Evaluation of an Epstein-Barr virus (EBV) immunoglobulin M enzyme-linked immunosorbent assay using a synthetic convergent peptide library, or mixotope, for diagnosis of primary EBV infection.

An immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed by using a 24-amino-acid peptide of the 18-kDa Epstein-Barr virus (EBV) viral capsid antigen (VCAp18). IgM detection was increased by 23% by using this antigen. Detection of IgM antibodies to the EBV proteins in the new ELISA was 100% specific and 95% sensitive.

Convergent peptide libraries, or mixotopes, to elicit or to identify specific immune responses.

Many recent studies have demonstrated the flexibility of epitope recognition by the immune system. This can be explored using a particular type of combinatorial peptide library, termed as 'convergent', consisting essentially of closely related molecular species; from this a fuzzy set can be constructed, which comprises several variants of a peptide that would act in synchrony to represent a model antigen and its recognition by the immune system.

Detection of human antibodies using "convergent" combinatorial peptide libraries or "mixotopes" designed from a nonvariable antigen: application to the EBV viral capsid antigen p18.

We have previously described the use of synthetic combinatorial "convergent" libraries, or "mixotopes" as immunogens or as antigens to represent naturally hypervariable sequences. The success of this approach suggests that such a mixture of closely related peptides could, at least in part, conveniently represent a nonvariable epitope during its multiple interactions with an antibody population. To address this possibility, we have designed from a non-variable immunodominant peptide of the EBV-viral capsid antigen of 18 kD (VCAp18) a series of three mixotopes containing from 65,000 to 16 million combinatorial sequences. The reactivity of VCAp18 and its three derived mixotopes was examined in ELISA towards a collection of 74 human sera from documented EBV-negative or EBV-positive donors, and analyzed in terms of sensitivity and specificity. Following the observation that the two least degenerated mixotopes could improve the sensitivity of detection of some sera of low reactivity for VCAp18, we decided to combine each mixotope with the VCAp18 peptide. In the case of the least degenerated mixotope in combination with VCAp18, sensitivity and specificity for immunoenzymatic EBV-serodiagnosis, were enhanced to 100%. Our results suggest that synthetic "convergent" combinatorial peptide libraries or "mixotopes," designed from nonvariable antigens, could be useful adjuncts to an antigenic single-sequence peptide in immunoenzymatic serodiagnosis.

Neuropeptide tyrosine in the brain of the African lungfish, Protopterus annectens: immunohistochemical localization and biochemical characterization.

Lungfishes, which share similarities with both fishes and amphibians, represent an interesting group in which to investigate the evolutionary transition from fishes to tetrapods. In the present study, we have investigated the localization and biochemical characteristics of neuropeptide Y (NPY)-immunoreactive material in the central nervous system of the African lungfish, Protopterus annectens. NPY-immunoreactive cell bodies were found in various regions of the brain, most notably in the telencephalon (septal area, ventral striatum, and nucleus accumbens), in the diencephalon (preoptic nucleus, periventricular region of the hypothalamus, and ventral thalamus), and in the tegmentum of the mesencephalon. A strong immunoreaction was also detected in cell bodies of the nervus terminalis. Immunoreactive nerve fibers were particularly abundant in the ventral striatum, the nucleus accumbens, the diagonal band of Broca, the hypothalamus, and the mesencephalic tegmentum. Positive fibers were also seen in the median eminence and in the neural lobe of the pituitary. The NPY-immunoreactive material localized in the brain and pituitary was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological quantitation. The displacement curves obtained with synthetic porcine and frog NPY and serial dilutions of brain and pituitary extracts were parallel. Reversed-phase HPLC analysis of telencephalon, diencephalon, and pituitary extracts resolved a major NPY-immunoreactive peak that coeluted with frog NPY. The similarity between the distribution of NPY-containing neurons and the biochemical characteristics of the immunoreactive peptide in the brain of lungfish and frog strongly favors a close phylogenetic relationship between dipnoans and amphibians.

