Vocal and territorial behavior in the Smith frog (Hypsiboas faber): Relationships with plasma levels of corticosterone and testosterone

The possible trade-off between the roles of glucocorticoids as facilitators of energy substrate mobilization and neural inhibitors of sexual behavior during breeding season is under debate. We studied the relationship between calling and territorial behavior with plasma levels of corticosterone (CORT) and plasma levels of testosterone (T) across the breeding season of Hypsiboas faber, a large and territorial Neotropical treefrog. We investigated these relationships through focal observations of males calling naturally, followed by blood sampling for hormonal radioimmunoassay. We additionally used an experimental approach, which consisted of broadcasting recorded advertisement calls for 10 min to simulate an invasion in the territory of the focal subjects, followed by behavioral observation and blood sampling for hormonal radioimmunoassay. Results showed a pattern of co-variation between CORT and T across the breeding season. Furthermore, individual variation in CORT and T was related to different aspects of behavior: individuals with higher CORT showed higher calling rates, and individuals with higher steroid levels, mainly T, showed higher responsivity to social stimulation by other males in the chorus. Experimental simulation of territorial intrusion by using playback of advertisement calls of this species did not elicit consistent changes in agonistic behavior and CORT, but decreased T in focal males. (C) 2012 Elsevier Inc. All rights reserved.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1β maturation

Abstract Background Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1β and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1β and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)α and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1β and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1β was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1β maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Changes in prolactin secretion in the short- and long-term after adrenalectomy

OBJECTIVE: To evaluate the modulation of the hypothalamus-pituitary-adrenal axis (HPA) on prolactin secretion in rats after adrenalectomy (ADX). MATERIALS AND METHODS: Plasma corticosterone, ACTH, and prolactin concentrations were measured by radioimmunoassay in rats after bilateral ADX in the short- (3 hours and 1day) and long-term (3, 7, and 14 days). RESULTS: Animals that underwent ADX showed undetectable corticosterone levels and a triphasic ACTH response with a transient increase (3h), a decrease (1d), and further increase in the long-term after ADX. Sham animals showed a marked increase in corticosterone and ACTH levels three hours after surgery, with a decrease to basal levels thereafter. Plasma prolactin levels were not changed after ADX. CONCLUSION: There are different points of equilibrium in the HPA axis after the glucocorticoid negative feedback is removed. Prolactin plasma secretion is not altered in the short or long- term after ADX, suggesting that the peptidergic neurons essential for prolactin release are not activated after ADX.

Pre-and post-puberty physiological plasma oxytocin concentrations in male domestic cats (Felis silvestris catus)

The hormone oxytocin is released by the neuropituitary gland through stimulation of the neurons of the supraoptic and paraventricular nuclei of the hypothalamus. In order to determine the physiological concentrations of this hormone in domestic cats, blood samples were collected from 15 male animals (Felis silvestris catus) during the pre- and post-puberty periods (at four and eight months of age, respectively). Oxytocin determination was accomplished by radioimmunoassay. The average oxytocin concentrations measured in the pre- and post-puberty periods were 2.54±0.24 (μg/dL) and 2.53±0.28 (μg/dL), respectively, and there were no statistical differences between these measurements. Because there are few literature on the analysis of this hormone, especially in the case of male Felis silvestris catus, more studies on the influence of oxytocin on the physiology and reproduction of this species should be conducted under maintenance and situations of stress (such as transportation), and other routine events.

Regulation of corticosterone function during early weaning and effects on gastric cell proliferation

