The detrimental effect of microplastics on critical periods of development in the neuroendocrine system.

As a result of human socio-economic activity, industrial wastes have increased alarmingly. Plastic pollution is globally distributed across the world due to its properties of buoyancy and durability. Two broad classes of plastic-related chemicals are of critical concern for human health-bisphenol-A or BPA, and additives used in the synthesis of plastics, which are known as phthalates. Our exposure to them is ubiquitous because they are used in the production of materials that we use daily such as polycarbonate plastics, epoxy resins, flooring, automotive parts, medical devices, dental sealants, and children's toys. Since these compounds are not covalently bound to the products, they easily leach from them, leading to high human exposure. Both, BPA and phthalates, are endocrine-disruptor compounds (EDCs) with steroidogenic activity, and can bind to different receptors, such as estrogen, androgen, PPAR-γ, and AhR. These pathways are part of the complex regulatory neuroendocrine network, since its cellular components not only express neuroendocrine receptors, but synthesize and respond to several hormones and other endocrine ligands. On the other hand, the effects of BPA and phthalates on neuroendocrine diseases have been poorly studied and the available data are inconclusive. This can be attributed to the enormous variety of animal models and the different doses used in experiments or levels found in humans. However, what is clear is that exposure to both EDCs during critical life stages induces many changes in the neuroendocrine system of exposed humans that are correlated with different reproductive and neurological diseases.

Galanin immunoreactivity is sexually polymorphic in neuroendocrine and vocal-acoustic systems in a teleost fish.

Galanin is a peptide that regulates pituitary hormone release, feeding, and reproductive and parental care behaviors. In teleost fish, increased galanin expression is associated with territorial, reproductively active males. Prior transcriptome studies of the plainfin midshipman (Porichthys notatus), a highly vocal teleost fish with two male morphs that follow alternative reproductive tactics, show that galanin is upregulated in the preoptic area-anterior hypothalamus (POA-AH) of nest-holding, courting type I males during spawning compared to cuckolding type II males. Here, we investigate possible differences in galanin immunoreactivity in the brain of both male morphs and females with a focus on vocal-acoustic and neuroendocrine networks. We find that females differ dramatically from both male morphs in the number of galanin-expressing somata and in the distribution of fibers, especially in brainstem vocal-acoustic nuclei and other sensory integration sites that also differ, though less extensively, between the male morphs. Double labeling shows that primarily separate populations of POA-AH neurons express galanin and the nonapeptides arginine-vasotocin or isotocin, homologues of mammalian arginine vasopressin and oxytocin that are broadly implicated in neural mechanisms of vertebrate social behavior including morph-specific actions on vocal neurophysiology in midshipman. Finally, we report a small population of POA-AH neurons that coexpress galanin and the neurotransmitter γ-aminobutyric acid. Together, the results indicate that galanin neurons in midshipman fish likely modulate brain activity at a broad scale, including targeted effects on vocal motor, sensory and neuroendocrine systems; are unique from nonapeptide-expressing populations; and play a role in male-specific behaviors.

Modeling neuroendocrine stress reactivity in salivary cortisol: adjusting for peak latency variability.

In this report, we present growth curve modeling (GCM) with landmark registration as an alternative statistical approach for the analysis of time series cortisol data. This approach addresses an often-ignored but critical source of variability in salivary cortisol analyses: individual and group differences in the time latency of post-stress peak concentrations. It allows for the simultaneous examination of cortisol changes before and after the peak while controlling for timing differences, and thus provides additional information that can help elucidate group differences in the underlying biological processes (e.g., intensity of response, regulatory capacity). We tested whether GCM with landmark registration is more sensitive than traditional statistical approaches (e.g., repeated measures ANOVA--rANOVA) in identifying sex differences in salivary cortisol responses to a psychosocial stressor (Trier Social Stress Test--TSST) in healthy adults (mean age 23). We used plasma ACTH measures as our "standard" and show that the new approach confirms in salivary cortisol the ACTH finding that males had longer peak latencies, higher post-stress peaks but a more intense post-peak decline. This finding would have been missed if only saliva cortisol was available and only more traditional analytic methods were used. This new approach may provide neuroendocrine researchers with a highly sensitive complementary tool to examine the dynamics of the cortisol response in a way that reduces risk of false negative findings when blood samples are not feasible.

