In vitro antibody secretion by peripheral blood mononuclear cells as an expression of the immune response to Brucella spp. in humans.

We developed an assay to detect antibodies spontaneously secreted in vitro by peripheral blood mononuclear cells (PBMC) against Brucella spp. High levels of anti-Brucella immunoglobulin G (IgG) and/or IgM and/or IgA antibodies were detected in the cell supernatant solution of PBMC cultures for 12 patients suffering from acute or focalized brucellosis and for 5 patients recently vaccinated against brucellosis. This spontaneous in vitro antibody production disappeared 5 to 20 months after onset of clinical signs and 20 to 27 days after vaccination. The transient character of this anti-Brucella antibody production by PBMC is consistent with a temporary in vivo stimulation of the immune system by Brucella antigens. Detection of this secretion could improve the diagnosis of evolutive brucellosis.     

Adult Celiac Disease Associated with HLA-DRw3 and -DRw7

Celiac disease (CD), which occurs in Europe with varying frequency, is known to be associated with HLA-B8 and even more strongly with HLA-Dw3 because of the high linkage disequilibrium between these two antigens. After typing for HLA-A, ∼B and -DR antigens in adult patients with CD, we can confirm the high prevalence of B8 (72.7%) and of DRw3 (63.7%). However we find that DRw7 is also significantly increased (54.5%); DRw3 and DRw7, taken together, represent 86% of our 22 patients.     

Plateau potentials and membrane oscillations in parasympathetic preganglionic neurones and intermediolateral neurones in the rat lumbosacral spinal cord

Whole-cell patch recordings were made from parasympathetic preganglionic neurones (P-PGNs) and unidentified intermediolateral (IML) neurones in thick slices of the lower lumbar and sacral spinal cord of 14- to 21-day-old rats. The P-PGNs and IML neurones examined were similar in terms of soma sizes, input resistance and capacitance, and displayed a sag conductance as well as rebound firing. In the absence of drugs, the neurones responded with either tonic or adapting firing to depolarizing current steps. However, in the presence of the group I metabotropic glutamate receptor agonist ( RS )-3,5-dihydroxyphenylglycine (DHPG), almost half of the neurones displayed accelerating firing rates during the constant current injection, followed by a sustained after-discharge. In the presence of TTX, plateau potentials were observed. The firing changes and plateaux were blocked by nifedipine, an L-type Ca²⁺ channel blocker, and ( S )-(−)-Bay K8644 was able to produce these firing changes and plateaux in the absence of DHPG, demonstrating the involvement of an L-type Ca²⁺ conductance. Ca²⁺-activated nonspecific cationic conductances also appear to contribute to the firing changes. A few neurones displayed membrane oscillations and burst firing in the presence of DHPG. The results suggest that the firing characteristics of both P-PGNs and other neurones likely to be involved in caudal spinal reflex control are not static but, rather, quite dynamic and under metabotropic glutamate receptor modulatory control. Such changes in firing patterns may be involved in normal pelvic parasympathetic reflex function during micturition, defaecation and sexual reflexes, and may contribute to the abnormal output patterns seen with loss of descending brainstem input and visceral or perineal sensory disturbances.     

Comparative distribution of nicotinic receptor subtypes during development, adulthood and aging: an autoradiographic study in the rat brain

The distribution in the rat brain of high affinity nicotinic heteromeric acetylcholine receptors and of low affinity nicotinic, alpha7-containing, homomeric receptors was studied using in vitro light microscopic autoradiography. As ligands, we used [3H]epibatidine, or [125I]epibatidine, and [125I]alpha-bungarotoxin, respectively. In adult animals, the two types of binding sites were widely distributed in many different brain structures, including the brainstem, cerebellum, mesencephalic structures, limbic system and cortex, but their anatomical distribution differed markedly. Only in rare instances could a co-localization be observed, for example in the superficial layer of the superior colliculus. In developing animals, both types of labeling were strongly expressed during embryonic and postnatal phases. Their distributions were qualitatively similar to those observed in adult animals, with a few noticeable exceptions in the cerebral cortex, hippocampus and brain stem. In aging animals, neither the distribution nor the density of nicotinic binding sites was significantly altered. Our conclusions are the following. (a) There is little overlap in the distribution of heteromeric and alpha7-containing homomeric nicotinic receptors in the rat brain. (b) The abundance of neuronal nicotinic receptors during embryonic and postnatal development suggests that they may play a role in the establishment of neuronal connectivity. (c) The expression of neuronal nicotinic receptors is unaltered in middle aged animals, suggesting that in the rat these receptors do not play any major role in aging process.     

