscopy. Acromegaly was apparent clinically in 11 patients and was equivocal in 1, and 3 lesions appeared to be nonfunctioning. Histology revealed various proportions of chromophobic PA and nervous tissue consisting of neuronlike cells and neuropil. Immunohistochemistry documented growth hormone (GH) in every PA, including those unassociated with clinical acromegaly. In contrast, the NCH component showed no consistent immunohis-tochemical profile. Most frequent reactivities were for the pituitary hormone a subunit, thyroid-stimulating hormone, and GH, whereas only a few cases displayed scattered positivity for GH-releasing hormone. Low-molecular weight keratin tested positive in PAs and in a few cells and processes of an NCH. A few fibrous bodies were immunoreactive for neurofilament protein. Electron microscopy revealed sparsely granulated GH cell adenoma, neurons, and neuropil. Cells intermediate between PA and neurons were numerous in 1 lesion. The present morphologic findings as well as lack of GH cell hyperplasia and the consistent association of NCH with but one type of PA do not support the causative role of NCH in the initiation of PA, as proposed previously. It appears that NCH is the result of neuronal differentiation within sparsely granulated GH cell adenomas.     

Pituitary Adenoma with Neuronal Choristoma (PANCH): Composite Lesion or Lineage Infidelity?

Fifteen cases of the rare association of pituitary adenoma and neuronal choristoma (PANCH) were investigated by histology, immunohistochemistry, and electron microscopy. Acromegaly was apparent clinically in 11 patients and was equivocal in 1, and 3 lesions appeared to be nonfunctioning. Histology revealed various proportions of chromophobic PA and nervous tissue consisting of neuronlike cells and neuropil. Immunohistochemistry documented growth hormone (GH) in every PA, including those unassociated with clinical acromegaly. In contrast, the NCH component showed no consistent immunohis-tochemical profile. Most frequent reactivities were for the pituitary hormone a subunit, thyroid-stimulating hormone, and GH, whereas only a few cases displayed scattered positivity for GH-releasing hormone. Low-molecular weight keratin tested positive in PAs and in a few cells and processes of an NCH. A few fibrous bodies were immunoreactive for neurofilament protein. Electron microscopy revealed sparsely granulated GH cell adenoma, neurons, and neuropil. Cells intermediate between PA and neurons were numerous in 1 lesion. The present morphologic findings as well as lack of GH cell hyperplasia and the consistent association of NCH with but one type of PA do not support the causative role of NCH in the initiation of PA, as proposed previously. It appears that NCH is the result of neuronal differentiation within sparsely granulated GH cell adenomas.     

Immunohistological studies on clinically silent pituitary adenomas

Clinically inactive adenomas represent 30.7% of all pituitary tumors in our surgical collection of 616 cases. Ninety-six tumors were studied immunohistologically with many antibodies for their hormone content. Morphological classification of these adenomas reveals oncocytic adenomas in 42 cases (44%), small cell chromophobe adenomas in 33 (34%), large cell chromophobe adenomas in 14 cases (15%), undifferentiated mucoid cell adenomas in 4 cases (4%) and undifferentiated acidophil adenomas in 3 cases (3%). Immunohistological studies performed with the six pituitary hormones GH, prolactin, ACTH, TSH, LH and FSH and additionally with α-subunit demonstrated nearly all possible combinations of hormones in adenoma cells. The most frequently occurring (29%) was LH (in 3% of adenomas alone); α-subunit followed in frequency (24%), with FSH present in 21%. Combinations of 2 hormones were detected in 16%, of 3 in 13% and of more than 3 hormones in 2%. All 6 hormones and α-subunit were negative in 48% of adenomas. It must be concluded 1) that many clinically silent adenomas are LH- or FSH- or α:-subunit-positive and therefore probably originate from gonadotropic cells, and 2) that clinically silent adenomas of acidophil cell type or mucoid cell type are rare. Although many of these adenomas apparently do not secrete the hormone which they immunohistologically contain, determining the plasma levels of the gonadotropins, and especially the α-subunit in clinical studies, may obtain a suitable and helpful clinical marker in the diagnosis of “endocrine inactive” adenomas, and especially of their recurrences.     