ExoSensor 517: a dual-analyte fluorescent chemosensor for visualizing neurotransmitter exocytosis.

A dual-analyte fluorescent chemosensor (ExoSensor 517) for the direct visualization of neurotransmitters released upon exocytosis is presented. The sensor exploits the high concentration of neurotransmitters (e.g., glutamate, norepinephrine, and dopamine) and the pH gradient between the vesicle and synaptic cleft. The cooperative recognition elements require both binding and a change in environmental pH to afford a fluorescence response which makes ExoSensor 517 one of the first integrated molecular logic gates to be used for biological applications.

Five functional adipokinetic peptides expressed in the corpus cardiacum of the moth genus Hippotion (Lepidoptera, Sphingidae).

This is the first study that finds five adipokinetic hormones (AKHs) in the corpus cardiacum of an insect. From two species of the sphingid moth genus Hippotion, eson and celerio, three novel and two known AKHs were isolated and sequenced by deduction from multiple MS(n) electrospray mass data: two octapeptides are pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp amide (denoted Hipes-AKH-I) and its Thr(7) analogue (Hipes-AKH-II); two nonapeptides are pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-Gly amide (Manse-AKH) and its Thr(7) analogue (Hipes-AKH-III), as well as a decapeptide pGlu-Leu-Thr-Phe-Ser-Ser-Gly-Trp-Gly-Gln amide (Manse-AKH-II). All sequences were confirmed by identical behaviour of natural and synthetic peptides in reversed-phase HPLC and liquid chromatography coupled to electrospray mass spectrometry, resulting in identical retention times and tandem mass spectral data. High resolution mass spectrometry and retention time data also confirmed that the amino acid at position 10 in Manse-AKH-II is Gln and not the isobaric Lys. Conspecific injections of all five peptides in synthetic form and low doses caused hyperlipaemia in H. eson. Our results and pertaining literature suggest that five genes code for the mature peptides, which are very likely released during flight to provide energy for long distance migration in this genus via lipid oxidation; as all five peptides are active at low doses in a conspecific bioassay, it may be speculated, but not proven, that there is only one AKH receptor present in Hippotion that can bind all five peptides with high affinity.

Dramatic changes in catestatin are associated with hemodynamics in acute myocardial infarction.

Acute myocardial infarction (AMI) is characterized by complex neuroendocrine activation. To investigate catestatin profiles, serial catestatin levels were determined by enzyme-linked immunosorbent assay in the first week after AMI in 50 patients. Catestatin levels reduced at admission and negatively correlated with heart rates; it increased significantly on the third day but remained decreased at 1 week and positively with blood pressure. In a subgroup of 20 patients admitted within 4 h after onset, circulating catestatin correlated inversely with norepinephrine. Catestatin might be involved in the course of AMI and act as a tool in monitoring the progression of AMI.

Mass spectrometric analysis of peptides in brain neurosecretory cells and neurohemal organs in the adult blowfly, Protophormia terraenovae.

Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum-hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg(7)]-corazonin and [Arg(7)]-corazonin(3)(-)(11)) in the PL, 13 peptides (including myosuppressin, [Arg(7)]-corazonin and [Arg(7)]-corazonin(3-11)) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 degrees C) and diapause-averting conditions (LD 18:6 at 25 degrees C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd-10th amino acids were sequenced as -YRKPPFNGS-, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils.

The distribution of vasotocin and mesotocin immunoreactivity in the hypothalamic magnocellular neurosecretory nuclei of the Saharan herbivorous lizard, Uromastix acanthinurus Bell, 1825 (Sauria-Agamidae).