Effect of chronic treatment with the antidepressant tianeptine on the hypothalamo-pituitary-adrenal axis.

The effects of acute and chronic administration of tianeptine, a novel antidepressant agent, on the hypothalamo-pituitary-adrenal axis were studied in the adult male rat. A single injection of tianeptine did not alter the activity of the hypothalamo-pituitary-adrenal axis. In contrast, chronic administration of tianeptine (10 mg/kg twice a day for 15 days) induced a significant decrease in the concentration of corticotropin-releasing factor (CRF) in the hypothalamus and adrenocorticotropin (ACTH) in the anterior lobe of the pituitary. Chronic tianeptine treatment did not modify CRF levels in the cerebral cortex and hippocampus, and did not alter alpha-melanocyte-stimulating hormone and beta-endorphin levels in the neurointermediate lobe of the pituitary. Using the in situ hybridization technique, we observed that chronic administration of tianeptine did not modify CRF mRNA levels in the paraventricular nucleus of the hypothalamus. The effect of chronic tianeptine treatment on the neuroendocrine response to stress was also investigated. Tube restraint stress for 30 min induced a significant depletion of hypothalamic CRF and a substantial increase of plasma ACTH and corticosterone. Tianeptine abolished the stress-induced reduction of hypothalamic CRF concentration and markedly reduced the stress-induced increase in plasma ACTH and corticosterone levels. Taken together, these results suggest that tianeptine acts primarily at the level of the hypothalamus: (1) in unstressed rats, tianeptine reduces hypothalamic CRF and pituitary ACTH contents; (2) in stressed animals, tianeptine attenuates the activation of the hypothalamo-pituitary-adrenal axis.

Neuropeptide Y inhibits alpha-MSH release from rat hypothalamic slices through a pertussis toxin-sensitive G protein.

The arcuate nucleus of the hypothalamus contains various types of peptidergic neurons. In particular, two distinct populations of neurosecretory neurons containing neuropeptide Y (NPY)- and alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactivity have been identified in the arcuate nucleus. Double-labeling immunocytochemical data have recently shown that NPY-containing fibers make synaptic contacts with proopiomelanocortin (POMC) immunoreactive neurons. We have thus investigated the possible effect of NPY on the release of alpha-MSH from rat hypothalamic slices in vitro, using the perifusion technique. NPY significantly inhibited KCl-stimulated alpha-MSH release in a dose-dependent manner. The inhibitory effect of NPY was mimicked by the Y2 agonist, NPY-(13-36), while the Y1 agonist, [Leu31,Pro34]NPY, was devoid of effect. Pretreatment of hypothalamic slices with pertussis toxin (PTX) blocked the inhibitory effect of NPY, suggesting that the action of NPY on POMC neurons is mediated through a PTX-sensitive G protein. These results support the notion that NPY may play a physiological role in the regulation of alpha-MSH release from hypothalamic neurons.

Alpha-melanocyte-stimulating hormone (alpha-MSH) in the brain of the African lungfish, Protopterus annectens: immunohistochemical localization and biochemical characterization.

The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) containing neurons and the molecular forms of alpha-MSH-related peptides exhibit substantial differences in the brains of fish and amphibians. Lungfishes, which share similarities with both fishes and tetrapods, represent a valuable group in which to investigate the neuroanatomical and neurochemical facets of evolution. In the present study, we have localized and characterized alpha-MSH-immunoreactive peptides in the central nervous system of the African lungfish Protopterus annectens. Perikarya exhibiting alpha-MSH-like immunoreactivity were observed in two distinct regions of the hypothalamus: the rostral part of the preoptic nucleus and the caudal part of the hypothalamus. In the caudal hypothalamus most alpha-MSH-immunopositive perikarya were located in both the subependymal and deepest layers of the ventral periventricular region. Scattered alpha-MSH-immunopositive cells were occasionally detected in the dorsal side of the caudal hypothalamus. The alpha-MSH-immunoreactive material localized in the brain was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. The displacement curves obtained with synthetic alpha-MSH and serial dilutions of brain and pituitary extracts were parallel. HPLC analysis of lungfish hypothalamic extracts showed that the major immunoreactive peak coeluted with synthetic desacetyl alpha-MSH and its sulfoxide derivative. An additional peak coeluted with synthetic sulfoxide alpha-MSH. In contrast, in the pituitary, the predominant form of alpha-MSH-like material coeluted with the N,O-diacetyl alpha-MSH standard. These results provide the first evidence for the presence of alpha-MSH-related peptides in the brain of a lungfish. The distribution of alpha-MSH neuronal systems in the lungfish is very similar to that reported in amphibians, supporting the existence of phylogenetic convergences between these two vertebrate groups.

