Association of GA genotype of SNP rs4680 in COMT gene with psoriasis.

Psoriasis is a multigene and multifactorial skin disease with heterogeneous genetic inheritance. Mental disorders participate in the development of psoriasis as predisposing factors; a correlation of dermatological diseases with pathological anxiety and stress was shown. Meanwhile, there are no studies describing molecular mechanisms of the linkages between psycho-emotional disorders and skin diseases. The aim of this study is to find the associations between SNP in genes COMT (rs4680), DBH (rs141116007), CCKAR (rs1800857) and CCKBR (rs1805002), and psoriasis. Patients were selected according to the 10th revision of International Classification of Diseases (L-40). The sample size was 88 patients. The size of the control sample (population control) was 365 people. Genotyping was performed using PCR-RFLP and real-time PCR. Statistical analysis was performed using WinPepi software. Identification of complex genotypes was performed by the Monte Carlo method using APSampler 3.6.1 algorithm. Among the studied genes, only GA genotype of COMT gene is significantly associated with psoriasis [χ2 = 19.163 (p = 1.3E-5), F (p) = 1.2E-5, OR 3.47 (CI 99% = 1.61-7.91)]. At the moment, the functional significance of this phenomenon is difficult to explain.

Essential role of hippocampal noradrenaline in the regulation of spatial working memory and TDP-43 tissue pathology.

Extensive loss of noradrenaline-containing neurons and fibers is a nearly invariant feature of Alzheimer's Disease (AD). However, the exact noradrenergic contribution to cognitive and histopathological changes in AD is still unclear. Here, this issue was addressed following selective lesioning and intrahippocampal implantation of embryonic noradrenergic progenitors in developing rats. Starting from about 3 months and up to 12 months post-surgery, animals underwent behavioral tests to evaluate sensory-motor, as well as spatial learning and memory, followed by post-mortem morphometric analyses. At 9 months, Control, Lesioned and Lesion + Transplant animals exhibited equally efficient sensory-motor and reference memory performance. Interestingly, working memory abilities were seen severely impaired in Lesion-only rats and fully recovered in Transplanted rats, and appeared partly lost again 2 months after ablation of the implanted neuroblasts. Morphological analyses confirmed the almost total lesion-induced noradrenergic neuronal and terminal fiber loss, the near-normal reinnervation of the hippocampus promoted by the transplants, and its complete removal by the second lesion. Notably, the noradrenergic-rich transplants normalized also the nuclear expression of the transactive response DNA-binding protein 43 (TDP-43) in various hippocampal subregions, whose cytoplasmic (i.e., pathological) occurrence appeared dramatically increased as a result of the lesions. Thus, integrity of ascending noradrenergic inputs to the hippocampus may be required for the regulation of specific aspects of learning and memory and to prevent TDP-43 tissue pathology.

Involvement of dopamine beta-hydroxylase in the neuroendocrine-immune regulatory network of white shrimp, Litopenaeus vannamei.

In shrimp, the biosynthesis of catecholamines, including dopamine and norepinephrine, is required for physiological and immunological responses against stress. Dopamine beta-hydroxylase (DBH), a copper-containing monooxygenase enzyme that plays an important role in catecholamine synthesis of the neuroendocrine regulatory network, was identified in Litopenaeus vannamei. In the present study, the potential role of DBH in the immunocompetence of L. vannamei was further estimated by depleting DBH by pharmaceutical inhibition of disulfiram and a gene silencing technique of L. vannamei DBH-double-stranded (ds)RNA (LvDBH-dsRNA). Immunocompetence was evaluated following the determination of the total hemocyte count, differential hemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, phagocytic activity, and the clearance efficiency as well as the susceptibility against Vibrio alginolyticus infection. At 30-120 min after shrimp had received disulfiram, they exhibited significantly reduced total hemocyte count, phenoloxidase activity of hemocytes in hemolymph, respiratory bursts of hemocytes in hemolymph and per hemocyte, phagocytic activity, clearance efficiency, and survival ratio against V. alginolyticus infection, compared to those injected with saline. In addition, the significantly lower total hemocyte count, phagocytic activity, clearance efficiency, and resistance to V. alginolyticus infection were observed in shrimp that received LvDBH-dsRNA at 3 days post injection compared to those injected with diethyl pyrocarbonate-water or non-targeting gene-dsRNA. The DBH depleted L. vannamei revealed immunosuppression and decreased the survival ratio to V. alginolyticus infection, which indicated that DBH played a crucial role in the neuroendocrine-immune regulatory network.

