[Membrane lipids in schizophrenia and early phases of psychosis: Potential biomarkers and therapeutic targets?] / Lipides membranaires dans la schizophrénie et la psychose débutante : de potentiels biomarqueurs et pistes thérapeutiques ?

The various roles of membrane lipids in human health has urged researchers to study their impact in neuropsychiatric diseases, especially in schizophrenia spectrum disorders and more recently in early stages of psychosis. The progress in mass spectrometry technologies now allows a more comprehensive analysis of phospholipids (PL) and their fatty acid (FA) molecular species. FA are defined by a carbon chain of variable length and are said to be unsaturated when their chain has one or more carbon-carbon double bonds. The PL are composed of a hydrophilic polar head with a phosphoric acid group and an hydrophobic part with FAs; they encompass glycerophospholipids and sphingolipids. The plasma membrane is a complex and dynamic structure consisting of a lipid bilayer composed of an outer layer and an inner layer of specific lipid composition. The permanent remodeling of membrane lipids involves phospholipases especially the phospholipase A2. Seventy percent of the brain consists of lipids from different classes and molecular species. Most of the brain lipids are composed of polyunsaturated fatty acid (PUFA)-enriched diacyl classes where omega-3 and omega-6 molecular species predominate. The balance between omega-3 and omega-6 is important for the neurodevelopment. PUFA are also involved in neurogenesis and neurotransmission. Sphingomyelin (SM) is a sphingolipid that influences inflammation, cell proliferation and lipid rafts formation. It is an important component of myelin sheaths of white matter and therefore is involved in cerebral connectivity. In rat models, deficiency in omega-3 causes abnormalities in dopaminergic neurotransmission, impacts on the functioning of some receptors (including cannabinoids CB1, glutamatergic N-methyl-D-aspartate receptor, NMDA), and increases sensitivity to hallucinogens. In contrast, omega-3 supplementation improves cognitive function and prevents psychotic-like behavior in some animal models for schizophrenia. It also reduces oxidative stress and prevents demyelination. The historical membrane hypothesis of schizophrenia has led to explore the lipids abnormality in this disorder. This hypothesis was initially based on the observation of an abnormal membrane prostaglandin production in schizophrenia caused by a membrane arachidonic acid deficiency. It has evolved emphasizing the various PUFA membrane's roles in particular regarding oxidative stress, inflammation and regulation of the NMDA receptors. In patients with mental disorders, low omega-3 index is more frequent than in the general population. This lipid abnormality could lead to myelination abnormalities and cognitive deficits observed in patients. It could also participate in oxidative stress abnormalities and inflammation reported in schizophrenia. On the other hand, low omega-3 index deficit was reported to be associated with an increased cardiovascular risk, and omega-3 supplementation may also have a positive cardiovascular impact in psychiatric patients, even more than in the general population. The presence of membrane lipid abnormalities is also found in patients during the first psychotic episode (FEP). The omega-3 supplementation improved the recovery rate and prevented the loss of gray matter in FEP. In patients at ultra-high risk to develop a psychotic disorder (UHR), omega-3 supplementation has been associated with a reduction of the rate of conversion to psychosis and with metabolic changes, such as decreased activity of phospholipase A2. However, this study has not as yet been replicated. Not all patients exhibit lipid abnormalities. Several studies, including studies from our team, have found a bimodal distribution of lipids in patients with schizophrenia. But some studies have found differences (in PUFA) in the acute phase whereas our studies (on phospholipids) are in chronic phases. It will be interesting to study in more depth the links between these two parameters. Furthermore, we identified a subgroup which was identified with a deficit in sphingomyelin and PUFA whereas others have found an increase of sphingomyelin. Individuals with this abnormal lipid cluster had more cognitive impairments and more severe clinical symptoms. Because the niacin test is an indirect reflection of arachidonic acid levels, it has been proposed to identify a subset of patients with membrane lipids anomalies. Niacin test response is influenced by several factors related to lipid metabolism, including cannabis use and phospholipase A2 activity. Despite progress, the function and impact of membrane lipids are still poorly understood in schizophrenia. They could serve as biomarkers for identifying biological subgroups among patients with schizophrenia. In UHR patients, their predictive value on the conversion to psychosis should be tested. Omega-3 supplementation could be a promising treatment thanks to its good tolerance and acceptability. It could be more appropriate for patients with PUFA anomalies in a more personalized medical approach.

