Bronchoalveolar lavage fluid from both lungs in horses: Diagnostic reliability of cytology from pooled samples.

Cytology of bronchoalveolar lavage fluid (BALF) from one lung may not predict findings in the contralateral lung of the same horse. The aim of this study was to determine whether a pooled BALF from both lungs was representative of corresponding individual samples. Fifty-one horses referred for poor performance and/or respiratory signs and for which a BALF was collected from both lungs, were included in the study. Cytology of pooled and individual BALF samples were performed using a masked protocol. Based on clinical signs and individual BALF cytologies, horses were classified as control (CTL), mild equine asthma (mEA), severe equine asthma (sEA) and/or exercise-induced pulmonary haemorrhage (EIPH). No significant difference was observed between pooled and individual BALF samples for all cell types (P>0.05). Correlations between pooled and individual BALF samples were good (r≥0.9) for neutrophil proportions and haemosiderophages/macrophages ratio, and moderate (r≥0.4) for metachromatic cell and eosinophil proportions. Similarly, intraclass correlation coefficient (ICC) were good (ICC≥0.9) for neutrophil proportions and haemosiderophages/macrophages ratio and substantial (ICC≥0.6) for metachromatic cell proportions. Based on threshold values for pooled samples as determined by receiver operating characteristic (ROC) analysis, categorical agreements were good (κ≥0.97) for diagnosis of mEA/sEA, and substantial (κ=0.74) for EIPH. Using a pooled BALF sample, only one horse was incorrectly classified as CTL instead of mEA and three horses were classified as EIPH instead of CTL. In conclusion, BALF cytology from pooled sample is representative of both individual lungs, and constitutes a valid method to diagnose EA.

Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines.

The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end-joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e.g., cancer-derived cells), obtaining a knockout usually requires lengthy screening and characterization procedures. Given the more precise type of modifications that can be introduced upon homology-directed repair (HDR), we have developed HDR-based gene-targeting strategies that greatly facilitate the process of knockout generation in cell lines. To generate reversible knockouts (R-KO), a selectable promoter-less STOP cassette is inserted in an intron, interrupting transcription. Loss-of-function can be validated by RT-qPCR and is removable, enabling subsequent restoration of gene function. A variant of the R-KO procedure can be used to introduce point mutations. To generate constitutive knockouts (C-KO), an exon is targeted, which makes use of HDR-based gene disruption together with NHEJ-induced indels on non-HDR targeted allele(s). Hence the C-KO procedure greatly facilitates simultaneous inactivation of multiple alleles. Overall these genome-editing tools offer superior precision and efficiency for functional genetic approaches. We provide detailed protocols guiding in the design of targeting vectors and in the analysis and validation of gene targeting experiments.

[Sleep disorders: Sociodemographics and psychiatric comorbidities in a sample of 14,734 adults in France (Baromètre santé INPES)]. / Troubles du sommeil : caractéristiques sociodémographiques et comorbidités anxiodépressives. Etude (Baromètre santé INPES) chez 14734 adultes en France.

AIM: Several studies have identified a high prevalence of sleep disorders and insomnia in the French population; however these studies especially focused on sleep did not give enough details on the sociodemographic and occupational characteristics of subjects with sleep disorders. The aim of this study was to characterize adults complaining of sleep disorders in a vast population-based health demographics study (Baromètre Santé, Institut national pour l'éducation à la santé [INPES]). METHOD: Fourteen thousand seven hundred and thirty-four adults aged from 18 to 65years old were interviewed by telephone and "subjects with severe sleep disorders in the last 8 days" were isolated and compared with the others regarding sociodemographic and occupational aspects and with regard to psychiatric disorders. The sleep items were extracted from the Duke and WHOQOL (brief form) questionnaires. Depressive disorders were recognized based on the Composite International Diagnostic Interview (CIDI) Short-Form (CIDI-SF) and the ISD 10. RESULTS: Among the subjects, 12.5% were concerned. Women, young or subjects after 45, low income, employees and unemployed categories had significantly more sleep disorders. Sleep disorders in the last 8 days were also significantly linked to previous depressive episodes, anxiety disorders and history of suicide. CONCLUSION: Assessing sleep disorders in the last 8 days seems to be a good method to identify severely affected subjects in general population surveys.

