IRF-1 responsiveness to IFN-γ predicts different cancer immune phenotypes.

BACKGROUND: Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes. METHODS: IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both. Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation. RESULTS: We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment. Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts. Functional interpretation analysis showed mTOR and Wnt/ß-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis. CONCLUSION: Our findings support the central role of IRF-1 in influencing different tumour phenotypes.

Encouraging effect of autologous bone marrow aspirate concentrate in rehabilitation of children with cerebral palsy.

OBJECTIVE: In this study, we used autologous bone marrow aspirate concentrate (BMAC) transplantation to treat children with cerebral palsy (CP) to improve their motor and cognitive functions. PATIENTS AND METHODS: Forty-two patients with CP received BMAC. The transplantation of stem cells via the intrathecal route includes three BMAC applications. The patients' examination was before the injection of stem cells, with follow-ups on 1, 3, 6, and 12 months after the injections. The assessments included the gross motor function classification scale, the Ashworth scale, and the Learning accomplishment profile-diagnostic scale. RESULTS: This study included 42 patients with CP who received three BMAC intrathecal administrations. A personalized home rehabilitation program was designed and included for each patient in the study. After the treatment, we observed a reduction of spasticity in 58% of patients and significant cognitive improvement in 35% of patients. CONCLUSIONS: The outcome of this study indicates that stem cell therapy and personalized training can improve the development of children with CP. The crucial goal of this therapeutic intervention is to substitute injured tissue with new tissues by activating the regenerative capacity of stem cells.

Genotypic and phenotypic evaluation of Rutilus spp. from Skadar, Ohrid and Prespa Lakes supports revision of endemic as well as taxonomic status of several taxa.

One mtDNA gene (cytochrome b), one nuclear DNA fragment, five microsatellites and a suite of morphological characters were evaluated in samples of Rutilus spp. from Skadar, Ohrid and Prespa Lakes. Both genetic and phenotypic data supported two sympatric taxa in Lake Skadar, whereby Prespa and Ohrid Lakes revealed only a single taxon each. One of the taxa from Lake Skadar was similar to samples from Lake Prespa, whereas the second taxon was the most divergent in the data set. The estimated time to the most recent common ancestor of these two sympatric taxa in Lake Skadar was between 125 000 and 500 000 years. The data did not support existing taxonomic schemes for Rutilus in these lakes. This study poses the following working hypothesis: (1) Rutilus prespensis lives both in Lake Prespa and Lake Skadar and therefore is not endemic to Lake Prespa, (2) Rutilus ohridanus lives in Lake Ohrid only and therefore could be considered an endemic if its species status is retained and (3) a third recently described taxon (Rutilus albus) sympatric to R. prespensis lives in Lake Skadar.

Psychological well-being in advanced NSCLC patients in Serbia: impact of sociodemographic and clinical factors.

UNLABELLED: Patients with advanced non-small cell lung cancer (NSCLC) usually undergo toxic treatment (chemotherapy and/or radiotherapy). They can experience devastating effects of illness and therapies on their psychological and emotional well-being. On the other hand, untreated psychological distress is associated with reduced quality of life and inadequate palliation of physical symptoms.
In order to estimate frequency of anxiety and depressive symptoms and influence of demographic, socioeconomic and clinical factors on psychological well-being, we performed this cross-sectional study in group of 100 patients with advanced stage of disease. Symptoms of anxiety and depression were assessed using the Hamilton Anxiety Rating Scale (HARS) and Hamilton Depression Rating scale (HDRS). Health-related quality of life data are obtained by EORTC QLC C30 and SF 36.
Patients with poor performance status (PS) experienced significantly more anxiety and depressive symptoms (p=0.001) and worse emotional (p=0.001) and mental functioning (p=0.001). Treated patients had significantly better mental (p=0.011) and emotional (p=0.001) functioning in compared to newly diagnosed ones. Somewhat unusual, unemployed participants reported significantly less anxiety (p=0.029) and depressive (p=0.002) symptoms, better mental (p=0.030) and emotional functioning (p=0.007). Additionally, nausea and vomiting adversely affected mental health and emotional functioning and correlated significantly positively with HARS and HDRS scores.
Our findings suggest significant impact of some disease-related factors (PS, active treatment) and treatment-related factors (chemotherapy -induced nausea and vomiting) on psychological well-being of patients with advanced NSCLC. This should be taking an account when appropriate interventions are planned. KEYWORDS: lung cancer, anxiety, depression, quality of life, chemotherapy, chemotherapy-induced nausea and vomiting.