Docosahexaenoic acid promotes micron scale liquid-ordered domains. A comparison study of docosahexaenoic versus oleic acid containing phosphatidylcholine in raft-like mixtures.

The understanding of the functional role of the lipid diversity in biological membranes is a major challenge. Lipid models have been developed to address this issue by using lipid mixtures generating liquid-ordered (Lo)/liquid-disordered (Ld) immiscibility. The present study examined mixtures comprising Egg sphingomyelin (SM), cholesterol (chol) and phosphatidylcholine (PC) either containing docosahexaenoic (PDPC) or oleic acid (POPC). The mixtures were examined in terms of their capability to induce phase separation at the micron- and nano-scales. Fluorescence microscopy, electron spin resonance (ESR), X-ray diffraction (XRD) and calorimetry methods were used to analyze the lateral organization of the mixtures. Fluorescence microscopy of giant vesicles could show that the temperature of the micron-scale Lo/Ld miscibility is higher for PDPC than for POPC ternary mixtures. At 37°C, no micron-scale Lo/Ld phase separation could be identified in the POPC containing mixtures while it was evident for PDPC. In contrast, a phase separation was distinguished for both PC mixtures by ESR and XRD, indicative that PDPC and POPC mixtures differed in micron vs nano domain organization. Compared to POPC, the higher line tension of the Lo domains observed in PDPC mixtures is assumed to result from the higher difference in Lo/Ld order parameter rather than hydrophobic mismatch.

[Understanding the use of self-evaluation questionnaires when assessing a patient's swallowing capacity and performing follow-up activities]. / Intérêt des echelles d'autoévaluation dans le bilan et le suivi des troubles de la déglutition.

The purpose of this study was to examine the possibility of improving the assessment protocol of dysphagia and show the interest of adding a self-assessment evaluation scale to the traditional functional assessment of the swallowing process. We have a comparison of the assessment of 30 dysphagic patients with two different scales (DHI and EAT-10). Results show that the self-evaluation scales selected led to quantitative data comparable to the objective results of the COLP-FR-G functional assessment tool. We may therefore conclude that the patients' own feelings do not differ from the results of the clinical dysphagia severity assessment. Results also highlight that both scales provide similar information in terms of content and also reflect the evolution of objective data. The EAT-10 scale, by its simplicity and limited number of questions, could be proposed as a screening test in neurological patients and geriatric population, where the DHI scale, with its three subdomains (physical, functional and emotional), would be a complementary tool for the functional swallowing evaluation. Indeed, in order to evaluate dysphagia, the association of DHI, EAT-10 and COLP-FR-G offers several advantages. This protocol is convenient and rapid to administrate, it takes the diversity of etiologies into account and its score is easy to transmit to the patients' medical practitioners. Consequently, its use in the assessment of swallowing functions should be encouraged as a consistent part of the clinical practice.

Effects of the association of aging and obesity on lipids, lipoproteins and oxidative stress biomarkers: a comparison of older with young men.

In this study, plasma lipids, lipoproteins and markers of oxidant/antioxidant status were investigated in young (n = 45) and older (n = 40) obese men and compared to those in young (n = 65) and older (n = 55) normal weight controls. The purpose was to determine whether obesity exacerbates or not lipid, lipoprotein abnormalities and oxidative stress in older men. Our findings showed that all obese patients had increased plasma triglyceride, cholesterol, LDL-cholesterol, -triglyceride and HDL-triglyceride levels concentrations compared to controls (P < 0.01). However, the younger obese men had relatively larger and accentuated changes in plasma lipids and lipoproteins than the older patients. Additionally, total antioxidant capacity (ORAC), vitamins C and E were lower while hydroperoxides and carbonyl proteins were higher in young and older obese patients compared to their respective controls (P < 0.001). Erythrocyte antioxidant SOD and catalase activities were enhanced in obese young patients, but reduced in obese older men. Glutathione peroxidase activity was low in obesity irrespective of age. In multiple regression analysis, BMI significantly predicted total cholesterol, LDL-C, LDL-TG and HDL-TG (P < 0.0001). These relationships were not modified by age. BMI alone was a not a significant predictor for ORAC, vitamins C, E, catalase and Glutathione peroxidase. However, the interaction BMI-age significantly predicted these parameters and explained 28-45% of their changes. BMI was a significant predictor of SOD, carbonyl proteins and hydroperoxides. This effect became more significant (P < 0.0001) and worsened with BMI-age interaction. In conclusion, lipoprotein metabolism and oxidant/antioxidant status are altered in obesity irrespective of age. However, obesity-related lipid and lipoprotein alterations were attenuated while oxidative stress was aggravated in older adults.