Identification of Candidate Tolerogenic CD8(+) T Cell Epitopes for Therapy of Type 1 Diabetes in the NOD Mouse Model.

Type 1 diabetes is an autoimmune disease in which insulin-producing pancreatic islet ß cells are the target of self-reactive B and T cells. T cells reactive with epitopes derived from insulin and/or IGRP are critical for the initiation and maintenance of disease, but T cells reactive with other islet antigens likely have an essential role in disease progression. We sought to identify candidate CD8(+) T cell epitopes that are pathogenic in type 1 diabetes. Proteins that elicit autoantibodies in human type 1 diabetes were analyzed by predictive algorithms for candidate epitopes. Using several different tolerizing regimes using synthetic peptides, two new predicted tolerogenic CD8(+) T cell epitopes were identified in the murine homolog of the major human islet autoantigen zinc transporter ZnT8 (aa 158-166 and 282-290) and one in a non-ß cell protein, dopamine ß-hydroxylase (aa 233-241). Tolerizing vaccination of NOD mice with a cDNA plasmid expressing full-length proinsulin prevented diabetes, whereas plasmids encoding ZnT8 and DßH did not. However, tolerizing vaccination of NOD mice with the proinsulin plasmid in combination with plasmids expressing ZnT8 and DßH decreased insulitis and enhanced prevention of disease compared to vaccination with the plasmid encoding proinsulin alone.

Orexin-A enhances feeding in male rats by activating hindbrain catecholamine neurons.

Both lateral hypothalamic orexinergic neurons and hindbrain catecholaminergic neurons contribute to control of feeding behavior. Orexin fibers and terminals are present in close proximity to hindbrain catecholaminergic neurons, and fourth ventricular (4V) orexin injections that increase food intake also increase c-Fos expression in hindbrain catecholamine neurons, suggesting that orexin neurons may stimulate feeding by activating catecholamine neurons. Here we examine that hypothesis in more detail. We found that 4V injection of orexin-A (0.5 nmol/rat) produced widespread activation of c-Fos in hindbrain catecholamine cell groups. In the A1 and C1 cell groups in the ventrolateral medulla, where most c-Fos-positive neurons were also dopamine ß hydroxylase (DBH) positive, direct injections of a lower dose (67 pmol/200 nl) of orexin-A also increased food intake in intact rats. Then, with the use of the retrogradely transported immunotoxin, anti-DBH conjugated to saporin (DSAP), which targets and destroys DBH-expressing catecholamine neurons, we examined the hypothesis that catecholamine neurons are required for orexin-induced feeding. Rats given paraventricular hypothalamic injections of DSAP, or unconjugated saporin (SAP) as control, were implanted with 4V or lateral ventricular (LV) cannulas and tested for feeding in response to ventricular injection of orexin-A (0.5 nmol/rat). Both LV and 4V orexin-A stimulated feeding in SAP controls, but DSAP abolished these responses. These results reveal for the first time that catecholamine neurons are required for feeding induced by injection of orexin-A into either LV or 4V.

The rate of fall of blood glucose determines the necessity of forebrain-projecting catecholaminergic neurons for male rat sympathoadrenal responses.

Different onset rates of insulin-induced hypoglycemia use distinct glucosensors to activate sympathoadrenal counterregulatory responses (CRRs). Glucosensory elements in the portal-mesenteric veins are dispensable with faster rates when brain elements predominate, but are essential for responses to the slower-onset hypoglycemia that is common with insulin therapy. Whether a similar rate-associated divergence exists within more expansive brain networks is unknown. Hindbrain catecholamine neurons distribute glycemia-related information throughout the forebrain. We tested in male rats whether catecholaminergic neurons that project to the medial and ventromedial hypothalamus are required for sympathoadrenal CRRs to rapid- and slow-onset hypoglycemia and whether these neurons are differentially engaged as onset rates change. Using a catecholamine-specific neurotoxin and hyperinsulinemic-hypoglycemic clamps, we found that sympathoadrenal CRRs to slow- but not rapid-onset hypoglycemia require hypothalamus-projecting catecholaminergic neurons, the majority of which originate in the ventrolateral medulla. As determined with Fos, these neurons are differentially activated by the two onset rates. We conclude that 1) catecholaminergic projections to the hypothalamus provide essential information for activating sympathoadrenal CRRs to slow- but not rapid-onset hypoglycemia, 2) hypoglycemia onset rates have a major impact on the hypothalamic mechanisms that enable sympathoadrenal CRRs, and 3) hypoglycemia-related sensory information activates hindbrain catecholaminergic neurons in a rate-dependent manner.