The osmotically and histamine-induced enhancement of the plasma vasopressin level is diminished by intracerebroventricularly administered orexin in rats

The effects of the centrally administered neuropeptides orexin-A on water intake and vasopressin (VP) secretion were studied in male Wistar rats (180–250 g). Different doses (10, 30, and 90 μg/10 μl) of the orexins and the specific orexin receptor-1 (OX₁) antagonist SB 408124 (30 μg/10 μl) were administered intracerebroventricularly (i.c.v.) under anaesthesia, and the water consumption was measured during 6 h. A plasma VP level elevation was induced by histamine (10 mg/kg) or 2.5% NaCl (10 ml/kg) administered intraperitoneally (i.p.). The plasma VP levels were measured by radioimmunoassay. Increased water consumption was observed after the administration of 30 μg/10 μl orexin-A. There were no changes in basal VP secretion after the administration of different doses of the orexins. A significant increase in plasma VP concentration was detected following histamine administration. After 2.5% NaCl administration, there was a moderate VP level enhancement. Intracerebroventricularly administered orexin-A (30 μg/10 μl) blocked the VP level increase induced by either histamine or 2.5% NaCl administration. The inhibitory effects were prevented by the specific OX₁ receptor antagonist. In conclusion, the orexins increased water consumption. After 30 μg/10 μl orexin-A administration, the polydipsia was more pronounced. The OX₁ receptor antagonist significantly decreased the polydipsia. Histamine or hyperosmotic VP release enhancement was blocked by previously administered orexin. This inhibition was not observed following OX₁ receptor antagonist administration. Our results suggest that the effects of the orexins on water consumption or blockade of the histamine and osmosis-induced VP level increase are mediated by the OX₁ receptor.     

垂体生长激素细胞腺瘤的电镜及免疫电镜观察

24 cases of growth hormone(GH)-producing pituitary adenomas were studied with electron microscopy and immunoelectron microscopy by protein A-gold complex, 6 cases were identified as densely granulated GH adenoma and 15 cases as sparsely granulated GH adenoma, among which 4 cases were proved by immunoelectron microscopy to be containing granules with prolactin(PRL) activity simultaneously. Intracytoplasmic fibrous bodies were often seen in the sparsely granulated cells anyhow, not all those cells with fibrous bodies possess the secretory granules with GH activity, and fibrous bodies were also detected in some PRL cells of certain mixed type adenoma. This suggests that fibrous bodies might not be the specific morphological feature of pituitary growth hormone cell adenomas.     

GH-, PRL-, POMC-, beta-TSH-, beta-LH-, beta-FSH-mRNA in gonadotroph adenomas of the pituitary by in situ hybridization in comparison with immunostaining and clinical data

In situ hybridization (ISH) enables the visualization of specific mRNA for pituitary hormones. Our collection consists of 40 surgically removed pituitary adenomas that were classified as follicle stimulating hormone/luteinizing hormone (FSH/LH) cell adenomas by structure and by immunostaining (IH) for all pituitary hormones. All forty adenomas were regarded as clinically inactive. The aim of our study was to examine nonfunctioning adenomas by ISH for demonostration of mRNAs for all pituitary hormones. The results were compared with proliferation markers, invasiveness and clinical data. ISH detected signals for all pituitary hormones at a range of 30% for prolactin (PRL) to 85% for proopiomelanocortin (POMC). mRNA for β-FSH was detected in 70% and β-LH mRNA in 43% of adenomas. Thirty-three percent of adenomas revealed negative mRNA detection for β-LH but positive hormone content. The majority of adenomas (75%) expressed more than two mRNAs simultaneously, mostly the combination of POMC mRNA together with β-FSH mRNA and one to four others. Comparison with clinical data showed no significant differences except for one adenoma with a high Ki-67 index (>2.1% positive nuclei). This adenoma showed very high signals for PRL and β-TSH mRNA.     

Pituitary adenomas in acromegaly: Comparison of different adenoma types with clinical data

Adenoma tissues from 309 patients with active acromegaly was examined by routine light microscopy and immunohistochemistry, and selectively by electron microscopy. All adenomas were immunoreactive for growth hormone. Eighty-seven adenomas were monohormonal (28%), 58 were bihormonal (immunoreactive for growth hormone and prolactin) (19%), and 157 adenomas were plurihormonal (51%), with positivity for glyco-proteins and/or their α-subunit as well. The mean tumor size was significantly greater in monohormonal adenomas than in other adenoma types. There was no difference in invasiveness among the various adenoma types. Younger patients showed invasive tumor growth more often. Patients with densely granulated GH cell adenomas had a significantly longer duration of symptoms compared to patients with other adenoma types. More than half of the patients with sparsely granulated GH cell adenomas had a duration of less than 5 yr. There was no correlation between duration of symptoms and tumor size. The preoperative mean GH level was significantly higher in patients with sparsely granulated GH cell adenomas than in those with mixed GH/PRL cell adenomas. The preoperative mean PRL level was significantly higher in patients with bihormonal adenomas than in those with plurihormonal adenomas. There was an inverse correlation between age and preoperative GH and PRL levels. No linear correlation was found between preoperative basal GH and PRL levels. Monohormonal adenomas presented more often with suprasellar and/or parasellar extension than other adenoma types. Our data suggest a positive correlation between tumor extension and preoperative GH and PRL levels. Patients with plurihormonal adenomas were significantly older than patients with sparsely granulated GH cell adenomas and mixed GH/PRL cell adenomas. No significant difference was found between the various adenoma types and the extent of surgical removal, which depends on the degree of invasiveness, tumor size, and extrasellar tumor extension.     