An immunohistochemical study of the magnocellular neurosecretory nuclei was performed in the hypothalamus of the desert lizard Uromastix acanthinurus using polyclonal antibodies against arginine vasotocin (AVT), mesotocin (MST) and neurophysins I and II (NpI, NpII). AVT- and MST-immunoreactivities were localized in individual neurons of the supraoptic, periventricular, and paraventricular nuclei and in scattered neurosecretory cells. The supraoptic nuclei (SONs) can be subdivided into rostral, medial and caudal portions. The rostral portion of the SONs was called the SON-ventral aggregation (V SON) because the neurosecretory neurons are present in the ventral part of the hypothalamus along the optic chiasma (OC). Their perikarya and fibres were only AVT-ir. The medial part of the SONs was constituted of two clusters of neurosecretory neurons located in the two lateral ends of the OC to form the SON-lateral aggregations (L SON). In the caudal end of the last one, some MST-ir perikarya appeared. The caudal part of the SONs was constituted of a dorso-lateral aggregation (D SON) of ir-neurons spreading over the lateral forebrain bundle (LFB). AVT- and MST- perikarya were observed in this caudal portion of the SONs, AVT-ir neurons being more numerous. AVTergic and MSTergic magnocellular neurons were present in the periventricular nuclei (PeVNs). Parvocellular and magnocellular AVT- and MST-ir were observed in the paraventricular nuclei (PVNs). The fibres emerging from the magnocellular neurons which belong to these nuclei and the scattered cells ran along the hypothalamic floor and entered the median eminence (ME) to end in the neural lobe of hypophysis. As a rule, immunoreactivity was also observed in all the regions of the forebrain with vasotocinergic and mesotocinergic perikarya and fibres. The immunoreactive distribution was similar to that described in other reptiles.

Defining global neuroendocrine gene expression patterns associated with reproductive seasonality in fish.

BACKGROUND: Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, by analyzing multiple female goldfish brain microarray datasets, we have characterized global gene expression patterns for a seasonal cycle. A core set of genes (873 genes) in the hypothalamus were identified to be differentially expressed between May, August and December, which correspond to physiologically distinct stages that are sexually mature (prespawning), sexual regression, and early gonadal redevelopment, respectively. Expression changes of these genes are also shared by another brain region, the telencephalon, as revealed by multivariate analysis. More importantly, by examining one dataset obtained from fish in October who were kept under long-daylength photoperiod (16 h) typical of the springtime breeding season (May), we observed that the expression of identified genes appears regulated by photoperiod, a major factor controlling vertebrate reproductive cyclicity. Gene ontology analysis revealed that hormone genes and genes functionally involved in G-protein coupled receptor signaling pathway and transmission of nerve impulses are significantly enriched in an expression pattern, whose transition is located between prespawning and sexually regressed stages. The existence of seasonal expression patterns was verified for several genes including isotocin, ependymin II, GABA(A) gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays. CONCLUSIONS/SIGNIFICANCE: Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development.

Peptidomic survey of the locust neuroendocrine system.

Neuropeptides are important controlling agents in animal physiology. In order to understand their role and the ways in which neuropeptides behave and interact with one another, information on their time and sites of expression is required. We here used a combination of MALDI-TOF and ESI-Q-TOF mass spectrometry to make an inventory of the peptidome of different parts (ganglia and nerves) of the central nervous system from the desert locust Schistocerca gregaria and the African migratory locust Locusta migratoria. This way, we analysed the brain, suboesophageal ganglion, retrocerebral complex, stomatogastric nervous system, thoracic ganglia, abdominal ganglia and abdominal neurohemal organs. The result is an overview of the distribution of sixteen neuropeptide families, i.e. pyrokinins, pyrokinin-like peptides, periviscerokinins, tachykinins, allatotropin, accessory gland myotropin, FLRFamide, (short) neuropeptide F, allatostatins, insulin-related peptide co-peptide, ion-transport peptide co-peptide, corazonin, sulfakinin, orcokinin, hypertrehalosaemic hormone and adipokinetic hormones (joining peptides) throughout the locust neuroendocrine system.

Obestatin in human neuroendocrine tissues and tumours: expression and effect on tumour growth.

The hormone obestatin, which is derived from the same precursor as ghrelin and whose receptor(s) is still unrecognized, possesses a variety of metabolic/modulatory functions mostly related to food intake suppression and reduction of gastrointestinal motility. The distribution of obestatin in normal and neoplastic human tissues is poorly understood. We report that in fetal tissue samples, obestatin peptide was detected in the thyroid, pituitary, lung, pancreas and gastrointestinal tract, usually being co-localized with chromogranin A. In adult tissues, obestatin protein expression was restricted to pituitary, lung, pancreas and gastrointestinal tract and was co-localized strictly with ghrelin. By contrast, in endocrine tumours obestatin was expressed in a small fraction of thyroid, parathyroid, gastrointestinal and pancreatic neoplasms, in most cases with a focal immunoreactivity and co-localized with ghrelin. Messenger RNA levels of the specific fragments of ghrelin and obestatin were comparable in both normal and tumour samples, confirming that post-translational mechanisms rather than alternative splicing events lead to ghrelin/obestatin production. Finally, in TT and BON-1 cell lines obestatin induced antiproliferative effects at pharmacological doses, opposite to those observed with ghrelin. In summary, our data demonstrate that obestatin is produced by the same endocrine cells that express ghrelin in normal tissues from fetal to adult life, whereas, as compared to ghrelin, in neoplastic conditions it is down-regulated by post-translational modulation and shows potential antiproliferative properties in vitro.