Health-related quality of life in lung cancer patients in Serbia: correlation with socio-economic and clinical parameters.

The objective of this study was to assess health-related quality of life (HRQoL) in patients with advanced non-small cell lung cancer (NSCLC). In Serbia, there is the lack of available data on HRQoL in lung cancer patients. The special attention in our study has been paid on relationships between socio-economic factors and HRQoL. This cross-sectional study was undertaken in group of 100 NSCLC patients with advanced stage diseases. HRQoL was measured using three standard instruments: 36-item Short Form Health Survey, EORTC QLQ-C30 and its Lung Cancer module (EORTC QLQ-LC13). Unexpected, highly educated patients reported significantly worse social functioning (P=0.044), and higher degree of financial difficulties (P=0.047), in comparison with less-educated. Also unusual, unemployed patients had significantly better HRQoL in all domains and significantly lower symptom distress. Significantly better overall HRQoL (P=0.043), social (P=0.024), emotional (P=0.001) and mental functioning (P=0.011) were observed in patients treated with chemotherapy in comparison with newly diagnosed ones. In addition, the most prominent side effects of chemotherapy were nausea and vomiting, and all QoL domains correlated significantly with them. Patients who undergo active treatment improve their HRQoL but chemotherapy-induced emesis adversely affects many HRQoL domains. Additionally, HRQoL is highly dependent on patient's socio-economic characteristic.

Gender and age trends of histological types of lung cancer in a 20-year period: pathological perspective.

PURPOSE: To find out the trends of distribution in different histological types of lung cancer in both genders in a period of 20 years. METHODS: The most frequent histological types of lung cancer in tissue specimens obtained by bronchoscopy or percutaneous needle biopsy were analysed in terms of age and gender. The studied population included 6289 patients (16.6% females and 83.1% males). Statistical significance was established by x(2) test at the level p<0.05. RESULTS: Squamous cell carcinoma (SCC) prevailed in the total number of patients in all investigated years (58.0%), and separately in male (60.4%) and female (45.7%) patients. This histological type was predominant in all age groups in both genders (41.6% in males and 38.1% in females). CONCLUSION: SCC has the highest incidence in Serbia. Continuous campaign against smoking and helping its cessation, improving working and socioeconomic conditions is a strategy for decreasing all histological types of lung cancer patients.

Autism treatment with stem cells: a case report.

OBJECTIVE: Autism Spectrum Disorder is a complex brain disorder and has multiple causes that occur in diverse combinations. There is a need to classify children with ASD at a very young age so that they can access evidence-based intervention that can significantly improve their outcomes. CASE REPORT: In this report we present a case of autism, which underwent intrathecal autologous bone marrow mononuclear cells transplantation along with neurorehabilitation. The primary goal of the treatment is to improve the quality of life of the patient. After the procedure, the child started to speak, therefore, the third communication subscale was scored within the GARS-2 assessment instrument. With these three subscales, a score of 91 has been achieved, representing an autism index of 27%, a significant improvement over the previous score. CONCLUSIONS: Our study demonstrated evidences to support the safety and effectiveness of BMAC transplantation in the management of autism.

p11 is up-regulated in the forebrain of stressed rats by glucocorticoid acting via two specific glucocorticoid response elements in the p11 promoter.