Omega-conotoxin- and nifedipine-insensitive voltage-operated calcium channels mediate K(+)-induced release of pro-thyrotropin-releasing hormone-connecting peptides Ps4 and Ps5 from perifused rat hypothalamic slices.

The rat thyrotropin-releasing hormone (TRH) precursor (prepro-TRH) contains five copies of the TRH progenitor sequence linked together by intervening sequences. Recently, we have shown that the connecting peptides prepro-TRH-(160-169) (Ps4) and prepro-TRH-(178-199) (Ps5) are released from rat hypothalamic neurones in response to elevated potassium concentrations, in a calcium-dependent manner. In the present study, the role of voltage-operated calcium channels in potassium-induced release of Ps4 and Ps5 was investigated, using a perifusion system for rat hypothalamic slices. The release of Ps4 and Ps5 stimulated by potassium (70 mM) was blocked by the inorganic ions Co2+ (2.6 mM) and Ni2+ (5 mM). In contrast, the stimulatory effect of KCl was insensitive to Cd2+ (100 microM). The dihydropyridine antagonist nifedipine (10 microM) had no effect on K(+)-evoked release of Ps4 and Ps5. Furthermore, the response to KCl was not affected by nifedipine (10 microM) in combination with diltiazem (1 microM), a benzothiazepine which increases the affinity of dihydropyridine antagonists for their receptor. The dihydropyridine agonist BAY K 8644, at concentrations as high as 1 mM, did not stimulate the basal secretion of Ps4 and Ps5. In addition, BAY K 8644 had no potentiating effect on K(+)-induced release of Ps4 and Ps5. The marine cone snail toxin omega-conotoxin, a blocker of both L- and N-type calcium channels had no effect on the release of Ps4 and Ps5 stimulated by potassium. Similarly, the omega-conopeptide SNX-111, a selective blocker of N-type calcium channels, did not inhibit the stimulatory effect of potassium. The release of Ps4 and Ps5 evoked by high K+ was insensitive to the non-selective calcium channel blocker verapamil (20 microM). Amiloride (1 microM), a putative blocker of T-type calcium channels, did not affect KCl-induced secretion of the two connecting peptides. Taken together, these results indicate that two connecting peptides derived from the pro-TRH, Ps4 and Ps5, are released by K(+)-induced depolarization through activation of voltage-sensitive calcium channels. The calcium channels appear to have a pharmacological profile different from that of L- and N-type channels. Although, their insensitivity to low Cd2+ concentrations and sensitivity to Ni2+ ions would support the involvement of T-type calcium channels, the lack of effect of amiloride suggests that they belong to a yet undefined class of calcium channels.

Central-type benzodiazepines inhibit release of alpha-melanocyte-stimulating hormone from the rat hypothalamus.

