Molecular analysis of three Clostridium difficile strain genomes isolated from pig farm-related samples.

Clostridium difficile is an anaerobic spore-forming bacillus that usually causes gastrointestinal disorders in man and other animal species. Most of the strains isolated from animals are toxigenic being the virulent ribotype (RT) 078 predominant in several animal species. Although C. difficile is pathogenic to both humans and animals, there is no direct evidence of zoonosis. Deep genome sequencing provides sufficient resolution to analyse which strains found in animals might be related to human pathogens. So far, there are only a few fully sequenced genomes of C. difficile strains isolated from domestic and wild animals. Using Illumina technology, we have sequenced the genome of three isolates; a strain isolated from the vagina of a sow (5754), one from rat (Rattus spp) intestinal content (RC10) and a third one isolated from environmental rat faeces (RF17). Both, rat and rat faeces were sampled in fattening pig farms. Our study reveals a close genetic relationship of two of these isolates with the virulent strain M120 (RT078) isolated from a human patient. The analysis of the sequences has revealed the presence of antibiotic resistance genes, mobile elements, including the transposon linked with virulence Tn6164, and the similarity of virulence factors between these isolates and human strains. This is the first study focused on the sequencing of C. difficile genomes obtained from wild animals like rats, which can be considered as potential reservoirs for humans and other animal species. This study can help to understand the genome composition and epidemiology of this bacterium species.

Comparison of autologous bone marrow and adipose tissue derived mesenchymal stem cells, and platelet rich plasma, for treating surgically induced lesions of the equine superficial digital flexor tendon.

Several therapies have been investigated for equine tendinopathies, but satisfactory long term results have not been achieved consistently and a better understanding of the healing mechanism elicited by regenerative therapies is needed. The aim of this study was to assess the separate effects of autologous bone marrow (BM) and adipose tissue (AT) derived mesenchymal stem cells (MSCs), and platelet rich plasma (PRP), for treating lesions induced in the superficial digital flexor tendon (SDFT) of horses. Lesions were created surgically in both SDFTs of the forelimbs of 12 horses and were treated with BM-MSCs (six tendons), AT-MSCs (six tendons) or PRP (six tendons). The remaining six tendons received lactated Ringer's solution as control. Serial ultrasound assessment was performed prior to treatment and at 2, 6, 10, 20 and 45 weeks post-treatment. At 45 weeks, histopathology and gene expression analyses were performed. At week 6, the ultrasound echogenicity score in tendons treated with BM-MSCs suggested earlier improvement, whilst all treatment groups reached the same level at week 10, which was superior to the control group. Collagen orientation scores on histological examination suggested a better outcome in treated tendons. Gene expression was indicative of better tissue regeneration after all treatments, especially for BM-MSCs, as suggested by upregulation of collagen type I, decorin, tenascin and matrix metalloproteinase III mRNA. Considering all findings, a clear beneficial effect was elicited by all treatments compared with the control group. Although differences between treatments were relatively small, BM-MSCs resulted in a better outcome than PRP and AT-MSCs.

Inflammatory response to the administration of mesenchymal stem cells in an equine experimental model: effect of autologous, and single and repeat doses of pooled allogeneic cells in healthy joints.

BACKGROUND: Mesenchymal stem cells (MSCs) transplantation has become a promising therapeutic choice for musculoskeletal injuries. Joint-related disorders are highly prevalent in horses. Therefore, these animals are considered as suitable models for testing MSC-based therapies for these diseases. The aim of this study was to investigate the clinical and inflammatory responses to intra-articular single and repeat dose administration of autologous or of pooled allogeneic MSCs in healthy equine healthy joints. Six horses were intra-articularly injected with a single autologous dose of bone marrow derived MSCs (BM-MSCs) and two separate doses of allogeneic BM-MSCs pooled from several donors. All contralateral joints were injected with Lactated Ringer's Solution (LRS) as the control vehicle. Signs of synovitis and lameness were evaluated at days 0, 1, 2, 3, 5 and 10 after injection. Total protein (TP), white blood cell count (WBC) and neutrophil count (NC) in synovial fluid were also measured at the same time-points. RESULTS: A mild synovial effusion without associated lameness was observed after all BM-MSCs injections. The second allogeneic injection caused the lowest signs of synovitis. Local temperature slightly increased after all BM-MSCs treatments compared to the controls. TP, WBC and NC in synovial fluids also increased during days 1 to 5 after all BM-MSCs injections. Both, clinical and synovial parameters were progressively normalized and by day 10 post-inoculation appeared indistinguishable from controls. CONCLUSIONS: Intra-articular administration of an allogeneic pool of BM-MSCs represents a safe therapeutic strategy to enhance MSCs availability. Importantly, the absence of hypersensitivity response to the second allogeneic BM-MSCs injection validates the use of repeat dose treatments to potentiate the therapeutic benefit of these cells. These results notably contribute to the development of stem cell based therapies for equine and human joint diseases.