Posttraumatic stress disorder (PTSD) is one of the most common psychiatric disorders. Despite the extensive study of the neurobiological correlates of this disorder, the underlying mechanisms of PTSD are still poorly understood. Recently, a study demonstrated that dexamethasone (Dex), a synthetic glucocorticoid, can up-regulate p11, known as S100A10-protein which is down-regulated in patients with depression, (Yao et al., 1999; Huang et al., 2003) a common comorbid disorder in PTSD. These observations led to our hypothesis that traumatic stress may alter expression of p11 mediated through a glucocorticoid receptor. Here, we demonstrate that inescapable tail shock increased both prefrontal cortical p11 mRNA levels and plasma corticosterone levels in rats. We also found that Dex up-regulated p11 expression in SH-SY5Y cells through glucocorticoid response elements (GREs) within the p11 promoter. This response was attenuated by either RU486, a glucocorticoid receptor (GR) antagonist or mutating two of three glucocorticoid response elements (GRE2 and GRE3) in the p11 promoter. Finally, we showed that p11 mRNA levels were increased in postmortem prefrontal cortical tissue (area 46) of patients with PTSD. The data obtained from our work in a rat model of inescapable tail shock, a p11-transfected cell line and postmortem brain tissue from PTSD patients outline a possible mechanism by which p11 is regulated by glucocorticoids elevated by traumatic stress.

The adaptive response of adult rat Leydig cells to repeated immobilization stress: the role of protein kinase A and steroidogenic acute regulatory protein.

The ability of immobilization stress (IMO) to decrease Leydig cell steroidogenesis and serum androgen concentration has been previously observed, but the possible mechanism(s) involved in the adaptation to prolonged or repeated stress have not been identified. In this study, we investigated whether the Leydig cells obtained from adult rats subjected to acute (15 min, 30 min or 2 h) and repeated (2 or 10 days, 2 h daily) IMO show adaptive mechanism(s) in response to stress-impaired steroidogenesis. The results showed that basal and human chorionic gonadotropin-stimulated cAMP production by Leydig cells isolated from rats exposed to both acute and repeated IMO was significantly reduced. Despite the reduced cAMP production, immunoblot analysis revealed increased immunoreactivity for both protein kinase A (PKA) and steroidogenic acute regulatory (StAR) protein in Leydig cells obtained from rats repeatedly exposed to IMO. Also, the phosphorylation and production of mature StAR protein was evident during exposure of rats to repeated IMO treatment. Treatment with cholesterol, the steroid substrate transported into mitochondria by StAR, significantly increased androgen and progesterone production by Leydig cells isolated from rats exposed to repeated IMO. In contrast, when other steroid substrates (22(R)-OH-cholesterol, pregnenolone, progesterone, Delta4-androstenedione) were present in the culture media, Leydig cell steroidogenesis was still reduced by IMO. Thus, PKA-mediated phosphorylation of StAR protein is an important mechanism in the adaptive response of Leydig cells to repeated IMO.

Secondary electron emission of carbonaceous dust particles.

In this paper we present measurements of the secondary electron emission yield (gamma) of a carbonaceous dust particle material, which was grown in argon diluted acetylene plasmas. One aim was to reach a better understanding of charging and discharging processes of dust particles in complex plasmas due to secondary electron emission and consequently to try to explain the anomalous behavior of electron density observed in afterglows of pulsed rf plasmas. We compared the results of a simple model and of a Monte Carlo simulation to the previously measured time dependence of the electron density in complex plasma afterglow. It was found that the value of the intrinsic secondary electron yield from the carbonaceous dust material is too low to explain the increase of electron density in the afterglow. It is, however, possible that the electrons charging the particles are weakly attached so that they may be released with high efficiency by ion bombardment due to field induced emission or by other mechanisms.

GABAA receptor subunit composition and functional properties of Cl- channels with differential sensitivity to zolpidem in embryonic rat hippocampal cells.