Systemic morphine administration causes gastric distention and hyperphagia in healthy horses.

BACKGROUND: There are no data investigating the effect of systemic morphine on the size of the stomach or the food consumption in horses. OBJECTIVES: To evaluate gastrointestinal side effects of morphine administered systemically in healthy horses by the means of clinical and ultrasonographic evaluations. STUDY DESIGN: In vivo experiment. METHODS: On day 1 of the experiment, six healthy French Trotter mares were evaluated clinically and an abdominal ultrasonography was performed three times 4 h apart to record the size of the stomach, the number of contractions per minute of the duodenum, jejunum, caecum, left and right ventral colons. On Day 2, morphine was administered three times 4 h apart at the dose of 0.1 mg/kg i.v. and the same ultrasonographic examinations performed. On Day 3, only clinical and ultrasonographic examinations were performed as on Day 1. Amounts of hay and water ingested, frequency and weight of faeces were recorded throughout the study. RESULTS: Number of contractions of the duodenum, caecum, left and right ventral colons were significantly decreased after morphine administration. Size of the stomach was increased significantly with a cumulative effect of repeated doses of morphine. Hay (+0.4 kg/h, P<0.001) and water (+1.1 L/h, P<0.001) consumption were significantly increased. MAIN LIMITATIONS: The study was performed in healthy horses. CONCLUSIONS: Systemic morphine administration causes gastrointestinal depression, gastric distention and hyperphagia in horses. Clinical and ultrasonographical examinations are valuable tools to identify side effects of morphine administration in horses. Further studies are needed to assess side effects and monitoring in clinically painful cases. Horses receiving systemic morphine administration should be closely monitored for signs of gastric distention and, specifically, the amount of food given while receiving treatment should be controlled to avoid complications.

[Cognitive rehabilitation of amusia]. / Vers une rééducation cognitive de l'amusie.

INTRODUCTION: The cognitive model of music processing has a modular architecture with two main pathways (a melody pathway and a time pathway) for processing the musical "message" and thus enabling music recognition. It also features a music-specific module for tonal encoding of pitch which stands apart from all other known cognitive systems (including language processing). To the best of our knowledge, rehabilitation therapy for amusia has not yet been reported. OBJECTIVE: We developed a therapeutic method (inspired by work on word deafness) in order to determine whether specific rehabilitation based on melody discrimination could prompt the regression of amusia. METHOD: We report the case of a patient having developed receptive, acquired amusia four years previously. His tone deafness disorder was assessed using the Montreal Battery of Evaluation of Amusia (MBEA), which revealed impairment of the melody pathway but no deficiency in the time pathway. A computer-assisted rehabilitation method was implemented; it used melody discrimination tasks and an errorless learning paradigm with progressively fading visual cues. RESULTS: After therapy, we noted an improvement in the overall MBEA score and its component subscores which could not be explained by spontaneous recovery (in view of the number of years since the neurological accident). The improvement was maintained at seven months post-therapy. Although post-therapy improvement in daily life was not systematically assessed, the patient started listening to his favourite music again. CONCLUSION: Specific amusia therapy has shown efficacy.

Intracellular uranium distribution: Comparison of cryogenic fixation versus chemical fixation methods for SIMS analysis.

Localization of uranium within cells is mandatory for the comprehension of its cellular mechanism of toxicity. Secondary Ion Mass Spectrometry (SIMS) has recently shown its interest to detect and localize uranium at very low levels within the cells. This technique requires a specific sample preparation similar to the one used for Transmission Electronic Microscopy, achieved by implementing different chemical treatments to preserve as much as possible the living configuration uranium distribution into the observed sample. This study aims to compare the bioaccumulation sites of uranium within liver or kidney cells after chemical fixation and cryomethods preparations of the samples: SIMS analysis of theses samples show the localization of uranium soluble forms in the cell cytoplasm and nucleus with a more homogenous distribution when using cryopreparation probably due to the diffusible portion of uranium inside the cytoplasm.

Fracture of the medial intercondylar eminence of the tibia in horses treated by arthroscopic fragment removal (21 horses).