Upscaling the diffusion equations in particulate media made of highly conductive particles. I. Theoretical aspects.

Many analytical and numerical works have been devoted to the prediction of macroscopic effective transport properties in particulate media. Usually, structure and properties of macroscopic balance and constitutive equations are stated a priori. In this paper, the upscaling of the transient diffusion equations in concentrated particulate media with possible particle-particle interfacial barriers, highly conductive particles, poorly conductive matrix, and temperature-dependent physical properties is revisited using the homogenization method based on multiple scale asymptotic expansions. This method uses no a priori assumptions on the physics at the macroscale. For the considered physics and microstructures and depending on the order of magnitude of dimensionless Biot and Fourier numbers, it is shown that some situations cannot be homogenized. For other situations, three different macroscopic models are identified, depending on the quality of particle-particle contacts. They are one-phase media, following the standard heat equation and Fourier's law. Calculations of the effective conductivity tensor and heat capacity are proved to be uncoupled. Linear and steady state continuous localization problems must be solved on representative elementary volumes to compute the effective conductivity tensors for the two first models. For the third model, i.e., for highly resistive contacts, the localization problem becomes simpler and discrete whatever the shape of particles. In paper II [Vassal, Phys. Rev. E 77, 011303 (2008)], diffusion through networks of slender, wavy, entangled, and oriented fibers is considered. Discrete localization problems can then be obtained for all models, as well as semianalytical or fully analytical expressions of the corresponding effective conductivity tensors.

Upscaling the diffusion equations in particulate media made of highly conductive particles. II. Application to fibrous materials.

In paper I [Vassal, Phys. Rev. E77, 011302 (2008)] of this contribution, the effective diffusion properties of particulate media with highly conductive particles and particle-particle interfacial barriers have been investigated with the homogenization method with multiple scale asymptotic expansions. Three different macroscopic models have been proposed depending on the quality of contacts between particles. However, depending on the nature and the geometry of particles contained in representative elementary volumes of the considered media, localization problems to be solved to compute the effective conductivity of the two first models can rapidly become cumbersome, time and memory consuming. In this second paper, the above problem is simplified and applied to networks made of slender, wavy and entangled fibers. For these types of media, discrete formulations of localization problems for all macroscopic models can be obtained leading to very efficient numerical calculations. Semianalytical expressions of the effective conductivity tensors are also proposed under simplifying assumptions. The case of straight monodisperse and homogeneously distributed slender fibers with a circular cross section is further explored. Compact semianalytical and analytical estimations are obtained when fiber-fiber contacts are perfect or very poor. Moreover, two discrete element codes have been developed and used to solve localization problems on representative elementary volumes for the same types of contacts. Numerical results underline the significant roles of the fiber content, the orientation of fibers as well as the relative position and orientation of contacting fibers on the effective conductivity tensors. Semianalytical and analytical predictions are discussed and compared with numerical results.

Increased levels of expression of an NMDARI splice variant in the superior temporal gyrus in schizophrenia.

Expression patterns of mRNAs for the NMDARI subunit (NRI) carboxy-terminus isoforms were investigated in postmortem brain tissue using isotopic in situ hybridization. Three brain regions (superior temporal, middle frontal and visual cortices) were examined in patients with schizophrenia (n = 6) and control subjects (n = 6). A 22% higher level of expression of the NRI isoform that contains neither spliced exon was observed in the superior temporal gyrus of patients with schizophrenia compared with controls (p = 0.01). No differences were observed in the expression of the other isoforms in the three regions studied. These data suggest that NRI alternative splicing might be abnormal in schizophrenia and reinforce previous findings implicating the superior temporal gyrus as a site of neural dysfunction in schizophrenia.