Using flow cytometry in conjunction with a voltage-sensitive fluorescent indicator dye (oxonol), we have identified and separated embryonic hippocampal cells according to the sensitivity of their functionally expressed GABAA receptors to zolpidem. Immunocytochemical and RT-PCR analysis of sorted zolpidem-sensitive (ZS) and zolpidem-insensitive (ZI) subpopulations identified ZS cells as postmitotic, differentiating neurons expressing alpha2, alpha4, alpha5, beta1, beta2, beta3, gamma1, gamma2, and gamma3 GABAA receptor subunits, whereas the ZI cells were neuroepithelial cells or newly postmitotic neurons, expressing predominantly alpha4, alpha5, beta1, and gamma2 subunits. Fluctuation analyses of macroscopic Cl- currents evoked by GABA revealed three kinetic components of GABAA receptor/Cl- channel activity in both subpopulations. We focused our study on ZI cells, which exhibited a limited number of subunits and functional channels, to directly correlate subunit composition with channel properties. Biophysical analyses of GABA-activated Cl- currents in ZI cells revealed two types of receptor-coupled channel properties: one comprising short-lasting openings, high affinity for GABA, and low sensitivity to diazepam, and the other with long-lasting openings, low affinity for GABA, and high sensitivity to diazepam. Both types of channel activity were found in the same cell. Channel kinetics were well modeled by fitting dwell time distributions to biliganded activation and included two open and five closed states. We propose that short- and long-lasting openings correspond to GABAA receptor/Cl- channels containing alpha4beta1gamma2 and alpha5beta1gamma2 subunits, respectively.

Glutamate acting at NMDA receptors stimulates embryonic cortical neuronal migration.

During cortical development, embryonic neurons migrate from germinal zones near the ventricle into the cortical plate, where they organize into layers. Mechanisms that direct neuronal migration may include molecules that act as chemoattractants. In rats, GABA, which localizes near the target destination for migrating cortical neurons, stimulates embryonic neuronal migration in vitro. In mice, glutamate is highly localized near the target destinations for migrating cortical neurons. Glutamate-induced migration of murine embryonic cortical cells was evaluated in cell dissociates and cortical slice cultures. In dissociates, the chemotropic effects of glutamate were 10-fold greater than the effects of GABA, demonstrating that for murine cortical cells, glutamate is a more potent chemoattractant than GABA. Thus, cortical chemoattractants appear to differ between species. Micromolar glutamate stimulated neuronal chemotaxis that was mimicked by microM NMDA but not by other ionotropic glutamate receptor agonists (AMPA, kainate, quisqualate). Responding cells were primarily derived from immature cortical regions [ventricular zone (vz)/subventricular zone (svz)]. Bromodeoxyuridine (BrdU) pulse labeling of cortical slices cultured in NMDA antagonists (microM MK801 or APV) revealed that antagonist exposure blocked the migration of BrdU-positive cells from the vz/svz into the cortical plate. PCR confirmed the presence of NMDA receptor expression in vz/svz cells, whereas electrophysiology and Ca2+ imaging demonstrated that vz/svz cells exhibited physiological responses to NMDA. These studies indicate that, in mice, glutamate may serve as a chemoattractant for neurons in the developing cortex, signaling cells to migrate into the cortical plate via NMDA receptor activation.

GABA expression dominates neuronal lineage progression in the embryonic rat neocortex and facilitates neurite outgrowth via GABA(A) autoreceptor/Cl- channels.