Study on growth hormone and insulin secretion in myotonic dystrophy

Growth hormone (GH) levels were measured in 12 patients with myotonic dystrophy (MD; 7 men and 5 women, aged 21–49 years) and 14 volunteers after administration of 100 g GH-releasing hormone (GHRH; 1–29). A 75-g oral glucose tolerance test was carried out to determine glucose, insulin, plasma C-peptide, and urinary C-peptide. The GH level in six MD patients responded normally to GHRH (group I), with a peak of 17.1 ± 1.46 g/l, compared withcontrols (27.8 ± 19.6 g/l, NS), and that in the other six patients responded subnormally, with a peak of 3.15 ± 1.46 g/l, lower than in controls and in group I patients (P < 0.001). In group I the insulin response to the glucose tolerance test showed hyperinsulinism and was lower than that in group II patients; stimulated C-peptide was also higher in group II than in group I and in controls; urinary C-peptide levels were parallel to those in previous data. In all MD patients there were a negative correlation between absolute values of GH response to GHRH and insulin response to glucose tolerance test (r = - 0.79, P < 0.001). Our data suggest that the failure in GH release and peripheral insulin action is due to a generalized defect in cellular membrane function in MD patients.Abbreviations BMI body mass index - GH Growth hormone - GHRH growth hormone releasing hormone - MD myotonic dystrophy Correspondence to: J. M. Gomez Saez     

In situ hybridization for different mRNA in GH-secreting and in inactive pituitary adenomas

In a series of 40 pituitary adenomas in acromegaly all tumors showed mRNA for GH by in situ hybridization (ISH). The signals were mostly very strong and found in more than 80% of adenoma cells by using frozen sections. In paraffin sections the number of positive cells and the intensity of signals are lower. Prolactin mRNA was found in 87% of adenomas. In 27% more than 80% of cells were marked. beta-HCGmRNA (with 90% hormology for LH-mRNA) was demonstrable in very sparse cells of 25% of adenomas. Comparing ISH with immunohistology (IH) we found a correlation between signals and hormone content in 100% of adenomas for GH, in 60% for Prolactin and in 10% for Gonadotropins. In 18% Prolactin mRNA but not the hormone was demonstrable and in 5% Prolactin was immunostained but no hybridization signals were detected. In a series of 40 clinically inactive adenomas sparse cells of three tumors expressed GHmRNA and two of these contained also the hormone, whereas in one adenoma GH but not GHmRNA was demonstrable. Prolactin mRNA was found in 8 adenomas. 7 of these also contained the hormone. In two cases Prolactin but not Prolactin mRNA was present. Beta-HCG(LH)mRNA and the respective hormones were shown in very sparse cells of 6 adenomas, whereas only beta-HCG(LH)-mRNA was found in 8 cases. The significance of the findings is discussed.     

Folliculostellate cells in pituitary adenomas: Studies of hormonal profile and tumor vascularity

The hormonal immunoreactivity and vascularity of pituitary adenomas containing folliculostellate (FS) cells have been compared with those of tumors in which such cells were not identified. FS cells were present in variable numbers in 36 of 92 tumors. Adenomas immunoreactive for growth hormone (GH), adrenocorticotropic hormone (ACTH), or prolactin (PRL) contained FS cells in 40–50% of cases. Those immunoreactive for glycoprotein hormones and alphasubunit contained FS cells in 67–85% of cases, a statistically significant correlation. When alpha-subunit was also present in GH-, GH/PRL-, and ACTH-immunoreactive tumors, a higher proportion contained FS cells (57–91%). These data suggest a correlation between the presence of FS cells and glycoprotein immunoreactivity in pituitary adenomas. Vascular channels identified by the binding of the lectinUlex europaeus were quantified in the two types of tumors. Those containing FS cells were not more vascular than those without FS cells, which suggests that FS cells do not play a significant role in the regulation of intratumoraf vascularization in human pituitary adenomas.     