Matching of digitised brain atlas to magnetic resonance images

A method has been developed to match a standard digitised brain atlas onto MR images for identification of cerebral structures in anatomical images. This method uses, first, a three-dimensional crude registration based on the proportional system of Talairach. Then, a two-dimensional refined registration is performed using a deformation function based on a set of homologous landmarks on both images (MR and atlas). Displacements vectors are computed between each corresponding landmark. These vectors are interpolated by thin-plate splines, generating an unwarping function defined on the whole image. This function can then be applied on any structure of the atlas. An evaluation of the matching procedure has been performed. First, the influence of the choice of the landmarks has been evaluated for the fine registration method. The latter has been then compared to the crude registration method considered as a classical reference method. These results show the advantages of the fine registration approach.     

Slow excitatory post-synaptic potentials in myenteric AH neurons of the guinea-pig ileum are reduced by the 5-hydroxytryptamine7 receptor antagonist SB 269970

Serotonin (5-HT) is a key modulator of neuronal excitability in the central and peripheral nervous system. In the enteric nervous system, 5-HT causes a slow depolarization in the intrinsic sensory neurons, but the receptor responsible for this has not been correlated with known gene products. The aim of this study was to determine whether the newly characterized 5-HT7 receptor may participate in the 5-HT-mediated depolarization of, and synaptic transmission to, the intrinsic sensory neurons of the guinea-pig ileum. Intracellular electrophysiological recordings were made from intrinsic sensory neurons identified as myenteric AH neurons from guinea-pig ileum. 5-HT (5 microM) applied to the cell body evoked both a fast depolarization (5-HT3 mediated) and/or a slow depolarization (5-HT1P-like). The 5-HT1/5/7 receptor agonist 5-carboxamidotryptamine (5-CT) (5 microM) evoked only a slow depolarization. When the fast depolarization evoked by 5-HT was blocked with granisetron (1 microM, 5-HT3 receptor antagonist), only a slow depolarization remained; this was abolished by the 5-HT7 receptor antagonist SB 269970 (1 microM, control: 14+/-2 mV, granisetron+SB 269970: -1+/-2 mV). The slow depolarization evoked by 5-CT was also significantly reduced by SB 269970 (control: 14+/-1 mV, SB 269970: 5+/-2 mV) suggesting a 5-HT7 receptor was activated by exogenous application of 5-CT and 5-HT. Slow excitatory postsynaptic potentials evoked by stimulating descending neural pathways (containing serotonergic fibers) were reduced by SB 269970 (control: 8+/-3 mV, SB 269970: 3+/-1 mV). However, SB 269970 had no effect on slow excitatory postsynaptic potentials evoked by stimulation of circumferential (tachykinergic) pathways (control: 7+/-1 mV, SB 269970: 6+/-1 mV). These data are consistent with the presence on enteric AH neurons of functional 5-HT7 receptors that participate in slow synaptic transmission.     

Genetic heterogeneity of the NS3 protease gene in hepatitis C virus genotype 1 from untreated infected patients

NS3 protease is essential for hepatitis C Virus (HCV) replication, and is one of the most promising targets for specific anti-HCV therapy. Its natural polymorphism has not been studied at the quasispecies level. In the present work, the genetic heterogeneity of the NS3 protease gene was analyzed in 17 HCV genotype 1 (5 subtypes 1a and 12 subtypes 1b) samples collected from infected patients before anti-viral therapy. A total of 294 clones were sequenced. Although the protease NS3 is considered to be one of the less variable genes in the HCV genome, variability of both nucleotide and amino acid sequences was found. In variants belonging to 1a and 1b subtypes, 224 and 267 of 543 positions showed one or more nucleotide substitutions, respectively. Forty and 74 of the 181 NS3 amino acid positions showed at least one mutation in HCV-1a and HCV-1b isolates, respectively. Most substitutions were conservative. This substantial polymorphism of the NS3 protease produced by HCV-1a and HCV-1b suggests that, despite the numerous functional and structural constraints, the enzyme is sufficiently flexible to tolerate substitutions.     

Allele variations in the OCA2 gene (pink-eyed-dilution locus) are associated with genetic susceptibility to melanoma

The occuloalbinism 2 (OCA2) gene, localized at 15q11, encodes a melanosomal transmembrane protein that is involved in the most common form of human occulo-cutaneous albinism, a human genetic disorder characterized by fair pigmentation and susceptibility to skin cancer. We wondered whether allele variations at this locus could influence susceptibility to malignant melanoma (MM). In all, 10 intragenic single-nucleotide polymorphisms (SNPs) were genotyped in 113 patients with melanomas and in 105 Caucasian control subjects with no personal or family history of skin cancer. By comparing allelic distribution between cases and controls, we show that MM and OCA2 are associated (p value=0.030 after correction for multiple testing). Then, a recently developed strategy, the 'combination test' enabled us to show that a combination formed by two SNPs was most strongly associated to MM, suggesting a possible interaction between intragenic SNPs. In addition, the role of OCA2 on MM risk was also detected using a logistic model taking into account the presence of variants of the melanocortin 1 receptor gene (MC1R, a key pigmentation gene) and all pigmentation characteristics as melanoma risk factors. Our data demonstrate that a second pigmentation gene, in addition to MC1R, is involved in genetic susceptibility to melanoma.