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"Antoine, Nadine"
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Tracking of autologous adipose-derived mesenchymal stromal/stem cells after intravenous administration: a pilot study in a dog with induced acute bladder injury
Regenerative therapy for bladder diseases has been studied in rodent to restore bladder function after a chronic and irreversible bladder wall deterioration. These studies rarely demonstrate the presence of stem cells in the bladder. Cell-tracking after intravenous (IV) administration of stem cells enables to confirm the homing potential of an injury. Our objective was to assess, in one dog, the homing capability of autologous adipose-derived mesenchymal stromal/stem cells (ADMSCs) injected intravenously to an acute bladder injury.
Adipose-derived mesenchymal stromal/stem cells were isolated from the subcutaneous tissue of a dog and labelled. As a homing signal, a full-thickness bladder biopsy representing an acute injury was created in this dog (day 0, control time). Twenty million autologous PKH26-labelled ADMSCs were injected in the cephalic vein on days 1, 4 and 8. Urinalysis was performed (day 5). Bladder biopsy was repeated at the location of the previous scar to assess the presence of labelled ADMSCs in the bladder wall (day 10).
Labelled ADMSCs were observed in the second bladder biopsy, not in the initial biopsy nor in urine. The only adverse event mild, self-limiting hematuria. Complete cell blood count, blood urea nitrogen and plasma creatinine were within normal limits (day 5).
The comparison of bladder biopsies before and after IV administration of autologous ADMSCs indicates that they reached the bladder injury. Our protocol was feasible and safe. Hematuria was probably due to the bladder biopsy. These results could encourage the evaluation of this protocol in larger cohorts of dogs.
Journal Article
Osteopontin and fibronectin in lung tissue, serum, and bronchoalveolar lavage fluid of dogs with idiopathic pulmonary fibrosis and control dogs
Abstract
Background
Idiopathic pulmonary fibrosis (IPF) affects West Highland white terriers (WHWTs). Osteopontin (SPP1) and fibronectin (FN1) are associated with human IPF and are overexpressed by bronchoalveolar lavage fluid (BALF) macrophages in dogs with IPF.
Objective
To investigate the value of these proteins as biomarkers of IPF.
Animals
West Highland white terriers (WHWTs) with IPF, control WHWTs, and terriers.
Methods
Cross-sectional observational study. Immunohistochemistry was used to localize SPP1 and FN1 in lung tissue. Serum and BALF SPP1 and FN1 concentrations were measured using canine ELISA kits and compared between groups.
Results
Osteopontin stained ciliated epithelial cells, smooth muscular cells, and macrophages of all included dogs, and type-II pneumocytes and extracellular matrix of all 12 diseased WHWTs, 4/6 control WHWTs, and none of the 3 terriers. Osteopontin serum concentration was higher in diseased WHWTs (n = 22; 2.15 ng/mL [0.74-5.30]) compared with control WHWTs (n = 13; 0.63 ng/mL [0.41-1.63]; P = .005) and terriers (n = 15; 0.31 ng/mL [0.19-0.51]; P < .0001), and in control WHWTs compared with terriers (P = .005). Osteopontin BALF concentrations were higher in diseased (0.27 ng/mL [0.14-0.43]) and control WHWTs (0.25 ng/mL [0.14-0.40]), compared with terriers (0.02 ng/mL [0.01-0.08]; P < .0001 and P = .003, respectively). Fibronectin (FN1) serum concentrations were lower in diseased dogs (1.03 ng/mL [0.35-1.48]) and control WHWTs (0.61 ng/mL [0.24-0.65]) compared with terriers (2.72 ng/mL [0.15-5.21]; P < .0001 and P = .0001, respectively). There was no difference in FN1 immunostaining and FN1 BALF concentrations between groups.
Conclusions
Results suggest that SPP1 is involved in pathogenesis of IPF and could predispose that breed to the disease. Osteopontin serum concentration could serve as a diagnostic biomarker of IPF.