GABA emerges as a trophic signal during rat neocortical development in which it modulates proliferation of neuronal progenitors in the ventricular/subventricular zone (VZ/SVZ) and mediates radial migration of neurons from the VZ/SVZ to the cortical plate/subplate (CP/SP) region. In this study we investigated the role of GABA in the earliest phases of neuronal differentiation in the CP/SP. GABAergic-signaling components emerging during neuronal lineage progression were comprehensively characterized using flow cytometry and immunophenotyping together with physiological indicator dyes. During migration from the VZ/SVZ to the CP/SP, differentiating cortical neurons became predominantly GABAergic, and their dominant GABA(A) receptor subunit expression pattern changed from alpha4beta1gamma1 to alpha3beta3gamma2gamma3 coincident with an increasing potency of GABA on GABA(A) receptor-mediated depolarization. GABA(A) autoreceptor/Cl(-) channel activity in cultured CP/SP neurons dominated their baseline potential and indirectly their cytosolic Ca(2+) (Ca(2+)c) levels via Ca(2+) entry through L-type Ca(2+) channels. Block of this autocrine circuit at the level of GABA synthesis, GABA(A) receptor activation, intracellular Cl(-) ion homeostasis, or L-type Ca(2+) channels attenuated neurite outgrowth in most GABAergic CP/SP neurons. In the absence of autocrine GABAergic signaling, neuritogenesis could be preserved by depolarizing cells and elevating Ca(2+)c. These results reveal a morphogenic role for GABA during embryonic neocortical neuron development that involves GABA(A) autoreceptors and L-type Ca(2+) channels.

Direct binding of estradiol enhances Slack (sequence like a calcium-activated potassium channel) channels' activity.

17Beta-estradiol (E2) is a major neuroregulator, exerting both genomic and non-genomic actions. E2 regulation of Slack (sequence like a calcium-activated potassium channel) potassium channels has not been identified in the CNS. We demonstrate E2-induced activation of Slack channels, which display a unitary conductance of about 60 pS, are inhibited by intracellular calcium, and are abundantly expressed in the nervous system. In lipid bilayers derived from rat cortical neuronal membranes, E2 increases Slack open probability and appears to decrease channel inactivation. Additionally, E2 binds to the Slack channel and activates outward currents in human embryonic kidney-293 cells that express Slack channels but not classical estrogen receptors (i.e. ERalpha or ERbeta). Neither E2-induced activation nor the binding intensity of E2 to the Slack channel is blocked by tamoxifen, an ER antagonist/agonist. Thus, E2 activates a potassium channel, Slack, through a non-traditional membrane binding site, adding to known non-genomic mechanisms by which E2 exerts pharmacological and toxicological effects in the CNS.

Neuroepithelial cells in the rat spinal cord express glutamate decarboxylase immunoreactivity in vivo and in vitro.

It is unknown whether neuroepithelial cells in the mammalian central nervous system express neurotransmitter-synthesizing enzymes. In this study, expression of glutamate decarboxylase (GAD), the gamma-aminobutyric acid (GABA)-synthesizing enzyme, was examined in proliferative cells and postmitotic neuroblasts in embryonic rat spinal cord. Immunostaining coronal sections of the embryonic spinal cord with K2 antiserum, which recognizes GAD proteins encoded by the GAD67 gene, revealed intensely stained neuroepithelial cells in the basal plate at embryonic day (E) 13, in the intermediate plate between E 13-16, and last seen in the alar plate at E 16. Nissl counterstaining demonstrated that a small number of these GAD-immunoreactive cells adjacent to the neural tube lumen were mitotic. The ventral-to-dorsal gradient of GAD expression in precursor cells and postmitotic neuroblasts correlates anatomically and temporally with the sequential generation of motoneurons, commissural neurons, and interneurons in the dorsal horn. Some of these GAD-immunoreactive neuroepithelial cells may re-enter the mitotic cycle, while others are postmitotic neuroblasts presumably migrating to the intermediate zone to differentiate into young neurons. Double-immunostaining cells acutely dissociated from E 11-18 spinal cords with K2 and anti-bromodeoxyuridine antisera, following a bromodeoxyuridine pulse in vivo, revealed considerable numbers of DNA-synthesizing cells immunoreactive for GAD. The absolute number of double-stained cells peaked during E 12-15, coinciding with terminal cell division in most spinal neurons. These observations suggest that spinal neuronal precursors can synthesize GAD-related proteins prior to, or during, the terminal cell cycle. Although GAD immunoreactivity revealed by K2 antiserum was detected in proliferative cells and in migrating postmitotic neuroblasts, GABA immunoreactivity was never detectable in these cells. These early embryonic GAD-immunoreactive neuroepithelial cells may either synthesize levels of GABA that cannot be detected immunocytochemically, and/or express enzymatically inactive GAD-related proteins.