Brain-region responsiveness to DT56a (Femarelle) administration on allopregnanolone and opioid content in ovariectomized rats.

OBJECTIVE: The natural selective estrogen receptor modulator DT56a (Femarelle), derived from soybean, has been shown to relieve menopausal vasomotor symptoms with no effect on sex steroid hormone levels or endometrial thickness.The purpose of the present study was to evaluate the neuroendocrine effect of DT56a administration through the evaluation of brain content of allopregnanolone (AP), an endogenous neurosteroid gamma-aminobutyric acid agonist with anxiolytic properties, and through the assessment of beta-endorphin (beta-END), the endogenous opioid implicated in pain mechanism, emotional state, and autonomic control. METHODS: Five groups of Wistar ovariectomized (OVX) rats received one of the following treatments: oral DT56a administration at doses of 6, 12, 60, and 120 mg kg(-1) day(-1) or estradiol valerate (E2V) at a dose of 0.05 mg kg(-1) day(-1) for 14 days. One group of fertile and one group of OVX rats receiving placebo were used as controls. The concentration of AP was assessed in the frontal and parietal cortex, hippocampus, hypothalamus, anterior pituitary, and serum, whereas the content of beta-END was evaluated in the frontal and parietal cortex, hippocampus, hypothalamus, neurointermediate lobe, anterior pituitary, and plasma. RESULTS: DT56a increased AP levels in all brain areas analyzed and in serum, with a classical dose-related curve in comparison with OVX rats. In some brain areas, such as the frontal cortex, the parietal cortex, and the anterior pituitary, positive results were found even with the administration of a lower DT56a dose of 60 mg kg(-1) day(-1), attaining AP levels in the range of those in animals treated with E2V. Similarly, beta-END levels were enhanced in selected brain areas such as the hippocampus, the hypothalamus, the neurointermediate lobe, and the anterior pituitary in comparison with those in OVX rats, in which the increase of the opioid was dose related and in the range of those in rats treated with E2V. CONCLUSIONS: This study demonstrated that DT56a positively affects brain neurosteroidogenesis and the opiatergic system: DT56a exerts an estrogen-like effect on selective areas related to mood, cognition, and homeostasis control, presenting a specific pattern of interaction with the brain function. These findings may, in part, explain the clinical effect of DT56a on menopausal symptoms.

Endopin serpin protease inhibitors localize with neuropeptides in secretory vesicles and neuroendocrine tissues.

BACKGROUND/AIMS: The endopin serpin protease inhibitors have been identified by molecular studies as components of secretory vesicles that produce neuropeptides. Endopin 1 inhibits trypsin-like serine proteases, and endopin 2 inhibits cathepsin L that produces neuropeptides in secretory vesicles. To assess the secretory vesicle and neuroendocrine tissue distribution of these endopins, the goal of this study was to define specific antisera for each endopin isoform and to examine their localization with neuropeptides and in neuroendocrine tissues. METHODS: This study utilized methods consisting of Western blots, immunoelectron microscopy, and immunofluorescence microscopy for evaluation of the localization of endopin protease inhibitors in neuroendocrine tissues. RESULTS: Immunoelectron microscopy with these selective antisera demonstrated the localization of endopins 1 and 2 within secretory vesicles of adrenal medulla (bovine). Cellular immunofluorescence confocal microscopy illustrated the high level of colocalization of endopins 1 and 2 with enkephalin and NPY neuropeptides that are present in secretory vesicles of adrenal medullary chromaffin cells in primary culture. Tissue distribution studies (by Western blots) showed the expression of endopins 1 and 2 in bovine brain, pituitary, adrenal medulla, and other neuroendocrine tissues. CONCLUSIONS: These results implicate endopins 1 and 2 as endogenous protease inhibitors in neuropeptide-containing secretory vesicles and neuroendocrine tissues.