Journal Article
Longitudinal Pilot Study of Progressive Urethral Sub-Obstruction in a Canine Model: Bladder Functional and Structural Changes and Exploratory Evaluation of Autologous Mesenchymal Stem Cells
Bladder outlet obstruction (BOO) may lead to detrusor decompensation through progressive bladder remodeling. Most experimental studies rely on acutely induced BOO in rodents. Since progressive obstruction better reflects the condition, non-lethal models are needed to investigate chronic obstruction pathophysiology and evaluate regenerative therapies. This exploratory study aimed to evaluate (1) a progressive BOO model induced by an artificial urethral sphincter (AUS) in 2 dogs and (2) the systemic administration of autologous adipose-derived mesenchymal stem cells (ADMSCs) after obstruction release. Two intact male dogs underwent progressive BOO through gradual AUS inflation. Longitudinal assessment included telemetric urodynamic monitoring, urethral pressure profilometry, ultrasonography, post-void residual measurement, oxidative stress markers in serial blood samples and serial bladder biopsies for histology, transmission electron microscopy, immunohistochemistry, RT-qPCR and RNA sequencing (CCL2, CCR2, GFAP, VEGF, HGF). After AUS removal, one dog received three intravenous injections of 20 × 106 PKH26-labelled autologous ADMSCs. BOO induced functional changes (increased detrusor pressure and urethral resistance, decreased urinary flow, prolonged voiding). No detrusor decompensation or fibrosis comparable to the human condition developed, encouraging refinement of this model. ADMSCs appeared to reach the bladder wall safely, but any influence on the glutathione redox system and CCL2 protein expression needs to be confirmed.
Journal Article
Apple Pomace and Performance, Intestinal Morphology and Microbiota of Weaned Piglets—A Weaning Strategy for Gut Health?
Apple pomace (AP) is known to be rich in biomolecules beneficial for health and it may advantageously be used to overcome the critical step of piglets’ weaning. The study aimed to determine the effect of two levels of incorporation of AP on the performance, intestinal morphology, and microbiota of weaned piglets and investigate this feed ingredient as a weaning strategy. An experiment was performed with 42 piglets from weaning (28 days old) over a five-week period, including three iso-energetic and iso-nitrogenous diets (0%, 2%, and 4% dried AP diets) with seven pen-repetitions per diet (two pigs per pen). AP diets were beneficial for the average daily gain calculated on week 3 (p = 0.038) and some parameters of the intestinal architecture on the 35 post-weaning day. The 4% AP diet was beneficial for the feed conversion ratio (p = 0.002) and the energetic feed efficiency (p = 0.004) on the 35 post-weaning day. AP tended to influence the consistency of feces (softer to liquid, p = 0.096) and increased the counts of excreted pathogens (p = 0.072). Four percent AP influenced the richness of the microbiota and the bacteria profile as observed for the phylum Bacteroidetes or the class Clostridia. The 4% AP diet appeared as an interesting weaning strategy that should be evaluated in a large cohort.
Journal Article
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.
Journal Article
Spreading of prions from the immune to the peripheral nervous system: a potential implication of dendritic cells
The implication of dendritic cells (DCs) in the peripheral spreading of prions has increased in the last few years. It has been recently described that DCs can transmit prions to primary neurons from the central nervous system. In order to improve the understanding of the earliest steps of prion peripheral neuroinvasion, we studied, using an in vitro model, the effect of exposing primary peripheral neurons to scrapie-infected lymphoid cells. Thanks to this system, there is evidence that bone marrow dendritic cells (BMDCs) are in connection with neurites of peripheral neurons via cytoplasmic extensions. BMDCs are competent to internalize prions independently from the expression of cellular prion protein (PrPC) and have the capacity to transmit detergent-insoluble, relatively proteinase K-resistant prion protein (PrPSc) to peripheral neurons after 96 h of coculture. Furthermore, we confirmed the special status of the peripheral nervous system in front of prion diseases. Contrary to central neurons, PrPSc infection does not disturb survival and neurite outgrowth. Our model demonstrates that PrPSc-loaded dendritic cells and peripheral nerve fibers that are included in neuroimmune interfaces can initiate and spread prion neuroinvasion.
Journal Article
Oral scrapie infection modifies the homeostasis of Peyer’s patches’ dendritic cells
In transmitted prion diseases the immune system supports the replication and the propagation of the pathogenic agent (PrPSc). DCs, which are mobile cells present in large numbers within lymph organs, are suspected to carry prions through the lymphoid system and to transfer them towards the peripheral nervous system. In this study, C57Bl/6 mice were orally inoculated with PrPSc (scrapie strain 139A) and sacrificed at the preclinical stages of the disease. Immunolabelled cryosections of Peyer's patches were analysed by confocal microscopy. Membrane prion protein expression was studied by flow cytometry. In Peyer's patches (PP), dissected at day one and day 105 after oral exposure to scrapie, we observed an increased population of DCs localised in the follicular-associated epithelium. On day 105, PrPSc was found in the follicles inside the PP of prion-infected mice. A subset of Peyer's patches DCs, which did not express cellular prion protein on their surface in non-infected mice conditions, was prion-positive in scrapie conditions. Within Peyer's patches oral scrapie exposure thus induced modifications of the homeostasis of DCs at the preclinical stages of the disease. These results give new arguments in favour of the implication of DCs in prion diseases.