BACKGROUND: Fractures of the medial intercondylar eminence of the tibia (MICET) are scarcely reported in horses. OBJECTIVES: To report the clinical and diagnostic findings, surgical treatment and outcome in a series of horses presented with MICET fracture and treated with arthroscopic fragment removal. STUDY DESIGN: Multicentre retrospective case series. METHODS: Case records of horses diagnosed with MICET fractures that had undergone surgical treatment were reviewed. Follow-up information was obtained from re-examination visits and/or owners. RESULTS: Twenty-one cases were identified at 9 equine hospitals between 2004 and 2016. A history of trauma and acute onset of lameness was reported in 12 horses. All cases underwent fracture removal via arthroscopy of the medial femorotibial joint. The cranial cruciate ligament was intact in 6 horses and damaged in 15 horses (damage was ≤25% [n = 9], 25-50% [n = 4] or ≥50% [n = 2] of the cross-sectional area). The cranial ligament of the medial meniscus was damaged in 11 horses (≤25% [n = 8], 25-50% [n = 3]). The medial meniscus was damaged in 5 horses and articular cartilage damage was identified in 14 horses (mild [n = 8], moderate [n = 6]). Follow-up information (median 14 months; 4 months-6 years) was available for 20 cases; 2 horses were sound but convalescing; 13 horses were sound and returned to their previous or expected use. Of the 4 horses with the most severe changes to the articular soft tissue structures, 2 remained lame and 2 were subjected to euthanasia because of persistent lameness. MAIN LIMITATIONS: The retrospective, multicentre nature of this study and the limited number of horses are the main limitations. CONCLUSIONS: Fractures of the MICET are commonly associated with a traumatic event. Prompt diagnosis and early arthroscopic removal of the fracture are recommended.

Contamination by Salmonella spp., Campylobacter spp. and Listeria spp. of most popular chicken- and pork-sausages sold in Reunion Island.

One of the most popular meat products of the local "cuisine" is sausage composed with 100% chicken or 100% pork. In this study, we aimed to determine the presence of Salmonella spp., Campylobacter spp. and Listeria spp. in chicken- and pork-sausages, quantify Salmonella spp. population and identify the factors that could be associated with contamination in the outlets. Two hundred and three batches of pork and chicken sausages were randomly collected from 67 local outlets (supermarkets, groceries and butcher shops). Salmonella spp. was detected in 11.8% (95% confidence interval (CI): [10.0; 13.5]) of samples, Campylobacter spp. in 1.5% [0.7; 4.2] and Listeria monocytogenes in 5.9% [4.4; 7.3]. Most probable number of Salmonella spp. varied between 6cfu per gram to 320cfu per gram. Salmonella serotypes isolated from pork and chicken sausages were S. Typhimurium (45.8%), S. London (20.8%), S. Derby (16.7%), S. Newport (8.33%), S. Blockley (4.2%) and S. Weltevreden (4.17%). Using a logistic (mixed-effect) regression model, we found that Salmonella spp. contamination was positively associated with sausages sold in papers or plastic bags and no control of rodents. Chicken sausages were associated with a decreasing risk of Salmonella contamination. Listeria monocytogenes contamination was positively associated with the presence of fresh rodent droppings in the outlet and negatively when the staff was cleaning regularly their hands with soap and water or water only. All the sampled outlets of Reunion Island were not equivalent in terms of food safety measures. Increasing awareness of these traders remains a cornerstone to limit the presence of Salmonella spp. and Listeria spp. in sausages, particularly in a tropical context (high temperature and humidity).

Absorption, accumulation and biological effects of depleted uranium in Peyer's patches of rats.

