Mitochondrial function is altered in horse atypical myopathy.

Equine atypical myopathy in Europe is a fatal rhabdomyolysis syndrome that results from the ingestion of hypoglycin A contained in seeds and seedlings of Acer pseudoplatanus (sycamore maple). Acylcarnitine concentrations in serum and muscle OXPHOS capacity were determined in 15 atypical myopathy cases. All but one acylcarnitine were out of reference range and mitochondrial respiratory capacity was severely decreased up to 49% as compared to 10 healthy controls. The hallmark of atypical myopathy thus consists of a severe alteration in the energy metabolism including a severe impairment in muscle mitochondrial respiration that could contribute to its high death rate.

Tissues from equine cadaver ligaments up to 72 hours of post-mortem: a promising reservoir of stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) harvested from cadaveric tissues represent a promising approach for regenerative medicine. To date, no study has investigated whether viable MSCs could survive in cadaveric tissues from tendon or ligament up to 72 hours of post-mortem. The purpose of the present work was to find out if viable MSCs could survive in cadaveric tissues from adult equine ligaments up to 72 hours of post-mortem, and to assess their ability (i) to remain in an undifferentiated state and (ii) to divide and proliferate in the absence of any specific stimulus. METHODS: MSCs were isolated from equine cadaver (EC) suspensory ligaments within 48-72 hours of post-mortem. They were evaluated for viability, proliferation, capacity for tri-lineage differentiation, expression of cell surface markers (CD90, CD105, CD73, CD45), pluripotent transcription factor (OCT-4), stage-specific embryonic antigen-1 (SSEA-1), neuron-specific class III beta-tubulin (TUJ-1), and glial fibrillary acidic protein (GFAP). As well, they were characterized by transmission electron microscope (TEM). RESULTS: EC-MSCs were successfully isolated and maintained for 20 passages with high cell viability and proliferation. Phase contrast microscopy revealed that cells with fibroblast-like appearance were predominant in the culture. Differentiation assays proved that EC-MSCs are able to differentiate towards mesodermal lineages (osteogenic, adipogenic, chondrogenic). Flow cytometry analysis demonstrated that EC-MSCs expressed CD90, CD105, and CD73, while being negative for the leukocyte common antigen CD45. Immunofluorescence analysis showed a high percentage of positive cells for OCT-4 and SSEA-1. Surprisingly, in absence of any stimuli, some adherent cells closely resembling neuronal and glial morphology were also observed. Interestingly, our results revealed that approximately 15 % of the cell populations were TUJ-1 positive, whereas GFAP expression was detected in only a few cells. Furthermore, TEM analysis confirmed the stemness of EC-MSCs and identified some cells with a typical neuronal morphology. CONCLUSIONS: Our findings raise the prospect that the tissues harvested from equine ligaments up to 72 hours of post-mortem represent an available reservoir of specific stem cells. EC-MSCs could be a promising alternative source for tissue engineering and stem cell therapy in equine medicine.

Quantification of hypoglycin A in serum using aTRAQ(®) assay.

BACKGROUND: Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine's biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication. METHODS: Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition. RESULTS: Outlined accuracy profiles demonstrated very reliable data. A good linearity was observed from 0.09 to 50µmol/L and precision was very good with coefficient of variation below 8%. Fifty-five samples collected from 25 confirmed AM horses revealed significant hypoglycin A concentrations, while toxin was not found in serum of 8 control animals. CONCLUSIONS: The described aTRAQ variant method has been analytically and clinically validated. The reliability of our approach is thus demonstrated into the workup of atypical myopathy.

Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells.

BACKGROUND: The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values. RESULTS: The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given. CONCLUSION: Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction.

Contribution of MX dynamin, oligoadenylate synthetase, and protein kinase R to anti-paramyxovirus activity of type 1 interferons in vitro.

OBJECTIVE: To determine the contribution of MX dynamin, oligoadenylate synthetase (OAS), and double-stranded RNA-dependent protein kinase R (PKR) to the antiviral effects of type 1 interferons (IFNs) against bovine parainfluenza-3 virus (PI-3V) infection of Vero cells. SAMPLE POPULATION: Vero cell cultures. PROCEDURES: PI-3V yield was first compared between control and transfected type 1 IFNs-incompetent Vero cells expressing recombinant OAS or MX proteins. Afterwards, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha) was used to scale the degree of PKR activation upon infection of Vero cells by PI-3V. RESULTS: Overexpression of OAS did not result in significantly decreased viral replication. Phosphorylated eIF2alpha forms, the hallmark of PKR activation, were not increased in IFNalpha-primed infected Vero cells. Although human MXA contributed to partial blockade of replication of bovine PI-3V, the antiviral effect was not as strong as that of IFNalpha. CONCLUSIONS AND CLINICAL RELEVANCE: The powerful anti-Paramyxovirus activity of type 1 IFNs is mediated by noncanonic pathways.

Molecular characterisation of the caprine (Capra hircus) lymphocyte function-associated antigen-1 alpha subunit-encoding cDNA.