Hindbrain noradrenergic input to the hypothalamic PVN mediates the activation of oxytocinergic neurons induced by the satiety factor oleoylethanolamide.

Oleoylethanolamide (OEA) is a gut-derived endogenous lipid that stimulates vagal fibers to induce satiety. Our previous work has shown that peripherally administered OEA activates c-fos transcription in the nucleus of the solitary tract (NST) and in the paraventricular nucleus (PVN), where it enhances oxytocin (OXY) expression. The anorexigenic action of OEA is prevented by the intracerebroventricular administration of a selective OXY receptor antagonist, suggesting a necessary role of OXYergic mediation of OEA's effect. The NST is the source of direct noradrenergic afferent input to hypothalamic OXY neurons, and therefore, we hypothesized that the activation of this pathway might mediate OEA effects on PVN neurons. To test this hypothesis, we subjected rats to intra-PVN administration of the toxin saporin (DSAP) conjugated to an antibody against dopamine-ß-hydroxylase (DBH) to destroy hindbrain noradrenergic neurons. In these rats we evaluated the effects of OEA (10 mg/kg, ip) on feeding behavior, on c-Fos and OXY immunoreactivity in the PVN, and on OXY immunoreactivity in the posterior pituitary gland. We found that the DSAP lesion completely prevented OEA's effects on food intake, on Fos and OXY expression in the PVN, and on OXY immunoreactivity of the posterior pituitary gland; all effects were maintained in sham-operated rats. These results support the hypothesis that noradrenergic NST-PVN projections are involved in the activation of the hypothalamic OXY system, which mediates OEA's prosatiety action.

Ascending caudal medullary catecholamine pathways drive sickness-induced deficits in exploratory behavior: brain substrates for fatigue?

Immune challenges can lead to marked behavioral changes, including fatigue, reduced social interest, anorexia, and somnolence, but the precise neuronal mechanisms that underlie sickness behavior remain elusive. Part of the neurocircuitry influencing behavior associated with illness likely includes viscerosensory nuclei located in the caudal brainstem, based on findings that inactivation of the dorsal vagal complex (DVC) can prevent social withdrawal. These brainstem nuclei contribute multiple neuronal projections that target different components of autonomic and stress-related neurocircuitry. In particular, catecholaminergic neurons in the ventrolateral medulla (VLM) and DVC target the hypothalamus and drive neuroendocrine responses to immune challenge, but their particular role in sickness behavior is not known. To test whether this catecholamine pathway also mediates sickness behavior, we compared effects of DVC inactivation with targeted lesion of the catecholamine pathway on exploratory behavior, which provides an index of motivation and fatigue, and associated patterns of brain activation assessed by immunohistochemical detection of c-Fos protein. LPS treatment dramatically reduced exploratory behavior, and produced a pattern of increased c-Fos expression in brain regions associated with stress and autonomic adjustments paraventricular hypothalamus (PVN), bed nucleus of the stria terminalis (BST), central amygdala (CEA), whereas activation was reduced in regions involved in exploratory behavior (hippocampus, dorsal striatum, ventral tuberomammillary nucleus, and ventral tegmental area). Both DVC inactivation and catecholamine lesion prevented reductions in exploratory behavior and completely blocked the inhibitory LPS effects on c-Fos expression in the behavior-associated regions. In contrast, LPS-induced activation in the CEA and BST was inhibited by DVC inactivation but not by catecholamine lesion. The findings support the idea that parallel pathways from immune-sensory caudal brainstem sources target distinct populations of forebrain neurons that likely mediate different aspects of sickness. The caudal medullary catecholaminergic projections to the hypothalamus may significantly contribute to brain mechanisms that induce behavioral "fatigue" in the context of physiological stressors.

Noradrenergic inputs to the paraventricular hypothalamus contribute to hypothalamic-pituitary-adrenal axis and central Fos activation in rats after acute systemic endotoxin exposure.