Objectives: The development of the gastrointestinal tract depends on many elements, including glucocorticoids. In the current study, we evaluated the effects of early weaning on corticosterone function and the growth of rat gastric mucosa. Methods: By using Wistar rats submitted to early weaning at 15 d, we analyzed plasma corticosterone, corticosteroid-binding globulin (CBG), and glucocorticoid receptor (GR) distribution in the gastric epithelium. Results: With the use of radioimmunoassay, we found that early weaning increased corticosterone concentration at day 16 and 17 in test subjects as compared with controls, whereas it was equivalent between groups at day 18. CBG binding capacity decreased during treatment, and it was significantly lower at day 18. At this age, GR levels and distribution in the gastric mucosa were also reduced as compared with suckling counterparts. To reduce corticosterone activity during early weaning and to explore cell proliferation responses, we administered RU486 to 15-d-old pups. We found that cytoplasmic GR reached a peak after 48 h, whereas nuclear levels remained constant, thereby confirming the inhibition of receptor function. Next, by checking gastric proliferative responses, we observed that RU486 induced higher DNA synthesis and mitotic indices in test subjects as compared with control groups. Conclusions: We demonstrated that early weaning changed corticosterone activity by increasing hormone levels, reducing CBG binding capacity, and decreasing GR distribution in the gastric epithelium. These modifications seem to be important to the reorganization of gastric growth after the abrupt interruption of suckling.

Intraneuronal angiotensinergic system in rat and human dorsal root ganglia

To elucidate the local formation of angiotensin II (Ang II) in the neurons of sensory dorsal root ganglia (DRG), we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of protein renin, Ang II, Substance P and calcitonin gene-related peptide (CGRP) in the rat and human thoracic DRG. Quantitative real time PCR (qRT-PCR) studies revealed that rat DRG expressed substantial amounts of Ang-N- and ACE mRNA, while renin mRNA as well as the protein renin were untraceable. Cathepsin D-mRNA and cathepsin D-protein were detected in the rat DRG indicating the possibility of existence of pathways alternative to renin for Ang I formation. Angiotensin peptides were successfully detected with high performance liquid chromatography and radioimmunoassay in human DRG extracts. In situ hybridization in rat DRG confirmed additionally expression of Ang-N mRNA in the cytoplasm of numerous neurons. Intracellular Ang II staining could be shown in number of neurons and their processes in both the rat and human DRG. Interestingly we observed neuronal processes with angiotensinergic synapses en passant, colocalized with synaptophysin, within the DRG. In the DRG, we also identified by qRT-PCR, expression of Ang II receptor AT(1A) and AT(2)-mRNA while AT(1B)-mRNA was not traceable. In some neurons Substance P and CGRP were found colocalized with Ang II. The intracellular localization and colocalization of Ang II with Substance P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the regulation of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human DRG and act as a neurotransmitter.

Colon cancer cells produce immunoregulatory glucocorticoids

Glucocorticoids (GC) have important anti-inflammatory and pro-apoptotic activities. Initially thought to be exclusively produced by the adrenal glands, there is now increasing evidence for extra-adrenal sources of GCs. We have previously shown that the intestinal epithelium produces immunoregulatory GCs and that intestinal steroidogenesis is regulated by the nuclear receptor liver receptor homolog-1 (LRH-1). As LRH-1 has been implicated in the development of colon cancer, we here investigated whether LRH-1 regulates GC synthesis in colorectal tumors and whether tumor-produced GCs suppress T-cell activation. Colorectal cancer cell lines and primary tumors were found to express steroidogenic enzymes and regulatory factors required for the de novo synthesis of cortisol. Both cell lines and primary tumors constitutively produced readily detectable levels of cortisol, as measured by radioimmunoassay, thin-layer chromatography and bioassay. Whereas overexpression of LRH-1 significantly increased the expression of steroidogenic enzymes and the synthesis of cortisol, downregulation or inhibition of LRH-1 effectively suppressed these processes, indicating an important role of LRH-1 in colorectal tumor GC synthesis. An immunoregulatory role of tumor-derived GCs could be further confirmed by demonstrating a suppression of T-cell activation. This study describes for the first time cortisol synthesis in a non-endocrine tumor in humans, and suggests that the synthesis of bioactive GCs in colon cancer cells may account as a novel mechanism of tumor immune escape.