BACKGROUND: Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alpha L beta 2) is required for many cellular adhesive interactions during the immune response. METHODS: We used SMART RACE technology to obtain caprine CD11a 5'- and 3'-ends and RT-PCR to amplify the full-length CDS. RESULTS: The Capra hircus CD11a-encoding cDNA was sequenced and compared with its human, murine, rat, bovine and ovine counterparts. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues. CONCLUSION: Therefore, along with the caprine CD18-encoding cDNA, which has been available for a few months, the sequence data revealed here will allow the Capra hircus LFA-1 expression in vitro as a tool to explore the specificities of inflammation in the caprine species.

Resistance of paramyxoviridae to type I interferon-induced Bos taurus Mx1 dynamin.

Typical targets of type I interferon (IFN)-induced antiviral Mx proteins known to date have been shown to share a common profile: single-stranded negative-sense RNA viruses. Among them, human MxA is known to interfere with the replication of measles, human, and bovine parainfluenza-3 viruses (BoPi3V), that is, three members of the Paramyxoviridae family. Recently, bovine Mx1 protein (BoMx1) was included in the group of Mx proteins with authenticated antiviral potential, as it dramatically represses the replication of vesicular stomatitis virus (VSV). As replication in bovine cells of Pi3, respiratory syncytial (RS), and Sendai (Se) viruses, all members of the same family, is known to be reduced on IFN-alpha incorporation into the culture medium, it was hypothesized that the BoMx1 pathway possibly was involved, its antiviral spectrum thus probably extending to Paramyxoviridae. In this study, probing of BoMx1-inhibiting effects was carried out by infecting a transgenic Vero cell line that allows tightly regulated conditional expression of BoMx1 after doxycycline treatment with a wide array of Paramyxoviridae. Expressing and nonexpressing cells displayed similar viability, cytopathic effects (CPEs), and amounts of infectious virus yields, whatever the infecting virus or the multiplicity of infection (moi) imposed. It is, therefore, concluded that BoMx1 does not interfere with Paramyxoviridae.

Conditional expression of type I interferon-induced bovine Mx1 GTPase in a stable transgenic vero cell line interferes with replication of vesicular stomatitis virus.

In some vertebrate species, type I interferon(IFN)-induced Mx gene expression has been shown to confer resistance to some single-stranded RNA (ssRNA) viruses in vitro. Because the bovine species is subject to an exceptionally wide array of infections caused by such viruses, it is anticipated that an antiviral allele should have been retained by evolution at the bovine Mx locus. The identification of such allele may help in evaluating the real significance of the Mx genotype for disease resistance in vivo, in deciphering host-virus molecular interactions involved, or in improving innate disease resistance of livestock through marker-assisted selection. We validated a double transgenic Vero cell clone in which the bovine Mx1 reference allele is placed under control of the human cytomegalovirus (CMV) enhancer-promoter sequence containing elements from the bacterial tetracycline resistance operon to regulate transcription. In the selected clone, transgene repression was very tight, and derepression by doxycycline led to homogeneous 48-h duration expression of physiologic levels of bovine Mx1. Expression of the transgene caused a dramatic decrease in cytopathic efficiency and a 500-5000-fold yield reduction of the Indiana and New Jersey serotypes of vesicular stomatitis virus (VSV). To our knowledge, the transgenic clone developed here is the first ever reported that allows conditional expression of an Mx protein, thus providing a valuable tool for studying functions of Mx proteins in general and that of bovine Mx1 in particular. This latter may henceforward be included in the group of Mx proteins with authenticated anti-VSV activity, which offers new research avenues into the field of host-virus interactions.

Genomic structure, organisation, and promoter analysis of the bovine (Bos taurus) Mx1 gene.

Some MX proteins are known to confer a specific resistance against a panel of single-stranded RNA viruses. Many diseases due to such viruses are known to affect cattle worldwide, raising the possibility that the identification of an antiviral isoform of a bovine MX protein would allow the implementation of genetic selection programs aimed at improving innate resistance of cattle. With this potential application in mind, the present study was designed to isolate the bovine Mx1 gene including its promoter region and to investigate its genomic organisation and promoter reactivity. The bovine Mx1 gene is made up of 15 exons. All exon-intron boundaries conformed to the consensus sequences. A PCR product that contained a approximately 1-kb, 5'-flanking region upstream from the putative transcription start site was sequenced. Unexpectedly, this DNA region did not contain TATA or CCAAT motifs. A computer scan of the region disclosed a series of putative binding sites for known cytokines and transcription factors. There was a GAAAN(1-2)GAAA(C/G) motif, typical of an interferon-sensitive responsive element, between -118 and -107 from the putative transcription start site. There were also a NF-kappaB, two interleukin-6 binding sites, two Sp1 sites and five GC-rich boxes. The region also contained 12 stretches of the GAAA type, as described in all IFN-inducible genes. Bovine Mx1 expression was assessed by Northern blotting and immunofluorescence in the Madin Darby bovine kidney cells (MDBK) cell line treated with several stimuli. In conclusion, the bovine Mx1 gene and promoter region share the major structural and functional characteristics displayed by their homologs described in the rainbow trout, chicken, mouse and man.