Noradrenergic (NA) neurons within the nucleus of the solitary tract (NST) and caudal ventrolateral medulla (VLM) innervate the hypothalamic paraventricular nucleus (PVN) to initiate and modulate hypothalamic-pituitary-adrenal (HPA) axis responses to interoceptive stress. Systemic endotoxin (i.e. bacterial lipopolysaccharide, LPS) activates NA neurons within the NST and VLM that project to the PVN and other brain regions that receive interoceptive signals. The present study examined whether NA neurons with axonal inputs to the PVN are necessary for LPS to activate Fos expression within the PVN and other interoceptive-related brain regions, and to increase plasma corticosterone. Male Sprague-Dawley rats received bilateral stereotaxic microinjections of DSAP (saporin toxin conjugated to an antibody against dopamine-beta-hydroxylase, DbH) into the PVN to destroy NA inputs. Control rats were microinjected with vehicle into the PVN or received no PVN injections. Two weeks later, DSAP and control rats were injected i.p. with LPS (200 microg/kg BW) or saline vehicle, and perfused with fixative 2.5-3 h later. Brain tissue sections were processed to reveal nuclear Fos protein and cytoplasmic DbH immunolabeling. DSAP lesions depleted NA terminals in the PVN and bed nucleus of the stria terminalis, reduced the number of NA cell bodies in the NST and VLM, attenuated PVN Fos activation after LPS, and attenuated LPS-induced increases in plasma corticosterone. These findings support the view that NA projections from hindbrain to hypothalamus are necessary for a full HPA axis response to systemic immune challenge.

Specificity and generality of the involvement of catecholaminergic afferents in hypothalamic responses to immune insults.

Catecholamine-containing projections from the medulla have been implicated in the mediation of activational responses of the paraventricular nucleus of the hypothalamus (PVH) provoked by moderate doses of interleukin-1 (IL-1). To test the generality of this mechanism, rats bearing unilateral transections of aminergic projections were challenged with intravenous IL-1 (2 microg/kg), bacterial lipopolysaccharide (LPS; 0.1, 2.0, or 100 microg/kg), or saline and perfused 3 hours later; their brains were then prepared for quantitative analysis of Fos induction and relative levels of corticotropin-releasing factor (CRF) mRNA. LPS provoked a robust and dose-related increase in Fos expression within the PVH on the intact side of the brain at all doses tested; the response to IL-1 approximated that to the lowest LPS dose. On the lesioned side, Fos induction was significantly reduced at all dosage levels but was eliminated only at the lowest dosage. The percentage reduction was greatest (75%) in IL-1-challenged rats and was progressively less in animals treated with increasing LPS doses (67, 59, and 46%, respectively). Specificity of aminergic involvement was tested by using intra-PVH administration of the axonally transported catecholamine immunotoxin, antiDBH-saporin. This treatment abolished IL-1-induced elevations of Fos-ir and CRF mRNA in the PVH but left intact comparable responses to restraint stress. These data support a specific involvement of ascending catecholaminergic projections in mediating PVH responses to IL-1 and LPS. Residual Fos induction seen in lesioned animals in response to higher doses of LPS provides a basis for probing additional circuits that may be recruited in a hierarchical manner in response to more strenuous or complex immune insults.

Interferon-alpha reduces the density of monoaminergic axons in the rat brain.

Interferon-alpha commonly induces depressive symptoms in clinical populations; however, the mechanism by which this occurs is unclear. Recent studies suggest that the degeneration of axons containing serotonin and noradrenaline is involved in the pathophysiology of depression. The present immunohistochemical study shows that the density of serotonergic axons decreased in the ventral medial prefrontal cortex and amygdala in the interferon-alpha-treated animals. Additionally, interferon-alpha induced decreases in the density of noradrenergic axons in the dorsal medial prefrontal cortex, ventral medial prefrontal cortex, and dentate gyrus. These results support the hypothesis that long-term administration of interferon-alpha causes the degeneration of monoaminergic axons in specific brain regions, which might be associated with depressive symptoms occurring in interferon-alpha-treated patients.

Immunolesions of glucoresponsive projections to the arcuate nucleus alter glucoprivic-induced alterations in food intake, luteinizing hormone secretion, and GALP mRNA, but not sex behavior in adult male rats.