Comparative quantitative assessment of GnRH- and LH-receptor mRNA expression in the urinary tract of sexually intact and spayed female dogs

Ovariectomy interrupts the regulatory loop in the hypothalamus-pituitary-gonad axis, leading to a several-fold increase in gonadotropin levels. This rise in hormonal secretion may play a causal role in ovariectomy-related urinary incontinence. The purpose of this study was to examine the effect of ovariectomy in bitches on the expression of GnRH- and LH-receptors in the lower urinary tract, and assess the relationship between receptor expression and plasma gonadotropin concentrations. Plasma gonadotropins were measured in 37 client-owned bitches. Biopsies were harvested from the mid-ventral bladder wall in all dogs, and from nine further locations within the lower urinary tract in 17 of the 37 animals. Messenger RNA of the LH and GnRH receptors was quantified using RT-PCR with the TaqMan Universal PCR Master Mix. Gonadotropins were measured with a canine-specific FSH-immunoradiometric assay and LH-radioimmunoassay. The hierarchical mixed ANOVA model using MINITAB, Mann-Whitney U-test, unpaired means comparison and linear regressions using StatView were applied for statistical analyses. Messenger RNA for both receptors was detected in all biopsy samples. Age was negatively correlated to mRNA expression of the LH and the GnRH receptors. A relationship between the mRNA values and the plasma gonadotropin concentrations was not established. Evaluation of results within each of the biopsy locations revealed greater LH-receptor expression in the proximal second quarter of the urethra in spayed bitches than in intact bitches (P=0.0481). Increased mRNA expression of LH receptors in this location could possibly play a role in the decrease in closing pressure of the urethra following ovariectomy.

Eicosanoids in exhaled breath condensates in the assessment of childhood asthma

The value of measurements of eicosanoids in exhaled breath condensate (EBC) for the evaluation of childhood asthma is still inconclusive most likely because of the limited value of the methods used. In this case-control study in 48 asthmatic and 20 healthy children, we aimed to characterize the baseline profile of the inflammatory mediators cysteinyl leukotrienes (cysLTs), 9(alpha)11(beta)PGF(2), PGE(2), PGF(2alpha), 8-isoprostane (8-iso-PGF(2alpha)) within EBC in asthmatic compared with healthy children using new methods. In addition, we investigated their relation to other inflammatory markers. The assessment included collection of EBC, measurement of fractional exhaled nitric oxide (FE(NO)) and evaluation of urinary excretion of leukotriene E(4.) cysLTs were measured directly in EBC by radioimmunoassay and prostanoids were measured using gas chromatography negative-ion chemical ionization mass spectrometry. Only cysLT levels were significantly higher in asthmatic compared with healthy children (p = 0.002). No significant differences in cysLTs were found between steroid naïve and patients receiving inhaled corticosteroids. In contrast, FE(NO) was significantly higher in steroid naïve compared with steroid-treated asthmatic and healthy children (p = 0.04 and 0.024, respectively). The diagnostic accuracy of cysLTs in EBC for asthma was 73.6% for the whole group and 78.2% for steroid-naïve asthmatic children. The accuracy to classify asthmatic for FE(NO) was poor (62.9%) for the whole group, but improved to 79.9% when only steroid-naïve asthmatic children were taken into consideration. cysLTs in EBC is an inflammatory marker which distinguishes asthmatics, as a whole group, from healthy children.

Selective increases in regional brain glucocorticoid: a novel effect of chronic alcohol

The hypothalamo-pituitary-adrenal axis shows functional changes in alcoholics, with raised glucocorticoid release during alcohol intake and during the initial phase of alcohol withdrawal. Raised glucocorticoid concentrations are known to cause neuronal damage after withdrawal from chronic alcohol consumption and in other conditions. The hypothesis for these studies was that chronic alcohol treatment would have differential effects on corticosterone concentrations in plasma and in brain regions. Effects of chronic alcohol and withdrawal on regional brain corticosterone concentrations were examined using a range of standard chronic alcohol treatments in two strains of mice and in rats. Corticosterone was measured by radioimmunoassay and the identity of the corticosterone extracted from brain was verified by high performance liquid chromatography and mass spectrometry. Withdrawal from long term (3 weeks to 8 months) alcohol consumption induced prolonged increases in glucocorticoid concentrations in specific regions of rodent brain, while plasma concentrations remained unchanged. This effect was seen after alcohol administration via drinking fluid or by liquid diet, in both mice and rats and in both genders. Shorter alcohol treatments did not show the selective effect on brain glucocorticoid levels. During the alcohol consumption the regional brain corticosterone concentrations paralleled the plasma concentrations. Type II glucocorticoid receptor availability in prefrontal cortex was decreased after withdrawal from chronic alcohol consumption and nuclear localization of glucocorticoid receptors was increased, a pattern that would be predicted from enhanced glucocorticoid type II receptor activation. This novel observation of prolonged selective increases in brain glucocorticoid activity could explain important consequences of long term alcohol consumption, including memory loss, dependence and lack of hypothalamo-pituitary responsiveness. Local changes in brain glucocorticoid levels may also need to be considered in the genesis of other mental disorders and could form a potential new therapeutic target.