Localization of 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptors in rabbit ocular and brain tissues.

Serotonin is thought to play a physiological role in various tissues of the rabbit eye, yet little is known about the relative distribution of the different serotonin receptors. Demonstration of the receptor subtypes present in the various ocular tissues is essential in order to understand the function of serotonin in the eye. Using a combination of in situ hybridization histochemistry, in vitro receptor autoradiography and polymerase chain reaction studies, we have explored the distribution of the 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptors in the rabbit eye. As these receptors have not been sequenced in the rabbit, we initially established the suitability of the oligonucleotide probes by analysis of brain tissue. The distributions of 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptor messenger RNAs in rabbit brain correlated well with those in other species, confirming the specificity of the probes for detection of the messenger RNAs in rabbit tissues. In the eye, the expression of 5-hydroxytryptamine1A receptors appears to be restricted to the epithelial cell layer of the ciliary processes, although very low levels may appear in the retina. In contrast, the expression of 5-hydroxytryptamine7 receptor messenger RNA is more widespread with positive signals evident in the ciliary processes, retina and iris. The results confirm the existence of 5-hydroxytryptamine1A receptors in the ciliary body and their localization in the ciliary epithelium supports the hypothesis that they are involved in the secretion of aqueous humour. Unexpectedly, there was little evidence to support the idea that 5-hydroxytryptamine1A receptors are present in the retina and iris sphincter. However, the subsequent finding of 5-hydroxytryptamine7 receptor messenger RNA in the retina and iris may explain the apparent absence of 5-hydroxytryptamine1A receptors in these tissues. The presence of both 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptors in the ciliary processes may account for the complex intraocular pressure response of the rabbit to serotonin.

Critical issues in the antisense inhibition of brain gene expression in vivo: experiences targetting the 5-HT1A receptor.

There have been many recent reports of receptor down-regulation in the brain by antisense oligodeoxynucleotides (ODNs) administered in vivo. However, the literature is inconsistent regarding the experimental criteria that are necessary or sufficient to demonstrate a true antisense effect. Here we review some of the critical conceptual and methodological issues. We highlight the problems of specificity and toxicity encountered in our attempts to down-regulate the 5-HT1A receptor using a phosphorothioate-modified ODN. We also present preliminary data suggestive of a decreased hippocampal 5-HT1AR expression induced by the antisense ODN, but it is a reduction which is of limited extent and which does not provide unequivocal evidence for an antisense-mediated effect. We conclude that antisense ODNs are not yet suitable as tools for routine in vivo neuropharmacological use, although they show considerable promise.

Increase of 5-HT7 (serotonin-7) and 5-HT1A (serotonin-1A) receptor mRNA expression in rat hippocampus after adrenalectomy.

The brain 5-HT (serotonin) system and circulating corticosteroids are in close interaction and both are implicated in the pathogenesis of affective disorders. The 5-HT1A receptor is thought to play a major role in this relationship. However, the recently cloned 5-HT7 receptor may also be involved, given its pharmacological similarities to the 5-HT1A receptor and its high expression in corticolimbic structures. Using in situ hybridization histochemistry, we have investigated 5-HT7 and 5-HT1A receptor mRNA expression in selected areas of the rat brain 7 days post-adrenalectomy. 5-HT7 receptor mRNA was increased in CA1 and CA3b after adrenalectomy, with no alterations in other hippocampal subfields or in retrosplenial cortex. Adrenalectomy was associated with a marked increase of 5-HT1A receptor mRNA in dentate gyrus, CA3 and CA2, but not in CA1, nor in the raphe. These data indicate that circulating adrenal steroids have a inhibitory role on the expression of hippocampal 5-HT7 receptors as well as 5-HT1A receptors, but the effect upon the two transcripts occurs in different subfields. The 5-HT7 receptor is an additional candidate for mediating the interactions between 5-HT and corticosteroids within the hippocampus.