Metabolic signals such as insulin, leptin and glucose are known to alter hypothalamic function. Although insulin and leptin are known to directly alter hypothalamic areas that regulate reproduction, the mechanisms by which glucose alters reproductive function are not as clear. Catecholaminergic neurons in the A1/C1 region in the hindbrain are glucose-responsive and project to the arcuate nucleus. To determine if this pathway is involved in the regulation of sex behavior and luteinizing hormone (LH) secretion, this catecholaminergic pathway was lesioned with injections of saporin conjugated with anti-dopamine-beta-hydroxylase (DSAP) or unconjugated saporin (SAP) in adult male rats. Rats were given glucoprivic challenges and feeding and sex behavior was observed. As was expected, the DSAP-treated rats showed decreased feeding during glucoprivation (250 mg/kg 2-deoxy-D-glucose, 2DG) compared to SAP controls. Glucoprivation caused a significant reduction in sex behavior in both SAP and DSAP animals equally, compared to saline treatments (p < 0.05). At the end of the experiment, animals were given a final challenge with 2DG or saline, euthanized by decapitation and trunk blood was assayed for plasma LH levels. In situ hybridization analysis revealed that 2DG treatment caused a significant reduction in GALP mRNA in SAP controls compared to saline treatment. This reduction in GALP mRNA was prevented with DSAP treatment. In SAP animals, 2DG elicited a significant decrease in plasma LH levels (p < 0.05); this reduction in plasma LH was absent in the DSAP-treated male rats. These data indicate that the A1/C1 efferents to the ventromedial hypothalamus are involved in the glucostatic regulation of GALP mRNA, feeding behavior and LH secretion, but not sex behavior in the adult male rat.

Retrograde axonal transport of dopamine beta hydroxylase antibodies by neurons in the trigeminal ganglion.

In this study we describe a population of neurons in the adult rat trigeminal ganglion (TG) that express dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH), and transport anti-DBH from their terminals. We have used NGF and NT3 labeled with biotin and anti-p75NTR labeled with FITC to examine the transport of neurotrophins and their receptors by these cells. In both the superior cervical ganglion (SCG) and the TG all neurons that transported anti-DBH transported NGF. While 100% of the DBH positive neurons in the TG also transported NT3, approximately 25% of these neurons in the SCG failed to transport NT3. In the SCG virtually all the neurons transported anti-p75NTR with the neurotrophins while in the TG more than 25% of these neurons failed to transport anti-p75NTR with the neurotrophins. These findings suggest that DBH positive neurons in the TG depend upon target-derived NGF and NT3 for their noradrenergic phenotype.

A structural basis for CD8+ T cell-dependent recognition of non-homologous peptide ligands: implications for molecular mimicry in autoreactivity.

Molecular mimicry of self-epitopes by viral antigens is one possible pathogenic mechanism underlying induction of autoimmunity. A self-epitope, mDBM, derived from mouse dopamine beta-mono-oxygenase (KALYDYAPI) sharing 44% sequence identity with the lymphocytic choriomeningitis virus-derived immunodominant epitope gp33 (KAVYNFATC/M), has previously been identified as a cross-reactive self-ligand, presentation of which results in autoimmunity. A rat peptide homologue, rDBM (KALYNYAPI, 56% identity to gp33), which displayed similar properties to mDBM, has also been identified. We herein report the crystal structure of H-2Db.rDBM and a comparison with the crystal structures of the cross-reactive H-2Db.gp33 and non-cross-reactive H-2Db.gp33 (V3L) escape variant (KALYNFATM, 88% identity to gp33). Despite the large sequence disparity, rDBM and gp33 peptides are presented in nearly identical manners by H-2Db, with a striking juxtaposition of the central sections of both peptides from residues p3 to p7. The structural similarity provides H-2Db in complex with either a virus-derived or a dopamine beta-mono-oxygenase-derived peptide with a shared antigenic identity that conserves the positioning of the heavy chain and peptide residues that interact with the T cell receptor (TCR). This stands in contrast to the structure of H-2Db.gp33 (V3L), in which a single conserved mutation, also present in rDBM, induces large movements of both the peptide backbone and the side chains that interact with the TCR. The TCR-interacting surfaces of the H-2Db.rDBM and H-2Db.gp33 major histocompatibility complexes are very similar with regard to shape, topology, and charge distribution, providing a structural basis for CD8 T cell activation by molecular mimicry and potential subsequent development of autoreactivity.