The digestive tract is the entry route for radionuclides following the ingestion of contaminated food and/or water wells. It was recently characterized that the small intestine was the main area of uranium absorption throughout the gastrointestinal tract. This study was designed to determine the role played by the Peyer's patches in the intestinal absorption of uranium, as well as the possible accumulation of this radionuclide in lymphoid follicles and the toxicological or pathological consequences on the Peyer's patch function subsequent to the passage and/or accumulation of uranium. Results of experiments performed in Ussing chambers indicate that the apparent permeability to uranium in the intestine was higher (10-fold) in the mucosa than in Peyer's patches ((6.21+/-1.21 to 0.55+/-0.35)x10(-6)cm/s, respectively), demonstrating that the small intestinal epithelium was the preferential pathway for the transmucosal passage of uranium. A quantitative analysis of uranium by ICP-MS following chronic contamination with depleted uranium during 3 or 9 months showed a preferential accumulation of uranium in Peyer's patches (1355% and 1266%, respectively, at 3 and 9 months) as compared with epithelium (890% and 747%, respectively, at 3 and 9 months). Uranium was also detected in the mesenteric lymph nodes ( approximately 5-fold after contamination with DU). The biological effects of this accumulation of depleted uranium after chronic contamination were investigated in Peyer's patches. There was no induction of the apoptosis pathway after chronic DU contamination in Peyer's patches. The results indicate no change in the cytokine expression (Il-10, TGF-beta, IFN-gamma, TNF-alpha, MCP-1) in Peyer's patches and in mesenteric lymph nodes, and no modification in the uptake of yeast cells by Peyer's patches. In conclusion, this study shows that the Peyer's patches were a site of retention for uranium following the chronic ingestion of this radionuclide, without any biological consequences of such accumulation on Peyer's patch functions.

Ligation of the ipsilateral common carotid artery and topical treatment for the prevention of epistaxis from guttural pouch mycosis in horses.

The objective of the study was to evaluate the effect of ligation of the ipsilateral common carotid artery (CCA) combined with various antimycotic treatments for the prevention of epistaxis in horses with guttural pouch mycosis. For each case, ipsilateral ligation of the CCA was performed, followed by application of various topical medications under endoscopic guidance. Frequency and number of treatments, outcome and recurrence of haemorrhage were retrospectively recorded. Twenty-four horses were included. Topical medication was administered by detachment of the diphtheric membrane and spraying (n=16) or by intralesional injection directly in the plaques using a transendoscopic needle (n=8). Epistaxis recurred in five horses (20.8 per cent), causing death of four horses (16.6 per cent). The mean number of treatments was 6.3±4.0 (range 2-14) for all topical treatments. Ligation of the ipsilateral CCA and topical medication carries a fair prognosis for avoidance of recurrent episodes of epistaxis, but fatal haemorrhage can occur. Removal of the fungal plaque and topical treatment of the underlying lesion appeared to speed up resolution of the mycotic mucosal lesions. The described technique is a salvage procedure when financial or technical constraints prevent the use of transarterial catheter occlusion techniques.

Membrane lipidomics in schizophrenia patients: a correlational study with clinical and cognitive manifestations.

Schizophrenia is a severe mental condition in which several lipid abnormalities-either structural or metabolic-have been described. We tested the hypothesis that an abnormality in membrane lipid composition may contribute to aberrant dopamine signaling, and thereby symptoms and cognitive impairment, in schizophrenia (SCZ) patients. Antipsychotic-medicated and clinically stable SCZ outpatients (n=74) were compared with matched healthy subjects (HC, n=40). A lipidomic analysis was performed in red blood cell (RBC) membranes examining the major phospholipid (PL) classes and their associated fatty acids (FAs). Clinical manifestations were examined using the positive and negative syndrome scale (PANSS). Cognitive function was assessed using the Continuous Performance Test, Salience Attribution Test and Wisconsin Card Sorting Test. Sphingomyelin (SM) percentage was the lipid abnormality most robustly associated with a schizophrenia diagnosis. Two groups of patients were defined. The first group (SCZ c/SM-) is characterized by a low SM membrane content. In this group, all other PL classes, plasmalogen and key polyunsaturated FAs known to be involved in brain function, were significantly modified, identifying a very specific membrane lipid cluster. The second patient group (SCZ c/SM+) was similar to HCs in terms of RBC membrane SM composition. Compared with SCZ c/SM+, SCZ c/SM- patients were characterized by significantly more severe PANSS total, positive, disorganized/cognitive and excited psychopathology. Cognitive performance was also significantly poorer in this subgroup. These data show that a specific RBC membrane lipid cluster is associated with clinical and cognitive manifestations of dopamine dysfunction in schizophrenia patients. We speculate that this membrane lipid abnormality influences presynaptic dopamine signaling.

Involvement of SOCS-1, the suppressor of cytokine signaling, in the prevention of prolactin-responsive gene expression in decidual cells.