Increased TUNEL staining in brains of autoimmune Fas-deficient mice.

Profound changes in brain morphology and behavior coincide with the spontaneous development of systemic autoimmune/inflammatory disease in Fas-deficient MRL-lpr mice. The dendrites atrophy, the density of hippocampal and cortical neurons decreases, and an anxious/depressive-like behavior emerges while lymphoid cells infiltrate into the choroid plexus of MRL-lpr mice. We hypothesized that the inherited lack of the Fas-dependent anti-inflammatory mechanism would lead to unsuppressed immune activity, characterized by reduced apoptosis in the MRL-lpr brain. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeled (TUNEL) method as an indicator of apoptosis, a surprisingly high incidence of TUNEL-positive cells was observed in the hippocampus, choroid plexus and periventricular regions of MRL-lpr mice, 5-10-fold higher than that found in the MRL +/+ control brain. Immunostaining with anti-CD3, CD4 and CD8 monoclonal antibodies showed limited overlap between CD-positive and TUNEL-positive cells, suggesting that the dying cells are for the most part (approximately 70%) not T-lymphocytes. Although further characterization of the phenotype of the dying cells and the mechanism of cell death are required, the present results suggest the involvement of a Fas-independent apoptotic process in neurodegeneration induced by systemic autoimmune disease.

Ethanol blocks both basic fibroblast growth factor- and carbachol-mediated neuroepithelial cell expansion with differential effects on carbachol-activated signaling pathways.

We have expanded neuroepithelial cells dissociated from the embryonic rat telencephalon in serum-free defined medium containing basic fibroblast growth factor (bFGF) in order to generate a model neuroepithelium to study the interaction of ethanol with both growth factor- and transmitter-stimulated proliferation. Ethanol blocked proliferation stimulated by bFGF and by carbachol, an agonist at muscarinic acetylcholine receptors, in a dose-dependent manner. In addition, ethanol attenuated autonomous expansion of neuroepithelial cells occurring following withdrawal of bFGF. The latter effect was associated with an increase in the number of apoptotic cells identified by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling labeling. We studied the effects of ethanol on carbachol-stimulated signaling pathways critical to its proliferative effects. Ethanol significantly reduced carbachol-stimulated Ca(2+) signaling, as well as Erk1/Erk2, Akt and cyclic AMP-response element-binding phosphorylations in a dose-dependent manner. Comparison of the potency of ethanol in attenuating carbachol-stimulated proliferation and signal transduction showed that mitogen-activated protein kinase phosphorylation was less sensitive to ethanol than the other parameters. The results indicate that ethanol's suppression of proliferation induced by carbachol in this model neuroepithelium likely involves multiple signaling pathways. These effects in vitro may help to explain the devastating effects of prenatal ethanol exposure in vivo, which contribute to the fetal alcohol syndrome.

Ethanol blocks cytosolic Ca2+ responses triggered by activation of GABA(A) receptor/Cl- channels in cultured proliferating rat neuroepithelial cells.