Immunohistochemical Expression of Neuroendocrine Secretory Protein-55 (NESP-55) in Pituitary Adenomas

Neuroendocrine secretory protein-55 (NESP-55) is a recently described member of the chromogranin family and appears to be a marker of the constitutive secretory pathway in certain neural, neuroendocrine, and endocrine cell types. It has been shown to be selectively expressed in tumors differentiating towards the adrenal chromaffin and pancreatic islet cell phenotypes. The highest levels of NESP-55 expression, at least in animals, appear to be in the adrenal medulla and the pituitary gland. However, very little is known about the status of NESP-55 expression in pituitary adenomas. We therefore studied the immunohistochemical profile of NESP-55 expression in a series of 30 well-characterized pituitary adenomas (five each of FSH/LH and ACTH, four GH, three TSH, seven prolactin, and six null cells). All tumors were positive for one or more generic marker(s) (chromogranin A, synaptophysin, neuron-specific enolase) of neuroendocrine differentiation. All pituitary adenomas selected for study were stained for NESP-55 with appropriate positive and negative controls. NESP-55 immunoreactivity, seen as brown finely granular cytoplasmic staining of the tumor cells with prominent perinuclear accentuation, was graded as focal (<10% tumor cells staining), moderate (10–50% tumor cells staining), and diffuse (>50% tumor cell staining). Four of seven prolactinomas were positive for NESP-55 (one focal, two moderate, and one diffuse). Two of four GH adenomas were also positive (one focal and one diffuse) while only 1/5 FSH tumors showed a moderately intense immunoreactivity. All other pituitary adenomas were completely negative for NESP-55. Our results indicate that, in human pituitary adenomas, NESP-55 has a more restricted pattern of expression than that of chromogranins A and B. Since immunohistochemical expression of NESP-55 is largely confined to prolactinomas and GH adenomas, it raises the possibility that NESP-55 may somehow be involved in the secretory pathways of these specific cell types.     

Downregulation of E-cadherin and its undercoat proteins in pituitary growth hormone cell adenomas with prominent fibrous bodies

The cadherin-catenin complex regulates cellular adhesion and motility, and genetic alterations in these molecules play a critical role in multistage tumorigenesis. In this study, the expression of three major type I classic cadherins—E-, N-, and P-cadherin—and their undercoat proteins—α-, β-, and γ-catenin, and pp120–was investigated in 127 pituitary adenomas and 10 normal adenohypophyseal glands using an immunohistochemical technique with highly specific monoclonal antibodies. In normal pituitary glands, E-cadherin, catenins, and pp 120 were strongly expressed on almost all hormone-producing cell-cell boundaries, N-cadherin was weakly immunoreactive on a few cell-cell boundaries, and P-cadherin was negative. In pituitary adenomas, a correlation was not identified among expression of E-cadherin, catenins, or pp120 with patient age, sex, hormone level, tumor size, and/or invasiveness, respectively. Expression of E-cadherin, catenins, and pp 120 was significantly reduced in 24 growth hormone (GH) cell adenomas with prominent fibrous bodies compared with the other subtypes of pituitary adenomas and normal pituitary glands (p<0.0001, respectively). Methylation-specific polymerase chain reaction analysis revealed that the E-cadherin gene promoter region was methylated in 6 of 16 (37.5%) GH cell adenomas with prominent fibrous bodies examined, 2 of which displayed total methylation, but not in 10 GH cell adenomas without fibrous bodies. No mutation of exon 3 of the β-catenin gene was found in 16 GH cell adenomas with prominent fibrous bodies or in 10 other subtypes of pituitary adenomas that showed unremarkable intracellular presence of β-catenin protein. In conclusion, the decreased expression of the E-cadherin-catenin complex and methylation of the E-cadherin gene promoter region only in GH cell adenomas with prominent fibrous bodies may be an event associated with the formation of fibrous bodies.     

Alpha-subunit immunoreactivity in plurihormonal pituitary adenomas of patients with acromegaly.

Twelve plurihormonal pituitary adenomas, removed surgically from 11 of 20 patients with acromegaly, were investigated. The mean age of the 11 patients was 45 yr. Seven patients with eight tumors were men. The tumors were immunostained for all known adenohypophysial hormones by the avidin-biotin-peroxidase complex (ABC) technique. All 12 adenomas were immunoreactive for growth hormone (GH) and alpha-subunit; prolactin (PRL) was detected in five tumors. Stains for the beta-subunits of glycoprotein hormones [thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH)] revealed immunopositivity for specific beta-subunits in ten adenomas. The close association between GH and alpha-subunit in pituitary tumors of acromegalic patients remains to be explained.     