Role of the vesicular chloride transporter ClC-3 in neuroendocrine tissue.

ClC-3 is an intracellular chloride transport protein known to reside on endosomes and synaptic vesicles. The endogenous protein has been notoriously difficult to detect in immunohistological experiments because of the lack of reliable antibodies. Using newly generated antibodies, we now examine its expression pattern at the cellular and subcellular level. In all tissues examined, immunostaining indicated that ClC-3 is a vesicular protein, with a prominent expression in endocrine cells like adrenal chromaffin cells and pancreatic islet cells. In line with a possible function of ClC-3 in regulating vesicle trafficking or exocytosis in those secretory cells, capacitance measurements and amperometry indicated that exocytosis of large dense-core vesicles (LDCVs) was decreased in chromaffin cells from ClC-3 knock-out mice. However, immunohistochemistry complemented with subcellular fractionation showed that ClC-3 is not detectable on LDCVs of endocrine cells, but localizes to endosomes and synaptic-like microvesicles in both adrenal chromaffin and pancreatic beta cells. This observation points to an indirect influence of ClC-3 on LDCV exocytosis in chromaffin cells, possibly by affecting an intracellular trafficking step.

Predicted versus expressed adipokinetic hormones, and other small peptides from the corpus cardiacum-corpus allatum: a case study with beetles and moths.

This mass spectrometric study confines itself to peptide masses in the range of 500-1500Da. Adipokinetic hormones (AKHs) that are predicted from the genome of the red flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori, are shown to exist as expressed peptides in the corpora cardiaca (CC) of the respective species as evidenced by various mass spectrometric methods. Additionally, some related species were included in this study, such as the tenebrionid beetles Tribolium brevicornis and Tenebrio molitor, as well as the moths Spodoptera frugiperda, Spodoptera littoralis, Mamestra brassicae and Lacanobia oleracea, to investigate whether AKH peptides are structurally conserved in the same genus or family. Interestingly, the AKH peptide of T. brevicornis is identical to that of T. molitor but not to the ones of its close relative T. castaneum. Moreover, other peptides in T. brevicornis, such as various FXPRL amides (=pyrokinins), also match the complement in T. molitor but differ from those in T. castaneum. All the CC of beetles lacked the signal for the mass of the peptide corazonin. All moths have the nonapeptide Manse-AKH expressed in their CC. In addition, whereas the silk moth has the decapeptide Bommo-AKH as a second peptide, all other moths (all noctuids) express the decapeptide Helze-HrTH. In M. brassicae and L. oleracea a novel amidated Gly-extended Manse-AKH is found as a possible third AKH. The noctuid moth species also all express the same FLRF amide-I, corazonin, and a group-specific isoform of a gamma-PGN-(=gamma-SGNP) peptide. In L. oleracea, however, the latter peptide has a novel sequence which is reported for the first time, and the peptide is code-named Lacol-PK.

The biological characterization of neuroendocrine tumors: the role of neuroendocrine markers.

Neuroendocrine tumors (NET) may originate in different organs, from cells embryologically different but expressing common phenotypic characteristics, such as: the immuno-reactivity for markers of neuroendocrine differentiation (defined as "pan-neuroendocrine"), the capacity to secrete specific or aspecific peptide and hormones and the expression of some receptors, that are at the basis of the current diagnostic and therapeutical approach, peculiar to these tumors. NET have been conventionally distinguished in functioning, when associated with a recognized clinical endocrine syndrome, and non-functioning. However, this terminology may be misleading, since the great majority of NET may secrete neuroendocrine peptides, which can be employed as clinical markers for both diagnosis and follow-up. On the other hand, tissue immuno-reactivity for specific hormones does not always reflect secretory activity of the tumor cells. Finally, receptors and genetic markers are acquiring a relevant role in the characterization of NET, both improving knowledge of biology and physiopathology of NET, as well as in developing specific strategies to establish an early diagnosis and targeted therapies, to adopt prophylactic strategies in familial forms, and to identify more efficacious targets for therapy in the future.

Chromogranins A and B and secretogranin II: evolutionary and functional aspects.