The cells forming the rat decidua produce PRL and PRL-related proteins and express both the long and short forms of the PRL receptor. Yet, only a defined subpopulation, the mesometrial cells, express the PRL-dependent alpha2-macroglobulin gene. This gene is silenced in vivo in the antimesometrial cells and in the GG-AD cell line, derived from antimesometrial cells. To examine whether the lack of alpha2-macroglobulin expression is due to defective components in the PRL signaling pathway, we compared the relative expression of Janus kinase 2 (Jak2), signal transducer and activator of transcription 5 a and b (Stat5 a and b), suppressor of cytokine signaling-1 (SOCS-1), and the tyrosine phosphatase SHP-2 mRNA in mesometrial and antimesometrial decidua on days 12 and 13 of pseudopregnancy, the time of maximal alpha2-macroglobulin expression. We found no significant differences in the relative expression of either Jak2, Stat5 (a and b), or SHP-2 in the two cell populations. However, we discovered a profound difference in the expression of SOCS-1, an inhibitor of the Jak/Stat pathway. This gene was highly expressed in the antimesometrial cells and in the GG-AD cells, which do not produce alpha2-macroglobulin. Immunoprecipitation experiments with GG-AD cells revealed that although Jak2 and Stat5 coprecipitate in response to PRL stimulation, no phosphorylation of Jak2 and Stat5 could be observed. To examine whether SOCS-1 plays a role in silencing the alpha2-macroglobulin gene, we cultured GG-AD cells in the presence of either a SOCS-1 antisense oligonucleotide or an irrelevant oligonucleotide for 4, 12, and 28 h. Cells were also treated with PRL. Within 4 h of SOCS-1 antisense treatment, alpha2-macroglobulin mRNA expression was initiated. After 28 h, only cells treated with PRL and SOCS-1 antisense oligonucleotide retained the ability to express the alpha2-macroglobulin gene. In summary, results of this study reveal that constitutive expression of SOCS-1 can prevent PRL signaling and that the lack of PRL-induced expression of alpha2-macroglobulin in a defined decidual cell population is largely due to SOCS-1 expression in these cells.

Electromyographic activity of the stylopharyngeus muscle in exercising horses.

REASONS FOR PERFORMING STUDY: There is a need to understand the process which leads to failure of recruitment of the stylopharyngeus muscle in clinical cases of nasopharygeal collapse. We therefore studied the timing and intensity of stylopharyngeus muscle activity during exercise in horses. OBJECTIVE: To measure the electromyographic (EMG) activity of the stylopharyngeus muscle in exercising horses and correlate it with the breathing pattern. METHODS: Five horses were equipped with a bipolar fine wire electrode placed on the stylopharyngeus muscle and a pharyngeal catheter. The horses exercised on a treadmill at speeds corresponding to 50 (HRmax50), 75 and 100% of maximum heart rate, and EMG activity of the stylopharyngeus muscle and upper airway pressures were recorded. The EMG activity of the stylopharyngeus muscle was then correlated to the breathing pattern and the activity quantified and reported as a percentage of the baseline activity measured at HRmax50. RESULTS: There was ongoing activity of the stylopharyngeus muscle throughout the breathing cycle; however, activity increased towards the end of expiration and peaked early during inspiration. Tonic activity was present during expiration. Peak, mean electrical and tonic EMG activity increased significantly (P<0.05) with exercise intensity. CONCLUSION: The stylopharyngeus muscle has inspiratory-related activity and tonic activity that increases with speed. POTENTIAL RELEVANCE: The stylopharyngeus muscle is one of a group of upper airway muscles that function to support and maintain the patency of the nasopharynx during inspiration. Failure of recruitment of the stylopharyngeus muscle during exercise is a potential explanation for clinical cases of dorsal pharyngeal collapse, but further work investigating the activity of the stylopharyngeus muscle in horses affected by this disease is needed.

The effect of bilateral glossopharyngeal nerve anaesthesia on swallowing in horses.