Effect of inflammatory environment on equine bone marrow derived mesenchymal stem cells immunogenicity and immunomodulatory properties.

Mesenchymal stem cells (MSCs) are being investigated for the treatment of equine joint diseases because of their regenerative potential. Recently, the focus mainly has addressed to their immunomodulatory capacities. Inflammation plays a central role in joint pathologies, since the release of proinflammatory mediators to the synovial fluid (SF) leads to the activation of enzymatic degradation of the cartilage. MSCs can modulate the local immune environment through direct or paracrine interaction with immune cells, suppressing their proliferation and re-addressing their functions. Proinflammatory molecules can induce MSC immunoregulatory potential, but they could also increase the expression of immunogenic molecules. Studying the effect of inflammatory environment on MSC immunomodulation and immunogenicity profiles is mandatory to improve cellular therapies. The aim of this study was to analyse the response of equine bone marrow MSCs (eBM-MSCs) to three inflammatory conditions. Equine BM-MSCs from three animals were exposed to: (a) 20% allogeneic inflammatory SF (SF); (b) 50 ng/ml of TNFα and IFNγ (CK50) and (c) 20 ng/ml of TNFα and IFNγ (CK20). After 72 h of exposure, expression of immunogenic and immunomodulation-related molecules, including cell-to-cell contact and paracrine signalling molecules, were analysed by RT-qPCR and flow cytometry. The gene expression of adhesion molecules was upregulated whereas MSC migration-related genes were downregulated by all inflammatory conditions tested. CK culture conditions significantly upregulated the expression of COX-2, iNOS, IDO and IL-6. MHC-I gene expression was upregulated by all conditions, whereas MHC-II was upregulated only after CK priming. The expression of CD40 did not significantly change, whereas the ligand, CD40L, was downregulated in CK conditions. Flow cytometry showed an increase in the percentage of positive cells and mean fluorescence intensity (MFI) of the MHC-I and MHC-II molecules at CK50 conditions, supporting the gene expression results. These outcomes reinforce the change of the immunophenotype of the eBM-MSCs according to the surrounding conditions. Inflammatory synovial environment did not lead to significant changes, so the environment found by eBM-MSCs when they are intraarticular administered may not be enough to activate their immunomodulatory potential. CK priming at tested doses enhances the immunoregulatory profile of eBM-MSCs, which may promote a therapeutic benefit. Even if CK priming induced an upregulation of MHC expression, costimulatory molecule expression however was not upregulated, suggesting that immunogenicity might not be increased. This study provides a better understanding about the behaviour of eBM-MSCs inside the inflamed joint and constitutes a first step to improve MSC-based therapies for equine joint diseases.

Donor-derived equine mesenchymal stem cells suppress proliferation of mismatched lymphocytes.

REASONS FOR PERFORMING STUDY: Recently, it has been shown that mesenchymal stem cells (MSCs) do not express the major histocompatibility complex (MHC) II antigen and are able to inhibit proliferation of MHC-mismatched stimulated lymphocytes, enabling their use as in vivo allogeneic transplants. However, prior to clinical application of allo-MSCs, in vitro tests are required to confirm the safety of treatment protocols. OBJECTIVES: To evaluate the immunosuppressive capabilities of equine bone-marrow-derived MSCs (BM-MSCs) on MHC-mismatched lymphocytes. STUDY DESIGN: In vitro experiment. METHODS: Phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) from 3 Thoroughbreds (recipients) were co-cultured with mismatched BM-MSCs from 3 Connemara ponies (donors). Proliferation of lymphocytes was monitored by carboxyfluorescein succinimidyl ester labelling and analysed by flow cytometry. In total, 6 horses were haplotyped using microsatellites to confirm mismatching. Optimisation of the conditions to stimulate Thoroughbred lymphocytes and titration of equine anti-CD4 and anti-CD8 antibodies were performed. Connemara pony and Thoroughbred BM-MSCs were isolated, expanded and characterised by tri-lineage differentiation. Finally, BM-MSCs from both breeds were set up in co-culture at different ratios with stimulated Thoroughbred lymphocytes. Proliferation of CD4(+) and CD8(+) cells was determined by flow cytometry. RESULTS: A high proportion of CD4/CD8 double-positive lymphocytes were found in freshly isolated PBMCs, although this percentage decreased after 4 days of culture. Mismatched BM-MSCs inhibited proliferation of stimulated lymphocytes in a dose-dependent manner, with the greatest suppression occurring at a 1:10 ratio of BM-MSCs to PBMCs. Proliferation of CD4(+) and CD8(+) subpopulations decreased in 1:10 co-culture, with statistical significance in the case of CD8(+) cells, while that of the CD4/CD8 double-positive population was similar to the phytohaemagglutinin control. CONCLUSIONS: The results demonstrate dose-dependent immunosuppression of stimulated lymphocytes by mismatched equine BM-MSCs, supporting their future application in allo-MSC clinical treatments.