Dopaminergic control of LH secretion by the A15 nucleus in anoestrous ewes.

Annual variations in the secretion of LH are responsible for seasonal changes in ovulatory activity in ewes. This hormonal pattern reflects an increase in the intensity of the negative feedback exerted by oestradiol under long days. Neuropharmacological studies have shown that this inhibition of LH secretion involves activation of catecholaminergic systems from preoptic and mediobasal hypothalamus (MBH) by oestradiol during anoestrus, and that 5-hydroxytryptamine inputs may also play a role. Within the MBH, the most important structures appear to be the retrochiasmatic region of the hypothalamus, which contains the A15 dopaminergic nucleus, and the median eminence, which contains the axon terminals of the GnRH cells controlling the pulsatile release of LH. In ovariectomized ewes in which oestradiol tonically inhibits LH secretion during the anoestrous season, LH pulse frequency is increased when the cells of the A15 nucleus are destroyed. The median eminence and other mediobasal structures contain more catecholamines and their metabolites under long days than under short days. Microdialysis of the A15 nucleus in vivo during long days revealed increased catecholaminergic activity under oestradiol treatment due to stimulation of tyrosine hydroxylase, the rate-limiting enzyme in the pathway of catecholaminergic synthesis. Tyrosine hydroxylase activity within the median eminence is increased under the various photoperiodic regimens that inhibit LH secretion. Neurochemical changes in the A15 nucleus and median eminence, in response to photoperiodic or oestradiol treatments, suggest a functional relationship which acts at the level of the GnRH axon terminals.

Differences in ketanserin binding in the ventromedial hypothalamus of ewes responsive or refractory to short days.

Participation of central 5HT receptors in the inhibition of LH pulsatility during refractoriness to short days (SD) in ewes has been suggested by previous in vivo studies using various 5HT-antagonist such as ketanserin. In the present study, binding of [3H]ketanserin in ewe brain sections was similar to that described in the brain of other species and could correspond with an interaction at 5HT2 receptors sites. Rosenthal analysis from the caudate nucleus was linear (Kd = 3 nM). The displacement studies from the cortex slices showed that the 5HT antagonists such as methysergide, ketanserin, cyproheptadine and spiperone competed with the labelled ligand at nanomolar concentrations whereas serotonin was less active. However, the first 3 drugs recognized different populations of binding sites. Prazosin, an alpha 1-adrenergic antagonist was inactive, but a slight inhibition of [3H]ketanserin binding was induced by pyrilamine, an H1 histaminic antagonist, within a nanomolar range. Methysergide (10(-6) M), which does not bind to H1 receptors, was therefore used to determine the nonspecific binding. Quantitative analysis of the binding of 3 nM [3H]ketanserin on sections of the ewe brain at the preopticohypothalamic level was then carried out by autoradiography. The highest binding densities were observed in the caudate nuclei (64.0 fmol/mg tissue Eq) and the mammillary bodies (52.7 fmol/mg tissue Eq) whereas intermediate or low densities were found in the other structures. The anatomical distribution of the labelling was similar to that described in other species for 5HT2 receptors. Ketanserin binding in these areas was compared between two groups of ovariectomized estradiol-treated Ile-de-France ewes, submitted to artificial short days (SD: 8L:16D), one group with a high LH pulsatility (responsive to SD) and the other one with a low LH pulsatility (photorefractory to SD). Binding densities were similar for each one of the studied regions between the two groups, except in the ventrolateral part of the mediobasal hypothalamus, where ewes exhibiting high LH pulsatility had a more than 2-fold higher binding density than those with a low LH pulsatility (mean +/- SEM, 14.6 +/- 1.4 vs. 5.7 +/- 1.0 fmol/mg tissue Eq, respectively; p < 0.0016). These results suggest that [3H]ketanserin binding sites in the ventromedial part of the mediobasal hypothalamus could be associated to the regulation of the photoperiodic inhibition of LH at the time of establishment of refractoriness to short days in the Ile-de-France ewe.