REASONS FOR PERFORMING STUDY: Dysfunction of the glossopharyngeal nerve has been implicated as a cause of dysphagia in horses. However, recent studies have indicated that this is not the case. OBJECTIVES: To determine whether bilateral glossopharyngeal nerve anaesthesia would cause dysphagia in horses or result in measurable alterations in the timing, function, or sequence of swallowing. METHODS: Swallowing was evaluated in 6 normal horses with and without bilateral glossopharyngeal nerve anaesthesia. Swallowing dynamics were assessed subjectively and objectively based on time from prehension of food until swallowing, number of tongue movements until initiation of swallowing, depth of bolus at the base of the tongue prior to initiation of swallow and evidence of tracheal aspiration using fluoroscopy and endoscopy. RESULTS: There was no evidence of aspiration or dysphagia in horses before or after bilateral glossopharyngeal nerve block. No observed or measured differences in swallowing sequence or function could be detected in blocked compared to unblocked horses. However, there was a trend in blocked horses for the number of tongue pushes and the time to swallowing to be increased. CONCLUSIONS: Glossopharyngeal nerve function may not be essential for normal swallowing function in otherwise healthy horses. POTENTIAL RELEVANCE: Clinically, normal swallowing is not an appropriate test of glossopharyngeal nerve function and dysphagic horses should not be assumed to have glossopharyngeal nerve dysfunction.

Genetics of upper and lower airway diseases in the horse.

Genetic predispositions for guttural pouch tympany, recurrent laryngeal neuropathy and recurrent airway obstruction (RAO) are well documented. There is also evidence that exercise-induced pulmonary haemorrhage and infectious diseases of the respiratory tract in horses have a genetic component. The clinical expression of equine respiratory diseases with a genetic basis results from complex interactions between the environment and the genetic make-up of each individual horse. The genetic effects are likely to be due to variations in several genes, i.e. they are polygenic. It is therefore unlikely that single gene tests will be diagnostically useful in these disorders. Genetic profiling panels, combining several genetic factors with an assessment of environmental risk factors, may have greater value, but much work is still needed to uncover diagnostically useful genetic markers or even causative variants for equine respiratory diseases. Nonetheless, chromosomal regions associated with guttural pouch tympany, recurrent laryngeal neuropathy and RAO have been identified. The association of RAO with other hypersensitivities and with resistance to intestinal parasites requires further study. This review aims to provide an overview of the available data and current thoughts on the genetics of equine airway diseases.

Characterization of a rat uterine cell line, U(III) cells: prolactin (PRL) expression and endogenous regulation of PRL-dependent genes; estrogen receptor beta, alpha(2)-macroglobulin, and decidual PRL involving the Jak2 and Stat5 pathway.

Decidualization of endometrial stroma in the rat induces the expression and secretion of rat decidual PRL (rdPRL). Recently, we have generated a nontransformed rat uterine stromal cell line (U(III)) that decidualizes spontaneously in culture. In this report, we have established by immunocytochemistry, RT-PCR, Western blot analysis, labeled amino acid incorporation and RIA that these cells express the rat PRL messenger RNA as well as synthesize and secrete PRL. We have also cloned by RT-PCR a 403-bp complementary DNA fragment whose sequence is identical with that of rat pituitary PRL. In addition, U(III) cells express the PRL receptor (PRL-R) long form, all the components involved in the PRL signal transduction pathway, estrogen receptor beta (ER beta) and alpha(2)-macroglobulin (alpha(2)-MG), which are known to be PRL-regulated genes. However, when U(III) cells were treated with PRL, no regulation of these genes was observed. Moreover, in these cells, the PRL signaling components: the tyrosine kinase Jak2 and the transcription factor Stat5 were endogenously phosphorylated and their phosphorylation states were not enhanced in the presence of exogenous PRL. To examine whether the endogenously secreted PRL affects the expression of PRL-regulated genes, U(III) cells were treated with either an anti-PRL receptor antibody or a Jak2 inhibitor, AG490. The anti-PRL receptor antibody decreased alpha(2)-MG expression. AG490 inhibited Jak2 and Stat5 phosphorylation, prevented Stat5 binding to its DNA consensus sequence, and also caused a dose-dependent down-regulation of alpha(2)-MG and ER beta expression. In contrast, AG490 enhanced PRL mRNA levels. In summary, we have established that the U(III) stromal cells of uterine origin produce PRL. Furthermore, we have shown for the first time that decidual PRL may act locally to activate the Jak2/Stat5 pathway and up-regulate important genes involved in decidual growth and placentation.

PRL antiapoptotic effect in the rat decidua involves the PI3K/protein kinase B-mediated inhibition of caspase-3 activity.