Increased lymphocyte dopamine beta-hydroxylase immunoreactivity in Alzheimer's disease: compensatory response to cholinergic deficit?

There is growing interest in the characterization of peripheral blood lymphocytes (PBL) as a biological tool with which to investigate changes in the neurotransmitter-receptor system in neurodegenerative disorders. Here we show a slight decrease in acetylcholinesterase (AChE) and a significant increase in dopamine beta-hydroxylase (DBH) immunoreactivity in the PBL of patients with probable Alzheimer's disease (AD). Therapy with AChE inhibitors completely reversed the increase in DBH immunoreactivity. We hypothesize that the increase in DBH immunoreactivity may represent a compensatory response to cholinergic impairment. Our findings suggest that neurochemical interactions between the noradrenergic and cholinergic systems may be measured at a peripheral level in AD.

Comparison of nerve terminal events in vivo effecting retrograde transport of vesicles containing neurotrophins or synaptic vesicle components.

Although vesicular retrograde transport of neurotrophins in vivo is well established, relatively little is known about the mechanisms that underlie vesicle endocytosis and formation before transport. We demonstrate that in vivo not all retrograde transport vesicles are alike, nor are they all formed using identical mechanisms. As characterized by density, there are at least two populations of vesicles present in the synaptic terminal that are retrogradely transported along the axon: those containing neurotrophins (NTs) and those resulting from synaptic vesicle recycling. Vesicles containing nerve growth factor (NGF), NT-3, or NT-4 had similar densities with peak values at about 1.05 g/ml. Synaptic-derived vesicles, labeled with anti-dopamine beta-hydroxylase (DBH), had densities with peak values at about 1.16 g/ml. We assayed the effects of pharmacologic agents in vivo on retrograde transport from the anterior eye chamber to the superior cervical ganglion. Inhibitors of phosphatidylinositol-3-OH (PI-3) kinase and actin function blocked transport of both anti-DBH and NGF, demonstrating an essential role for these molecules in retrograde transport of both vesicle types. Dynamin, a key element in synaptic vesicle recycling, was axonally transported in retrograde and anterograde directions, and compounds able to interfere with dynamin function had a differential effect on retrograde transport of NTs and anti-DBH. Okadaic acid significantly decreased retrograde axonal transport of anti-DBH and increased NGF retrograde transport. We conclude that there are both different and common proteins involved in endocytosis and targeting of retrograde transport of these two populations of vesicles.

Immunotoxic destruction of distinct catecholaminergic neuron populations disrupts the reproductive response to glucoprivation in female rats.

We tested the hypothesis that hindbrain catecholamine (norepinephrine or epinephrine) neurons, in addition to their essential role in glucoprivic feeding, are responsible for suppressing estrous cycles during chronic glucoprivation. Normally cycling female rats were given bilateral injections of the retrogradely transported ribosomal toxin, saporin, conjugated to monoclonal dopamine beta-hydroxylase antibody (DSAP) into the paraventricular nucleus (PVN) of the hypothalamus to selectively destroy norepinephrine and epinephrine neurons projecting to the PVN. Controls were injected with unconjugated saporin. After recovery, we assessed the lesion effects on estrous cyclicity under basal conditions and found that DSAP did not alter estrous cycle length. Subsequently, we examined effects of chronic 2-deoxy-d-glucose-induced glucoprivation on cycle length. After two normal 4- to 5-d cycles, rats were injected with 2-deoxy-d-glucose (200 mg/kg every 6 h for 72 h) beginning 24 h after detection of estrus. Chronic glucoprivation increased cycle length in seven of eight unconjugated saporin rats but in only one of eight DSAP rats. Immunohistochemical results confirmed loss of dopamine beta-hydroxylase immunoreactivity in PVN. Thus, hindbrain catecholamine neurons with projections to the PVN are required for inhibition of reproductive function during chronic glucose deficit but are not required for normal estrous cyclicity when metabolic fuels are in abundance.

Weak agonist self-peptides promote selection and tuning of virus-specific T cells.