GABA(A) receptor/Cl- channels and voltage-gated Ca2+ channels are believed to be important sites of ethanol action in the CNS. Acute exposure of ethanol potentiates GABA(A) receptor/Cl- channel activity and inhibits voltage-gated Ca2+ channels in a number of preparations, mostly post-mitotic neurons. The effects of ethanol on these channels in primary cultures of undifferentiated neural precursor cells remain unknown. To address this issue, we examined the effects of ethanol on GABA(A) agonist-activated elevation of cytosolic Ca2+ in an in vitro model of the cortical neuroepithelium derived from rat basic fibroblast growth factor-expanded neural precursor cells. We found a potent inhibition of GABA(A)-activated elevation of cytosolic Ca2+ by ethanol in actively proliferating cells. Since we had recently demonstrated that GABA(A) receptor activation depolarizes these cells and elevates their cytosolic Ca2+, we tested whether the effects of ethanol involved both GABA(A) receptors and voltage-gated Ca2+ channels. Both extracellular K+- and muscimol-induced cytosolic Ca2+ elevations were abolished by nitrendipine, indicating that both depolarizing stimuli triggered Ca2+ influx through L-type voltage-gated Ca2+ channels. Exposure of proliferating cells to different concentrations of ethanol revealed that the drug was more potent in blocking muscimol-induced compared to K+-evoked cytosolic Ca2+ elevations. These results raise the possibility that ethanol blocks GABAergic stimulation of cytosolic Ca2+ levels in proliferating precursors primarily by interacting with GABA(A) receptor/Cl- channels and secondarily with voltage-gated Ca2+ channels.

Enkephalins and anaphylactic shock: modulation and prevention of shock in the rat.

Wistar rats were sensitized for anaphylactic shock with ovalbumin and treated intraperitoneally with 10 injections of methionine-enkephalin and leucine-enkephalin (4 mg/kg of body weight). Both pentapeptides suppressed the development of systemic anaphylaxis after intravenous injection of a shocking dose of ovalbumin. Methionine-enkephalin completely protected the animals from fatal shock. The level of circulating IgE and precipitating anti-ovalbumin antibodies decreased in rats given 10 injections of enkephalins. Mast cell degranulation was significantly inhibited in these animals as observed at autopsy. A single intraperitoneal injection of 4 mg/kg body weight of methionine-enkephalin given 30 min before the challenge with shock-inducing dose of antigen was also effective, although to a lesser extent, in protecting animals from anaphylactic shock. One injection of leucine-enkephalin did not exhibit significant antishock activity. The results suggest that enkephalins, and methionine-enkephalin in particular, are potent modulators of the complex biochemical processes underlying the anaphylactic shock in the rat.

Involvement of inducible nitric oxide synthase in stress-impaired testicular steroidogenesis.

The immobilization stress induces an acute inhibition of testicular steroidogenesis that is mediated by the nitric oxide (NO) signaling pathway. Here we compared the effects of 2-h immobilization stress on in vivo and in vitro rat steroidogenesis at two time points, 0 h and 6 h after the end of the stress session. As expected, serum androgens and human chorionic gonadotropin (hCG)-stimulated progesterone and testosterone production by testicular tissue were inhibited at 0 h, and also at the 6-h time point. Both the acute and sustained inhibitions of in vitro steroidogenesis were accompanied by a significant increase in nitrite, a stable oxidation product of NO. To clarify which subtype of NO synthase (NOS) (constitutive (cNOS) or inducible (iNOS)) participates in down-regulation of testicular steroidogenesis, aminoguanidine hydrochloride (AG), a selective iNOS inhibitor, was employed. Intratesticular injection of AG prevented the sustained, but not the acute, stress-induced decrease in serum testosterone. When added in vitro, it also prevented the sustained decrease in steroid production and increase in nitrite production by testicular tissue, both in a dose-dependent manner and with EC microM. Furthermore, AG added in vivo and in vitro effectively blocked the sustained decrease in 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17alpha-hydroxylase/C17-20 lyase (P450c17) activities. In all concentrations employed, AG did not affect serum androgens and in vitro steroid and nitrite production in unstressed animals. These results indicate that the NO signaling pathway participates in acute and sustained stress-induced down-regulation of testicular steroidogenesis, presumably through its direct action on 3betaHSD and P450c17. The acute NO production is controlled by cNOS and the sustained production of this messenger is controlled by iNOS.