In a previous work, we have shown that GABA inhibits the release of alpha-melanocyte-stimulating hormone (alpha-melanotropin) from hypothalamic neurons through activation of GABAA receptors [Delbende et al. (1989) Brain Res. 497, 86-93]. Since GABA-gated channel activity can be allosterically modulated by a variety of compounds including benzodiazepines, we have investigated the effect of benzodiazepines in the control of alpha-melanotropin release by the rat basal hypothalamus. This study was conducted in vitro using perifused rat hypothalamic slices and the amount of alpha-melanotropin release was monitored with a sensitive and highly specific radioimmunoassay. Infusion of clonazepam (50 microM), a selective agonist for central-type benzodiazepine binding sites, induced an inhibition of KCl (50 mM)-evoked alpha-melanotropin release. The inhibitory effect of clonazepam was rapid and reversible. Administration of Ro 15-1788 (100 microM), a specific antagonist for central-type benzodiazepine receptors or SR 95531, a GABAA receptor antagonist, completely reversed the inhibitory effect of clonazepam. In addition, Ro 15-1788 and SR 95531 both enhanced the amplitude of the response observed during prolonged KCl infusion on alpha-melanotropin neurons, suggesting the existence of a tonic inhibitory effect of endogenous GABA and/or benzodiazepines in the release of alpha-melanotropin by hypothalamic neurons. To investigate further the effect of benzodiazepines in the regulation of alpha-melanotropin neurons, rats were treated in vivo with clonazepam (5 mg/kg) or the non-selective benzodiazepine receptor agonist diazepam (3 mg/kg). Both compounds caused a significant increase in the content of alpha-melanotropin and beta-endorphin in the rat hypothalamus within 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

gamma-Aminobutyric acid inhibits the release of alpha-melanocyte-stimulating hormone from rat hypothalamic slices.

The effect of gamma-aminobutyric acid (GABA) on release of alpha-melanocyte-stimulating hormone (alpha-MSH) from hypothalamic neurons was investigated in vitro using the perifusion technique. Rat hypothalamic slices were continuously superfused with Krebs-Ringer medium and the release of alpha-MSH in the effluent perifusate was monitored by means of a sensitive and specific radioimmunoassay method. Infusion of 50 mM K+ for 15 min induced a transient increase of alpha-MSH release (5- to 8-fold above the spontaneous level). Infusion of the same dose of K+ for 75 min caused a brief discharge of alpha-MSH during the first 30 min followed by sustained release of the neuropeptide. The effect of GABA was investigated 27 min after the onset of KCl infusion. Application of GABA (5 x 10(-5) M) resulted in a significant and reversible inhibition of K+-induced alpha-MSH release. The GABAA agonist, muscimol (10(-4) M), produced a prolonged inhibition of K+-evoked alpha-MSH release, while the GABAB agonist, baclofen (10(-4) M), was devoid of effect on hypothalamic alpha-MSH release. Bicuculline (10(-4) M), a specific GABAA antagonist, had no effect when added alone to the medium but totally reversed the inhibitory effect of GABA on K+-induced alpha-MSH release. Taken together, these data suggest that exogenous GABA exerts an inhibitory control on alpha-MSH neurons. Our data also show that the effect of GABA on alpha-MSH release by hypothalamic neurons is mediated through GABAA-type receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of voltage-operated calcium channels in alpha-melanocyte-stimulating hormone (alpha-MSH) release from perifused rat hypothalamic slices.

The contribution of voltage-operated calcium (VOC) channels in the mechanism of release of alpha-melanocyte-stimulating hormone (alpha-MSH) from hypothalamic neurons was investigated using perifused rat hypothalamic slices. The stimulatory effect of potassium (50 mM) on alpha-MSH release was completely blocked by cadmium (1 mM) a calcium competitor which indifferently blocks T-, L-and N-type VOC channels. To determine the nature of calcium conductances involved in K+-evoked alpha-MSH release, we have investigated the effect of a VOC channel agonist and 3 antagonists on the secretion of the neuropeptide. Administration of synthetic omega-conotoxin fraction GVIA (1 microM), a peptide toxin which blocks both N- and L-type VOC channels, reduced by 33% K+-induced alpha-MSH release. In contrast, the 1,4-dihydropyridine (DHP) antagonist nifedipine, at concentrations up to 100 microM, did not affect the response of hypothalamic alpha-MSH neurons to depolarizing concentrations of KCl. In addition, the secretion of alpha-MSH induced by high K+ concentrations was not reduced by nifedipine (10 microM) in the presence of diltiazem (1 microM), a benzothiazepine derivative which increases the affinity of the DHP antagonist for L-type VOC channels. The DHP agonist BAY K 8644 (0.1-10 microM) did not modify the early phase of the response of alpha-MSH neurons to K+-induced depolarization. In contrast BAY K 8644 (1 or 10 microM) significantly prolonged the duration of K+-induced alpha-MSH release. This sustained release of alpha-MSH induced by BAY K 8644 (10 microM) was totally suppressed by nifedipine (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of alpha-MSH-related peptides released from rat hypothalamic neurons in vitro.