Journal Article
Serum and bronchoalveolar lavage fluid concentration of osteopontin and fibronectin in West Highland white terriers either affected with canine idiopathic pulmonary fibrosis or healthy and other terriers non predisposed to the disease
Overexpression of osteopontin (SPP1) and fibronectin (FN1), two molecules associated with pulmonary fibrosis in humans and mice, was recently identified in pro-fibrotic bronchoalveolar lavage fluid (BALF) macrophage populations in West Highland white terriers (WHWTs) affected with canine idiopathic pulmonary fibrosis (CIPF) compared to healthy WHWTs. The aims of the present study were to confirm the overexpression of SPP1 and FN1 genes at the protein level and to assess the potential utility of those proteins as serum and BALF biomarkers of CIPF severity.
Serum and BALF concentrations of SPP1 and FN1 were measured using commercially available canine ELISA kits in CIPF WHWTs at diagnosis (n=24), healthy aged-matched WHWTs (n=13) and healthy terriers from other breeds (n=15). Values obtained were compared between groups using Kruskal-Wallis test. Correlations between SPP1 and FN1 concentrations and markers of disease severity (arterial partial pressure in oxygen (PaO2) and 6-minute walked distance (6MWD)) were performed using Spearman test.
SPP1 serum concentrations were higher in CIPF WHWTs (median [interquartile range]: 2.15 ng/mL [0.87-5.13]) compared with healthy WHWTs (0.63 ng/mL [0.41-1.63]; P=0.013) and healthy terriers (0.31 ng/mL [0.19-0.51]; P<0.0001), and higher in healthy WHWTs compared with healthy terriers (P=0.002). Higher SPP1 BALF concentrations were found in CIPF (0.34 ng/mL [0.15-0.52]) and healthy WHWTs (0.25 ng/mL [0.14-0.40]) compared with heathy terriers (0.02 ng/mL [0.01-0.08]; P<0.0001 and P=0.002, respectively), while no difference was shown between CIPF and healthy WHWTs (P=0.962). SPP1 serum concentrations negatively correlated with PaO2 (r=-0.502; P=0.007), but not with 6MWD. FN1 serum concentrations were lower in WHWTs either affected with CIPF or healthy (0.81 ng/mL [0.36-1.47] and 0.61 ng/mL [0.24-0.65]) compared with healthy terriers (2.72 ng/mL [2.15-5.21]; P<0.0001 and P=0.001, respectively). No difference was found between groups in BALF FN1 concentrations (P=0.077). No correlation was identified between serum and BALF FN1 concentrations and parameters of CIPF severity.
Results of the present study did not confirm the FN1 overexpression found in BALF pro-fibrotic macrophages neither in serum nor in BALF. Serum and BALF FN1 concentrations were not associated with CIPF severity parameters. SPP1 gene overexpression was confirmed at the protein level in serum but not in BALF in CIPF compared with healthy WHWTs. Moreover, SPP1 serum concentration could serve as a biomarker of disease severity as it correlates with PaO2. Finally, the higher serum and BALF SPP1 concentrations found in the WHWT breed compared to other terriers could be linked to their predisposition to CIPF.
Conference Proceeding
Guanine nucleotide exchange factor RABGEF1 regulates keratinocyte-intrinsic signaling to maintain skin homeostasis
Epidermal keratinocytes form a structural and immune barrier that is essential for skin homeostasis. However, the mechanisms that regulate epidermal barrier function are incompletely understood. Here we have found that keratinocyte-specific deletion of the gene encoding RAB guanine nucleotide exchange factor 1 (RABGEF1, also known as RABEX-5) severely impairs epidermal barrier function in mice and induces an allergic cutaneous and systemic phenotype. RABGEF1-deficient keratinocytes exhibited aberrant activation of the intrinsic IL-1R/MYD88/NF-κB signaling pathway and MYD88-dependent abnormalities in expression of structural proteins that contribute to skin barrier function. Moreover, ablation of MYD88 signaling in RABGEF1-deficient keratinocytes or deletion of Il1r1 restored skin homeostasis and prevented development of skin inflammation. We further demonstrated that epidermal RABGEF1 expression is reduced in skin lesions of humans diagnosed with either atopic dermatitis or allergic contact dermatitis as well as in an inducible mouse model of allergic dermatitis. Our findings reveal a key role for RABGEF1 in dampening keratinocyte-intrinsic MYD88 signaling and sustaining epidermal barrier function in mice, and suggest that dysregulation of RABGEF1 expression may contribute to epidermal barrier dysfunction in allergic skin disorders in mice and humans. Thus, RABGEF1-mediated regulation of IL-1R/MYD88 signaling might represent a potential therapeutic target.
Journal Article