During gestation, the uterus undergoes severe changes to accommodate and protect the developing conceptus. In particular, stromal endometrial cells proliferate and differentiate to form the decidual tissue, which produces PRL. Once the conceptus begins to grow, extensive regression by apoptosis take place in the decidua coincident with the loss of the PRL receptor in this tissue. In this report we have established for the first time that PRL, acting through the long form of the PRL receptor and the PI3K pathway, exerts an antiapoptotic effect in rat decidua. We have also shown that protein kinase B phosphorylation on serine 473 as well as its nuclear translocation are stimulated by PRL in decidual cells. Moreover, we have found that caspase-3, a well known effector of apoptosis, becomes expressed and active in the rat decidua just at a time when this tissue undergoes extensive apoptosis. PRL was able to down-regulate both caspase-3 mRNA levels as well as activity. Furthermore, using a protein kinase B dominant-negative expression vector, we provide evidence that PRL inhibition of caspase-3 requires an intact protein kinase B pathway. Finally, we have also found that rat placental lactogen I and II dose-dependently inhibit caspase-3 mRNA, suggesting multiple sources of PRL in the hormonal control of rat decidual regression. In summary, the results of this study have defined an important role for decidual PRL in the normal progress of pregnancy, specifically in the regression and reorganization of the decidua.

Estrogen receptors alpha and beta in rat decidua cells: cell-specific expression and differential regulation by steroid hormones and prolactin.

Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), alpha and beta. In this investigation, we analyzed the expression of decidual ERalpha and ERbeta, studied their regulation by PRL and steroid hormones and examined the ability of decidual ERp to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ERalpha and ERbeta by PRL and ovarian steroid hormones. Whereas PRL, estradiol, and progesterone all increased ERbeta messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ERa. Estradiol had no effect on ERalpha expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ERalpha and had no synergistic effect on ERbeta expression. The use of GG-AD cells, which express only ERbeta, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ERbeta is expressed in addition to ERalpha in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ERalpha, but concomitantly increases the expression of a functional ERbeta that mediates estradiol up-regulation of the decidual progesterone receptor.

The expression of interleukin-6 (IL-6), IL-6 receptor, and gp130-kilodalton glycoprotein in the rat decidua and a decidual cell line: regulation by 17beta-estradiol and prolactin.

The cytokine interleukin 6 (IL-6), a major mediator of immune and acute phase responses of the liver, has been implicated in the termination of pregnancy once expressed in the uterus. This study was undertaken to investigate the expression and regulation of genes encoding IL-6 and IL-6 receptor (IL-6R) in rat decidual tissue. Total RNA obtained from rat decidual tissue on different days of pseudopregnancy was analyzed by RT-PCR using specific primers for IL-6, IL-6R, and 130-kDa glycoprotein (gp130). Ribosomal L19 primers served as an internal control. IL-6R and gp130 were found to be expressed in the decidua throughout development, while no messenger RNA (mRNA) for IL-6 was detected. Interestingly, within several hours of culture, decidual explants acquired the ability to express IL-6. The apparent ability of decidual cells to express IL-6 and its lack of expression in vivo led us to examine whether the IL-6 gene is actively inhibited. Primary decidual cells were cultured in the presence of estradiol, progesterone, or PRL. Progesterone showed no effect, whereas estradiol and PRL reduced the level of IL-6 mRNA expression. To examine the mechanism by which these hormones inhibit IL-6 expression, we used a simian virus 40-transformed decidual cell line (GG-AD), which expresses only estrogen receptor-beta (ERbeta). Like primary decidual cells in culture, GG-AD cells express IL-6, IL-6R, and gp130 mRNA. When cultured in the presence of estradiol (0-100 ng/ml), mRNA for IL-6 and its receptor components were down-regulated in a dose-dependent manner. Estradiol also caused a dose-dependent decrease in IL-6 protein secretion into the culture medium. The inhibitory effect of estradiol on IL-6 mRNA expression was reversed by the antiestrogen ICI-164,384. Similar inhibition of IL-6 and gp130 mRNA expression was observed with PRL treatment. However, PRL had no effect on IL-6R mRNA levels. PRL inhibition of IL-6 expression was totally reversed by tyrphostin AG490, a JAK2 inhibitor. In summary, the results of this investigation indicate that IL-6 expression, which is detrimental to the maintenance of pregnancy, is inhibited in the rat decidual tissue. This inhibition is induced by PRL and estradiol, which down-regulate not only IL-6 expression, but also the expression of IL-6 receptor and signaling proteins. The results also suggest that PRL signaling to the IL-6 gene is mediated through the long form of PRL receptor and involves JAK2 activation, whereas that of estradiol can be transduced by estrogen receptor-beta.