Effects of minor laboratory procedures, adrenalectomy, social defeat or acute alcohol on regional brain concentrations of corticosterone

Concentrations of corticosterone in brain areas of TO strain mice were measured by radioimmunoassay. The studies examined the effects of routine laboratory maneuvers, variation during the circadian peak, adrenalectomy, social defeat and acute injections of alcohol on these concentrations. Brief handling of mice increased corticosterone levels in plasma but not in striatum and reduced those in the hippocampus. Single injections of isotonic saline raised the plasma concentrations to a similar extent as the handling, but markedly elevated concentrations in the three brain regions. Five minutes exposure to a novel environment increased hippocampal and cerebral cortical corticosterone levels and striatal concentrations showed a larger rise. However, by 30 min in the novel environment, plasma concentrations rose further while those in striatum and cerebral cortex fell to control levels and hippocampal corticosterone remained elevated. Over the period of the circadian peak the hippocampal and striatal concentrations paralleled the plasma concentrations but cerebral cortical concentrations showed only small changes. Adrenalectomy reduced plasma corticosterone concentrations to below detectable levels after 48 h but corticosterone levels were only partially reduced in the hippocampus and striatum and remained unchanged in the cerebral cortex. Single or repeated social defeat increased both brain and plasma concentrations after 1 h. Acute injections of alcohol raised the regional brain levels in parallel with plasma concentrations. The results show that measurements of plasma concentrations do not necessarily reflect the levels in brain. The data also demonstrate that corticosterone levels can change differentially in specific brain regions. These results, and the residual hormone seen in the brain after adrenalectomy, are suggestive evidence for a local origin of central corticosterone.

Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia

To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N- and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.

Brain Neuropeptides That Control Prolactin Secretion in Cattle

The objective was to test the hypothesis that dopamine regulates prolactin (PRL) secretion by determining acute changes in catecholamine concentrations in hypophyseal portal blood of cattle and their relation to peripheral blood concentration of PRL in hypophyseal stalk-transected (HST) and sham-operated control (SOC). Holstein heifers were subjected to neurosurgery to collect hypophyseal portal blood with a stainless steel cannula designed with a cuff placed under the pituitary stalk and peripheral blood via a jugular vein catheter. PRL plasma concentration was measured by radioimmunoassay, and dopamine and norepinephrine in portal plasma by radioenzymatic assay. During anesthesia before HST or SOC, PRL plasma concentration ranged from 20–40 ng/ml throughout 255 minutes. PRL abruptly increased and remained above 90 ng/ml after HST, compared with a steady decrease to <20 ng/ml in SOC heifers throughout 440 minutes. Within 5 minutes after severing of the hypophyseal stalk, dopamine in portal blood (>8 ng/ml) was significantly increased (P<0.05) compared with peripheral blood (<2 ng/ml). Norepinephrine concentration in portal blood was significantly greater (P<0.05) than in peripheral blood during the first 60 minutes. The sustained high PRL level in peripheral plasma after severing the hypophyseal stalk stimulated hypothalamic dopamine secretion from hypophyseal portal vessels during the prolonged period of blood collection. Norepinephrine concentration in these cattle was greater in hypophyseal portal blood than in peripheral blood, implicating both an important hypothalamic source of the catecholamine as well as an adrenal gland contribution during anesthesia.