Chromogranins/secretogranins or granins are a class of acidic, secretory proteins that occur in endocrine, neuroendocrine, and neuronal cells. Granins are the precursors of several bioactive peptides and may be involved in secretory granule formation and neurotransmitter/hormone release. Characterization and analysis of chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) in distant vertebrate species confirmed that CgA and CgB belong to related monophyletic groups, probably evolving from a common ancestral precursor, while SgII sequences constitute a distinct monophyletic group. In particular, selective sequences within these proteins, bounded by potential processing sites, have been remarkably conserved during evolution. Peptides named vasostatin, secretolytin and secretoneurin, which occur in these regions, have been shown to exert various biological activities. These conserved domains may also be involved in the formation of secretory granules in different vertebrates. Other peptides such as catestatin and pancreastatin may have appeared late during evolution. The function of granins as propeptide precursors and granulogenic factors is discussed in the light of recent data obtained in various model species and using knockout mice strains.

Morphometric and immunohistochemical study of the abomasum of red deer during prenatal development.

The red deer is well suited to scientific study, given its economic importance as an animal to be hunted, and because it has a rich genetic heritage. However, there has been little research into the prenatal development of the stomach of ruminants in general, and none for the red deer. For this reason, we undertook histological evaluation of the ontogenesis of the abomasum in red deer. Histomorphometric and immunohistochemical analyses were carried out on 50 embryos and fetuses from the initial stages of prenatal life until birth. The animals were divided for test purposes into five experimental groups: group I [1.4-3.6 cm crown-rump length (CRL); 30-60 days, 1-25% of gestation]; group II (4.5-7.2 cm CRL; 67-90 days, 25-35% of gestation); group III (8-19 cm CRL; 97-135 days, 35-50% of gestation); group IV (21-33 cm CRL; 142-191 days, 50-70% of gestation) group V (36-40 cm CRL; 205-235 days, 75-100% of gestation). In the organogenesis of the primitive gastric tube of red deer, differentiation of the abomasum took place at 67 days, forming a three-layered structure: the epithelial layer (pseudostratified), pluripotential blastemic tissue and serosa. The abomasal wall displayed the primitive folds of the abomasum and by 97 days abomasal peak areas were observed on the fold surface. At 135 days the abomasal surface showed a single mucous cylindrical epithelium, and gastric pits were observed in the spaces between abomasal areas. At the bottom of these pits the first outlines of glands could be observed. The histodifferentiation of the lamina propria-submucosa, tunica muscularis and serosa showed patterns similar to those described for the forestomach of red deer. The abomasum of red deer during prenatal life, especially from 67 days of gestation, was shown to be an active structure with full secretory capacity. Its histological development, its secretory capacity (as revealed by the presence of neutral mucopolysaccharides) and its neuroendocrine nature (as revealed by the presence of positive non-neuronal enolase cells and the neuropeptides vasoactive intestinal peptide and neuropeptide Y) were in line with the development of the rumen, reticulum and omasum. Gastrin-immunoreactive cells first appeared in the abomasum at 142 days, and the number of positive cells increased during development. As for the number of gastrin cells, plasma gastrin concentrations increased throughout prenatal life. However, its prenatal development was later than that of the abomasum in sheep, goat and cow.

Localization of immunoreactive HIF-1alpha and HIF-2alpha in neuroendocrine cells of both benign and malignant prostate glands.

BACKGROUND: Hypoxia induces increased tumor growth by promoting angiogenic and glycolytic pathways. Tumors expressing hypoxia-inducible factor-1alpha (HIF-1alpha), an important transcriptional activator of oxygen-regulated genes, are resistant to chemotherapy and radiotherapy. The major challenge in prostate cancer therapy today is to gain a better understanding of the development of hormone-refractory tumors, which is often characterized by neuroendocrine differentiation. Here we studied the expression of HIF-1alpha and HIF-2alpha in neuroendocrine cells of the benign prostate and in prostate cancer. METHODS: Tissue sections from 30 patients who underwent radical prostatectomy and from 21 patients operated by transurethral resection of the prostate were selected for immunohistochemical analysis for expression of HIF-1alpha, HIF-2alpha, androgen receptor (AR), neuroendocrine markers (chromogranin A, synaptophysin), and two gene products downstream of HIF-1alpha: VEGF and GAPDH. RESULTS: Immunoreactive HIF-1alpha was detected in a subpopulation of AR-negative neuroendocrine cells in benign and malignant prostate tissue. Analysis of serial sections showed that the levels of expression of GAPDH and VEGF proteins are increased in AR-negative malignant neuroendocrine cells expressing HIF-1alpha. In situ-hybridization indicated that HIF-1alpha mRNA levels are not higher in neuroendocrine prostate cancer cells relative to corresponding non-neuroendocrine tumor cells. We also demonstrated induced stabilization of nuclear HIF-1alpha in LNCaP cells by hypoxia and long-term stimulation with interleukin-6. Focal HIF-2 expression was detected in benign neuroendocrine-like cells and in malignant prostatic cells. CONCLUSIONS: The expression of HIF-1alpha and HIF-2alpha in prostate cancer has been confirmed, but we also identified immunoreactive HIF-1alpha and downstream gene products in benign and malignant prostate neuroendocrine cells.