Recent progress has begun to define the interactions and signaling pathways that are triggered during positive selection. To identify and further examine self-peptides that can mediate positive selection, we searched a protein-database to find peptides that have minimal homology with the viral peptide (p33) that activates a defined P14 transgenic TCR. We identified four peptides that could bind the restriction element H-2D(b) and induce proliferation of P14 transgenic splenocytes at high concentration. Two of the four peptides (DBM and RPP) were able to positively select the virus-specific TCR in fetal thymic organ culture but were unable to induce clonal deletion. Reverse-phase HPLC and mass spectrometry demonstrated that these peptides were presented by H-2D(b) molecules on thymic epithelial cell lines. We also examined whether the selecting ligands altered T cell responsiveness in vitro. DBM-selected T cells lost their ability to respond to the positively selecting ligand DBM, whereas RPP-selected T cells only retained their ability to respond to high concentrations of RPP. These results demonstrate that self-peptides that mediate positive selection can differentially "tune" the activation threshold of T cells and alter the functional repertoire of T cells.

Immunolesion of norepinephrine and epinephrine afferents to medial hypothalamus alters basal and 2-deoxy-D-glucose-induced neuropeptide Y and agouti gene-related protein messenger ribonucleic acid expression in the arcuate nucleus.

Neuropeptide Y (NPY) and agouti gene-related protein (AGRP) are orexigenic peptides of special importance for control of food intake. In situ hybridization studies have shown that NPY and AGRP mRNAs are increased in the arcuate nucleus of the hypothalamus (ARC) by glucoprivation. Other work has shown that glucoprivation stimulates food intake by activation of hindbrain glucoreceptor cells and requires the participation of rostrally projecting norepinephrine (NE) or epinephrine (E) neurons. Here we determine the role of hindbrain catecholamine afferents in glucoprivation-induced increase in ARC NPY and AGRP gene expression. The selective NE/E immunotoxin saporin-conjugated antidopamine-beta-hydroxylase (anti-dbetah) was microinjected into the medial hypothalamus and expression of AGRP and NPY mRNA was analyzed subsequently in the ARC under basal and glucoprivic conditions using (33)P-labeled in situ hybridization. Saporin-conjugated anti-dbetah virtually eliminated dbetah-immunoreactive terminals in the ARC without causing nonspecific damage. These lesions significantly increased basal but eliminated 2-deoxy-D-glucose-induced increases in AGRP and NPY mRNA expression. Results indicate that hindbrain catecholaminergic neurons contribute to basal NPY and AGRP gene expression and mediate the responsiveness of NPY and AGRP neurons to glucose deficit. Our results also suggest that catecholamine neurons couple potent orexigenic neural circuitry within the hypothalamus with hindbrain glucose sensors that monitor brain glucose supply.

Intrinsic choroidal neurons in the duck eye receive sympathetic input: anatomical evidence for adrenergic modulation of nitrergic functions in the choroid.

Intrinsic choroidal neurons (ICN) in the duck eye form an intramural ganglionic plexus that may subserve complex integrative functions. A key feature of such ganglia is an innervation by sympathetic postganglionic neurons. The present study was thus aimed at determining the sympathetic postganglionic innervation of ICN. Choroids were processed for double immunofluorescence labelling with the following markers: tyrosine-hydroxylase (TH)/nitric oxide synthase (nNOS), TH/galanin (GAL), dopamine-beta-hydroxylase (DBH)/vasoactive intestinal polypeptide (VIP), TH/DBH and DBH/alpha-smooth-muscle actin (alphaSMA), and for triple immunofluorescence labelling with VIP/DBH/TH. Epifluorescence and confocal laser scanning microscopy were used for evaluation. Immunoperoxidase staining for TH or DBH in combination with NADPH-diaphorase histochemistry was applied for electron microscopy. ICN spread over the entire choroid but were concentrated in an equatorial zone passing obliquely from naso-cranial to temporocaudal. More than 80% of nNOS-positive ICN showed close appositions of TH/DBH-immunoreactive varicose nerve fibres at the light-microscopic level, as could be confirmed by confocal laser scanning microscopy. Ultrastructurally, these appositions could be defined as both synapses or close contacts without synaptic specialisation. Vascular and non-vascular smooth muscle fibres also received TH/DBH-immunopositive innervation. Our findings suggest that most ICN receive a sympathetic input that might modulate their nitrergic effects upon vascular and non-vascular smooth muscle fibres in the choroid and that they may have more complex functions than merely being a simple parasympathetic relay.