Isolation and {\it in vitro}-immunosuppressive characterization of a novel cyclosporine metabolite

Cyclosporine (CsA) has shown great benefit to organ transplant recipients, as an immunosuppressive drug. To optimize CsA immunosuppressive therapy, pharmacodynamic evaluation of serial patient serum samples after CsA administration, using mixed lymphocyte culture (MLC) assays, revealed in vitro serum immunosuppressive activity of a CsA-like, ether-extractable component, associated with good clinical outcome in vivo. Since the in vitro immunosuppressive CsA metabolites, M-17 and M-1, are erythrocyte-bound, the immunosuppressive activity demonstrated in patient serum suggests that other immunosuppressive metabolites need exist. To test this hypothesis and obtain CsA metabolites for study, ether-extracted bile from tritiated and nonradioactive CsA-treated pigs was processed by novel high performance liquid and thin-layer chromatography (HPLC and HPTLC) techniques. Initial MLC screening of potential metabolites revealed a component, designated M-E, to have immunosuppressive activity. Pig bile-derived M-E was characterized as a CsA metabolite, by radioactive CsA tracer studies, by 56% crossreactivity in CsA radioimmunoassay, and by mass spectrometric (MS) analysis. MS revealed a CsA ring structure, hydroxylated at a site other than at amino acid one. M-E was different than M-1 and M-17, as demonstrated by different retention properties for each metabolite, using HPTLC and a novel rhodamine B/ $\alpha$-cyclodextrin stain, and using HPLC, performed by Sandoz, that revealed M-E to be different than previously characterized metabolites. The immunosuppressive activity of M-E was quantified by determination of mean metabolite potency ratio in human MLC assays, which was found to be 0.79 $\pm$ 0.23 (CsA, 1.0). Similar to parent drug, M-E revealed inter-individual differences in its immunosuppressive activity. M-E demonstrates inhibition of IL-2 production by concanavalin A stimulated C3H mouse spleen cells, similar to CsA, as determined with an IL-2 dependent mouse cytotoxic T-cell line. ^

EXPRESSION OF TUMOR ASSOCIATED ANTIGENS ON HUMAN COLON CARCINOMA CELLS (HUMAN COLON CANCER, MONOCLONAL ANTIBODY)

Human colon cancer cells, LS180 and 174T, exhibit monoclonal antibody (mAb) 1083-17-1A and 5E113 defined tumor associated antigens. By radioimmunoassay, LS180 cells expressed the highest amount of mAb1083 defined antigens among the cell lines tested. Another mAb, 5E113, competed with mAb1083 for binding to LS180 cells, suggesting that both mAbs might bind onto identical (or adjacent) epitopes. By Scatchard analysis, about one million copies of the epitopes were present on LS180 colon cancer cells. The affinity of mAb1083 binding to the cells was 2.97 x 10('10) M('-1); the Sipsian heteroclonality value of mAb1083 was 0.9, thus approximating a single clone of reactive antibody. The qualitative studies showed that the epitopes were probably not carbohydrate because of their sensitivity to proteinases and not to mixed glucosidases and neuraminidase. The tunicamycin homologue B(,2) inhibited the incoporation of ('3)H-labeled galactose but not uptake of ('35)S-labeled methionine, nor expression of monoclonal antibody defined antigens providing further evidence to exclude the possibility of carbohydrate epitopes. There was evidence that the epitope might be partially masked in its "native" conformation, since short exposure or low dose treatment with proteases increased mAbs binding. The best detergent for antigen extraction, as detected by dot blotting and competitive inhibition assays, was octylglucoside at 30 mM concentration. Three methods, immunoprecipitation, Western blotting and photoaffinity labeling, were used to determine the molecular nature of the antigens. These results demonstrated that the antibody bound both 43 K daltons (KD) and 22 KD proteins.^ An in vitro cell-mediated immune approach was also used to attempt identifying function for the antigens. The strategy was to use mAbs to block cytotoxic effector cell killing. However, instead of blocking, the mAb1083 and 5E113 showed strong antibody-dependent cell-mediated cytotoxicities (ADCCs) in the in vitro xenoimmune assay system. In addition, cytotoxic T lymphocytes (CTLs), natural killer cells, and K cell activity were found. Since even the F(ab')2 fragment of mAbs did not inhibit the cytolytic effect, the mAbs defined antigens may not be major target molecules for CTLs. In summary, two molecular species of tumor antigen(s) were identified by mAbs to be present on colon tumor cell lines, LS180 and LS174T. (Abstract shortened with permission of author.) ^