Comparative analysis of alternative and traditional immunohistochemical markers for the distinction of ovarian sertoli cell tumor from endometrioid tumors and carcinoid tumor: A study of 160 cases.

The main neoplasms in the differential diagnosis for primary ovarian tumors with a tubule-rich pattern are pure Sertoli cell tumor, endometrioid tumors (including borderline tumor, well-differentiated carcinoma, and the sertoliform variant of endometrioid carcinoma), and carcinoid tumor. Because traditional immunohistochemical markers [pan-cytokeratin (pan-CK), low molecular weight cytokeratin (CK8/18), epithelial membrane antigen (EMA), inhibin, calretinin, CD99, chromogranin, and synaptophysin] can occasionally have diagnostic limitations, the goal of this study was to determine whether or not any alternative markers [cytokeratin 7 (CK7), estrogen receptor (ER), progesterone receptor (PR), CD10, and CD56] have better diagnostic utility when compared with traditional markers for this differential diagnosis. Immunohistochemical stains for alternative, as well as traditional, markers were performed on the following primary ovarian tumors: pure Sertoli cell tumor (n = 40), endometrioid borderline tumor (n = 38), sertoliform endometrioid carcinoma (n = 13), well-differentiated endometrioid carcinoma (n = 27), and carcinoid tumor (n = 42). Extent and intensity of immunostaining were semiquantitatively scored. In addition, immunohistochemical composite scores (ICSs) in positive cases were calculated on the basis of the combination of extent and intensity scores. Cytokeratin 7 (CK7) was positive in 97% of endometrioid tumors, 13% of Sertoli cell tumors, and 24% of carcinoid tumors. The differences in the mean ICSs for endometrioid tumors versus Sertoli cell tumor or carcinoid tumor were statistically significant (P values ranging from <0.001 to 0.018). ER and PR were positive in 87% and 86% of endometrioid tumors, 8% and 13% of Sertoli cell tumors, and 2% each of carcinoid tumors, respectively. The differences in the mean ICSs for endometrioid tumors versus Sertoli cell tumor were statistically significant (P values ranging from <0.001 to 0.012). Among the epithelial markers, EMA seemed to be the most discriminatory but only slightly better than CK7, ER, or PR. Pan-CK and CK8/18 were not helpful. CD10 showed overlapping patterns of expression in all categories of tumors. Among the sex cord markers, CD10 was markedly less useful than inhibin or calretinin; CD99 was not discriminatory. CD56 showed overlapping patterns of expression in all categories of tumors. Among the neuroendocrine markers, CD56 was less useful than chromogranin or synaptophysin. When traditional immunohistochemical markers are problematic for the differential diagnosis of ovarian Sertoli cell tumor versus endometrioid tumors versus carcinoid tumor, adding CK7, ER, and/or PR to a panel of markers can be helpful. Endometrioid tumors more frequently express CK7, ER, and PR and show a greater extent of immunostaining in contrast to Sertoli cell tumor and carcinoid tumor. Compared with traditional epithelial markers, CK7, ER, and PR are nearly as advantageous as EMA. Inhibin is the most discriminatory sex cord marker, and CD10 is not helpful in the differential diagnosis. Chromogranin and synaptophysin are excellent discriminatory markers for carcinoid tumor, and CD56 is neither sufficiently sensitive nor specific enough for this differential diagnosis to warrant its use in routine practice.