The potential of mesenchymal stem cell in prion research.

Scrapie and bovine spongiform encephalopathy are fatal neurodegenerative diseases caused by the accumulation of a misfolded protein (PrP(res)), the pathological form of the cellular prion protein (PrP(C)). For the last decades, prion research has greatly progressed, but many questions need to be solved about prion replication mechanisms, cell toxicity, differences in genetic susceptibility, species barrier or the nature of prion strains. These studies can be developed in murine models of transmissible spongiform encephalopathies, although development of cell models for prion replication and sample titration could reduce economic and timing costs and also serve for basic research and treatment testing. Some murine cell lines can replicate scrapie strains previously adapted in mice and very few show the toxic effects of prion accumulation. Brain cell primary cultures can be more accurate models but are difficult to develop in naturally susceptible species like humans or domestic ruminants. Stem cells can be differentiated into neuron-like cells and be infected by prions. However, the use of embryo stem cells causes ethical problems in humans. Mesenchymal stem cells (MSCs) can be isolated from many adult tissues, including bone marrow, adipose tissue or even peripheral blood. These cells differentiate into neuronal cells, express PrP(C) and can be infected by prions in vitro. In addition, in the last years, these cells are being used to develop therapies for many diseases, including neurodegenerative diseases. We review here the use of cell models in prion research with a special interest in the potential use of MSCs.

Comparative study of equine bone marrow and adipose tissue-derived mesenchymal stromal cells.

REASONS FOR PERFORMING STUDY: Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. OBJECTIVES: To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. METHODS: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT-qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. RESULTS: High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT- and BM-MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM-MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. CONCLUSIONS: Proliferation ability is different in AT-MSCs and BM-MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT-MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. POTENTIAL RELEVANCE: To provide data to better understand AT-MSCs and BM-MSCs behaviour in vitro.

Effect of scrapie on the stability of housekeeping genes.

Scrapie is the archetype of prion diseases, fatal neurodegenerative disorders that affect humans and animals. Gene expression analysis of normal and infected sheep may provide clues to clarify the molecular mechanisms involved in the neuropathology of these diseases. Real time quantitative PCR has become a powerful and accurate technique for examination of transcription patterns in different biological conditions. One of the critical steps in the comparison of transcription profiles is the selection of stable genes for normalization of expression data. In this work, we have investigated the effect of scrapie on the stability of eight commonly used housekeeping genes in the central nervous system of sheep. We found that their stability decreased in scrapie-infected tissues, with the effect of the disease most evident in the medulla oblongata, a highly affected area of the brain stem. The risk of choosing inappropriate housekeeping genes for expression analysis was evaluated. Although the stability of each reference gene was suitable, a wide variation in expression of target genes (BAX and BCL2) was observed when only one or two housekeeping genes were used to normalize. However, reliable results were obtained with a normalization factor based on three reference genes, regardless of their position in a stability ranking.

PRNP haplotype distribution in Moroccan goats.

Susceptibility/resistance to scrapie in sheep and goats is influenced by host prion protein gene (PRNP) genotype. In this study, we report the analysis of prion protein gene polymorphisms in 137 goats of two Moroccan populations: D'man and Chaouni. We found seven previously described amino acid polymorphisms at codons 37, 127, 137, 142, 154, 222 and 240, as well as three known silent mutations. In addition, we identified three new allelic variants: 101R and 139S in D'man goats and 145D in D'man and Chaouni individuals. The high frequency of the resistant allele 154H could offer genetic protection against the disease to the analysed animals. A total of 12 haplotypes and 28 genotypes were found, the distribution of which shows significant differences between both groups. Moreover, haplotype frequencies were compared with bibliographic data showing that the haplotype distribution of PRNP in Moroccan populations is genetically similar to Southern Italian and Greek goats.