Comparative immunohistochemical analysis of estrogen receptor and chromogranin-A reactivity in plurihormonal human prolactinomas

The aim of the study was to compare the immunoreactivity of estrogen receptors (ER) and chromogranin-A (CHR-A) in human prolactinomas with verified plurihormonality. Eleven cases of prolactinomas, nine found in women aged from 15-32 and two found in two men both aged 54 years, were analyzed for possible colocalization of other hormones produced by adenohypophysis, i.e. growth hormone (GH), thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH) and adrenocorticotropic hormone (ACTH). All evaluated cases of prolactinomas were clinically manifested by elevated values of prolactin (PRL) in patient serum, while the values of other assayed hormones were within the normal range. Although biopsy material is not routinely submitted to immunohistochemical analysis for plurihormonality, these eleven cases of operated prolactinomas were randomly examined to the presence of plurihormonality. In six cases of prolactin-producing adenomas, the coexistence of growth hormone was detected. Colocalization of follicle-stimulating hormone and weak expression of adrenocorticotropic hormone were found in two cases each. Thus, bihormonal activity (PRL + GH) was found in six, and trihormonal activity (PRL + GH + FSH and PRL/GH + ACTH) in three cases of prolactinoma. In addition, the presence of prolactin and growth hormone was demonstrated in morphologically different cells. Eight of these eleven pituitary adenomas were tested for estrogen receptors (ER), which play an important role as growth stimulating factors and secretory factors for prolactin-producing cells. We tried to determine if there was a difference in the intensity of expression of estrogen receptors and chromogranin-A between pure prolactinomas and mixed, plurihormonal prolactinomas. By use of monoclonal antibodies, chromogranin-A found to be reactive in seven of eleven prolactinomas, i.e. in plurihormonal prolactinomas. Estrogen receptors were markedly expressed in all the eight prolactinomas analyzed, which may prove significant in the treatment of these hypophyseal tumors.     

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10⁻² ng/μl (278-bp, S. enterica), 11.2 × 10⁻² ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10⁻² ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10⁴ copies/μl, 3.58 × 10⁴ copies/μl, and 1.79 × 10⁴ copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.     

Morphologically unclassified GH-producing adenoma showing galactorrhea without acromegaly

A 24-year-old woman with a large pituitary adenoma had amenorrhea and galactorrhea, but no physical stigmata of acromegaly despite slightly elevated serum growth hormone (GH) and normal serum prolactin (PRL) levels. Subtotal removal of the tumor cured galactorrhea and resulted in normalization of serum GH concentration. The question is raised whether amenorrhea and galactorrhea were related to excessive GH production in this patient. Absence of acromegaly might have been due to the short duration of the disease. The tumor was a chromophobic, periodic acid-Schiff-negative adenoma. Immunocytochemistry and in situ hybridization revealed focal GH immunoreactivity and diffuse, weak signal for GH messenger RNA. By electron microscopy, the tumor showed no features of GH or PRL-producing adenomas. Two different cell types could be distinguished: the majority were similar to null cells, whereas a small number of cells resembled somatotrophs and lactotrophs, possessing many secretory granules and exhibiting exocytosis. On the basis of its ultrastructure, this tumor can be classified as an atypical acidophil cell line adenoma in which adenomatous null cells transformed to the differentiated cells capable of producing GH.     

Light bodies in human pituitary adenomas

Summary Light bodies are large cytoplasmic granules originally described in the gonadotrophic cells of the rat pituitary gland. In order to determine whether similar bodies occur in the human anterior pituitary gland, 89 pituitary adenomas and periadenomatous tissue from 20 cases were examined by transmission electron microscopy. Double membrane bound bodies with filamentous internal structure identical to rodent light bodies were identified in 10 hormone-producing adenomas: 5 PRL, 1 PRL-GH, 2 GH, and 2 ACTH-producing tumours. No light bodies were found in the remaining 79 tumours nor in the pituitary cells in periadenomatous tissue from 20 cases. These results show that some human pituitary adenomas may contain light bodies identical to those seen in gonadotrophs of rat pituitary.Abbreviations PRL prolactin - GH growth hormone - ACTH adenocorticotropic hormone - FSH follicle-stimulating hormone - LH luteinizing hormone