Reverse-phase high-performance liquid chromatography analysis, coupled with a sensitive radioimmunoassay for alpha-melanocyte-stimulating hormone (alpha-MSH), was used to characterize the alpha-MSH-related peptides stored in the rat hypothalamus or released from perifused hypothalamic slices. Four peaks of alpha-MSH-like immunoreactivity (alpha-MSH-LI) co-eluting with synthetic des-N alpha-acetyl alpha-MSH, alpha-MSH and their respective sulfoxide derivatives were resolved and quantified. In hypothalamic extract, deacetyl alpha-MSH which was the predominant peptide represented 94.4% of total alpha-MSH-LI content, while the relative amount of alpha-MSH was only 5.6%. Analysis of alpha-MSH-related peptides contained in effluent perifusates showed that deacetyl alpha-MSH and its oxidized form were the major peptides released from neurons in basal conditions or under KCl-induced depolarization (50 mM KCl for 75 min). However, the proportion of acetylated peptide was 3-4 times higher in the perifusion medium than in hypothalamic extracts. Our data indicate that acetylation of des-N alpha-acetyl alpha-MSH may occur during the process of exocytosis. Since acetylation of alpha-MSH markedly increases the behavioural potency of the peptide, these results suggest that regulation of the acetyltransferase activity could be a key mechanism to modulate the bioactivity of alpha-MSH-related peptides in the brain.

Immunocytochemical Localization and Biochemical Characterization of alpha-Melanocyte-Stimulating Hormon in the Brain of the Rainbow Trout, Salmo gairdneri.

Abstract The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH)-like immunoreactivity in the central nervous system of the rainbow trout Salmo gairdneri was investigated by indirect immunofluorescence and peroxidase-antiperoxidase techniques, using a highly specific antiserum generated in rabbits against synthetic alpha-MSH. Immunoreactive perikarya were exclusively observed in the basal hypothalamus within the pars anterioris of the nucleus lateralis tuberis. In this region, a moderate number of small stained cell bodies were observed surrounding the dorsal wall of the anterior infundibular recess. These immunoreactive cells were organized in rostro-caudal rows extending over the whole portion of the nucleus. Positive fibres originating from these perikarya were visualized in the dorsal posterior lobe and the ventral hypothalamus. A dense tract of immunoreactive fibres projected ventrally through the pituitary stalk and terminated in the neurohypophysis. The concentrations of alpha-MSH in different regions of the brain were measured by means of a sensitive and specific radioimmunoassay. The dilution curves obtained with synthetic alpha-MSH and serial dilutions of diencephalon, mesencephalon, medulla oblongata, telencephalon or pituitary extracts were strictly parallel. The highest concentration of alpha-MSH in brain was found in the diencephalon (1.31 +/- 0.07 ng/mg protein). In contrast alpha-MSH was not detectable in cerebellar extracts. Reverse-phase high-performance liquid chromatography and radioimmunoassay were used to characterize alpha-MSH-like peptides in the trout brain and pituitary. Two major forms of immunoreactive alpha-MSH were resolved by high performance liquid chromatography in hypothalamic extracts; these peptides exhibited the same retention times as des-Na-acetyl alpha-MSH and its sulfoxide derivative, respectively. Additional peaks of alpha-MSH immunoreactive material were detected in pituitary extract. These latter peptides coeluted with authentic alpha-MSH, diacetyl alpha-MSH and their sulfoxide forms. These results provide the first evidence for the presence of alpha-MSH in the brain of a teleostean fish. Our data indicate that, in the brain, the immunoreactivity corresponds to the non-acetylated form of alpha-MSH, while three different types of alpha-MSH-like molecules (namely deacetylated, monoacetylated, and diacetylated forms) coexist in the pituitary. It thus appears that, in salmonoid fish, mono- or diacetylation of the N-terminal serine residue of aL-MSH only occurs at the pituitary level.