Serotonergic 5HT2 receptors mediate the inhibitory action of serotonin on luteinizing hormone secretion in ovariectomized, estradiol-treated ewes that are refractory to short days.

Previous studies with high doses of cyproheptadine suggested that serotonergic (5HT) neural mechanisms participate in the photoperiodic inhibition of secretion of LH in ovariectomized estradiol-treated ewes. In the present study, we tested this hypothesis further with lower doses of more specific 5HT2 receptor antagonists, including the highly specific ketanserin. Eight ovariectomized estradiol-treated ewes were subjected to 90 long days (16L:8D) followed by 207 short days (8L:16D) in order to induce photorefractoriness. Plasma LH concentrations were measured in blood sampled twice weekly. Changes in LH pulse frequency were measured after intrajugular injections of cyproheptadine or ketanserin (0.1, 0.25, 0.6, and 1.5 mg/kg) before the increase in LH secretion induced by the short days (SD ewes). After the ewes became refractory to short days (RSD ewes), they were again treated with the three lowest doses of cyproheptadine and ketanserin and, in addition, methysergide at the same doses. In the period following the transition from long to short photoperiod, LH secretion increased above basal levels after 42.0 +/- 0.0 days (mean +/- SEM) and then decreased after 137.0 +/- 7.4 days. In SD ewes, cyproheptadine significantly increased the number of LH pulses at the lowest dose tested (0.13 vs. 1.13 pulses/3 h at 0.1 mg/kg). Similar responses were observed at 0.25 mg/kg (0.13 vs. 0.88 pulses/3 h), at 0.6 mg/kg (0.13 vs. 0.75 pulses/3 h) and at 1.5 mg/kg (0.13 vs. 0.88 pulses/3 h). Ketanserin induced a dose-dependent increase in the number of LH pulses at 0.6 mg/kg (0 vs. 0.63 pulses/3 h) and at 1.5 mg/kg (0 vs. 0.75 pulses/3 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Control of photoperiodic inhibition of luteinizing hormone secretion by dopaminergic and serotonergic systems in ovariectomized Ile-de-France ewes supplemented with oestradiol.

The role of dopaminergic and serotonergic systems on LH secretion was investigated in Ile-de-France ewes under different artificial inhibitory photoperiodic regimens. All animals were ovariectomized at the end of the breeding season, chronically treated with an oestradiol implant, and subjected to various changes in daylength for 9 months to inhibit or stimulate their LH secretion. Plasma LH concentration was assessed by taking blood samples twice a week throughout the experiment. The effects of acute intravenous injections of the dopaminergic2 receptor antagonist pimozide (0.08 mg kg-1) and the 5-hydroxytryptamine2 (5HT2) receptor antagonist cyproheptadine (3 mg kg-1) on LH pulsatility were assessed during challenges in four different situations: (1) long days (LD); (2) before short-day response (SD); (3) during refractoriness to short days (RSD); and (4) during inhibition by long days (ILD). LH in blood samples collected twice a week remained low during long days (0.59 +/- 0.03; mean +/- SEM), increased 45 +/- 1.5 days after the onset of short days and decreased 132 +/- 4.9 days later when ewes became refractory to short days, whereas ewes subjected to long days after 91 short days stopped their neuroendocrine activity 19 days earlier (113 +/- 4.7) (P < 0.01). In comparison with the pre-injection period, pimozide significantly increased the mean number of pulses in SD and RSD ewes, but not in LD and ILD ewes: SD: 0 versus 0.45 pulses in 4 h (P < 0.02); RSD: 0 versus 0.9 (P = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)