Regional distribution of vasoactive intestinal peptide in brains from normal and parkinsonian subjects.

The distribution of vasoactive intestinal peptide (VIP) in the post-mortem human brain was determined by radioimmunoassay using a highly specific antiserum. The detection limit of the assay was 4 fmol/tube. The highest concentrations of VIP were found in the cerebral cortex, amygdala, hypothalamus and hippocampus. The lowest levels of peptide were detected in basal ganglia including caudate nucleus, external pallidum, putamen and substantia nigra. All dilution curves of acetic acid extracts from different brain areas were strictly parallel to the standard curve. Sephadex G-50 gel filtration of frontal cortex extract showed that VIP-like immunoreactivity (VIP-LI) eluted as a major peak comigrating with synthetic hVIP. Detailed mapping of VIP in the human cerebral cortex showed the existence of a rostro-caudal gradient of VIP-LI concentrations: the frontal cortex exhibited the highest VIP levels, the parietal and temporal cortex contained medium values and the occipital cortex contained the lowest VIP levels. The concentrations of VIP-LI were compared in various regions of the human brain from normal and parkinsonian subjects. No significant changes in VIP-LI levels occurred in the brains of patients dying with Parkinson's disease. No difference in VIP levels could be found either when the parkinsonian group was subdivided into nondemented and demented patients. These data indicate that VIP-containing neurons are not affected in parkinsonian patients. Our results also suggest that VIP neuronal systems are not involved in the course of dementing process in Parkinson's disease.

Hypothalamic alpha-melanocyte-stimulating hormone (alpha-MSH) is not under dopaminergic control.

A possible dopaminergic regulation of hypothalamic proopiomelanocortin (POMC)-containing neurons has been investigated in rats by means of in vivo and in vitro approaches. Acute or 3-weeks chronic in vivo treatments with the dopaminergic agonists apomorphine (1 mg/kg: s.c.) and 2-Br-alpha-ergocriptine (2.5 mg/kg; s.c.) or the dopaminergic antagonist haloperidol (0.15-3 mg/kg; i.p.) had no significant effect on the concentration of alpha-melanocyte-stimulating hormone (alpha-MSH) in two hypothalamic regions: arcuate nucleus (AN) and dorsolateral area (DLH). In the same way, chronic administration of the dopaminergic agonists or antagonist did not induce any change in hypothalamic contents of beta-endorphin, another peptide derived from POMC. Reverse-phase high-performance liquid chromatographic analysis revealed that acetic acid extracts of AN and DLH both contained two major forms of alpha-MSH-like peptides: deacetylated alpha-MSH and authentic alpha-MSH. The ratio between these two forms was not altered after acute haloperidol treatment (3 mg/kg, i.p.). The possible effect of dopamine on the release of hypothalamic alpha-MSH was studied in vitro using perifused rat hypothalamic slices. Infusion of dopamine (10(-7)-10(-5)M) or its antagonist haloperidol (10(-5)M) had no effect on spontaneous alpha-MSH release from hypothalamic tissue. In addition, none of these drugs had any effect on potassium (50 mM)-induced alpha-MSH release. It is concluded that dopaminergic neurons are not involved in the regulation of synthesis, post-translational processing (acetylation) or release of hypothalamic alpha-MSH.