Low-dose xenogeneic mesenchymal stem cells target canine osteoarthritis through systemic immunomodulation and homing.

BACKGROUND: As current therapies for canine osteoarthritis (OA) provide mainly symptomatic improvement and fail to address the complex pathology of the disease, mesenchymal stem cells (MSCs) offer a promising biological approach to address both aspects of OA through their immunomodulatory properties. METHODS: This study aimed to investigate the safety and efficacy of xenogeneic MSCs in dogs with OA at different dose levels after intravenous injection. OA was surgically induced in the right stifle joint. Thirty-two male and female dogs were divided into three treatment groups and a control group. Regular general physical examinations; lameness, joint, radiographic, and animal caretaker assessments; pressure plate analyses; and blood analyses were performed over 42 days. At study end, joint tissues were evaluated regarding gross pathology, histopathology, and immunohistochemistry. In a follow-up study, the biodistribution of intravenously injected 99mTc-labeled equine peripheral blood-derived MSCs was evaluated over 24h in three dogs after the cruciate ligament section. RESULTS: The dose determination study showed the systemic administration of ePB-MSCs in a canine OA model resulted in an analgesic, anti-inflammatory, and joint tissue protective effect associated with improved clinical signs and improved cartilage structure, as well as a good safety profile. Furthermore, a clear dose effect was found with 0.3 × 106 ePB-MSCs as the most effective dose. In addition, this treatment was demonstrated to home specifically towards the injury zone in a biodistribution study. CONCLUSION: This model-based study is the first to confirm the efficacy and safety of systemically administered xenogeneic MSCs in dogs with OA. The systemic administration of a low dose of xenogeneic MSCs could offer a widely accessible, safe, and efficacious treatment to address the complex pathology of canine OA and potentially slow down the disease progression by its joint tissue protective effect.

The quorum sensing peptide EntF* promotes colorectal cancer metastasis in mice: a new factor in the host-microbiome interaction.

BACKGROUND: Colorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide association analyses of healthy individuals and cancer patients suggest a role for the human intestinal microbiota in tumor progression. However, the microbial molecules involved in host-microbe communication are largely unknown, with current studies mainly focusing on short-chain fatty acids and amino acid metabolites as potential mediators. Quorum sensing peptides are not yet considered in this context since their presence in vivo and their ability to affect host cells have not been reported so far. RESULTS: Here, we show that EntF*, a metabolite of the quorum sensing peptide EntF produced by Enterococcus faecium, is naturally present in mice bloodstream. Moreover, by using an orthotopic mouse model, we show that EntF* promotes colorectal cancer metastasis in vivo, with metastatic lesions in liver and lung tissues. In vitro tests suggest that EntF* regulates E-cadherin expression and consequently the epithelial-mesenchymal transition, via the CXCR4 receptor. In addition, alanine-scanning analysis indicates that the first, second, sixth, and tenth amino acid of EntF* are critical for epithelial-mesenchymal transition and tumor metastasis. CONCLUSION: Our work identifies a new class of molecules, quorum sensing peptides, as potential regulators of host-microbe interactions. We prove, for the first time, the presence of a selected quorum sensing peptide metabolite in a mouse model, and we demonstrate its effects on colorectal cancer metastasis. We believe that our work represents a starting point for future investigations on the role of microbiome in colorectal cancer metastasis and for the development of novel bio-therapeutics in other disease areas.

Scintigraphic tracking of 99mTechnetium-labelled equine peripheral blood-derived mesenchymal stem cells after intravenous, intramuscular, and subcutaneous injection in healthy dogs.

BACKGROUND: Mesenchymal stem cell treatments in dogs have been investigated as a potential innovative alternative to current conventional therapies for a variety of conditions. So far, the precise mode of action of the MSCs has yet to be determined. The aim of this study was to gain more insights into the pharmacokinetics of MSCs by evaluating their biodistribution in healthy dogs after different injection routes. METHODS: Three different studies were performed in healthy dogs to evaluate the biodistribution pattern of radiolabelled equine peripheral blood-derived mesenchymal stem cells following intravenous, intramuscular and subcutaneous administration in comparison with free 99mTechnetium. The labelling of the equine peripheral blood-derived mesenchymal stem cells was performed using stannous chloride as a reducing agent. Whole-body scans were obtained using a gamma camera during a 24-h follow-up. RESULTS: The labelling efficiency ranged between 59.58 and 83.82%. Free 99mTechnetium accumulation was predominantly observed in the stomach, thyroid, bladder and salivary glands, while following intravenous injection, the 99mTechnetium-labelled equine peripheral blood-derived mesenchymal stem cells majorly accumulated in the liver throughout the follow-up period. After intramuscular and subcutaneous injection, the injected dose percentage remained very high at the injection site. CONCLUSIONS: A distinct difference was noted in the biodistribution pattern of the radiolabelled equine peripheral blood-derived mesenchymal stem cells compared to free 99mTechnetium indicating equine peripheral blood-derived mesenchymal stem cells have a specific pharmacokinetic pattern after systemic administration in healthy dogs. Furthermore, the biodistribution pattern of the used xenogeneic equine peripheral blood-derived mesenchymal stem cells appeared to be different from previously reported experiments using different sources of mesenchymal stem cells.

Safety and immunomodulatory properties of equine peripheral blood-derived mesenchymal stem cells in healthy cats.

OBJECTIVE: Due to the immunomodulatory properties of mesenchymal stem cells (MSCs) through stimulation of endogenous immune cells by paracrine signals and cell contact, they have been proposed as alternative treatment option for many inflammatory and immune-mediated diseases in veterinary medicine. However, the long-term cultivation possibilities of feline MSCs are currently compromised due to a restricted proliferation capacity. Therefore, the xenogeneic use of equine peripheral blood-derived MSCs (ePB-MSCs) would present an interesting alternative thanks to their superior cultivation properties. To the authors' knowledge, there are currently no safety reports concerning the xenogeneic use of ePB-MSCs in cats. Therefore, the overall goal of this preliminary study was to investigate if ePB-MSCs can safely be administered in healthy cats and by extension evaluating their immunogenic and immunomodulatory properties. METHODS: Ten healthy cats were intravenously (i.v.) injected with 3 × 105 ePB-MSCs at three time points (T0, T1, T2). All cats were daily inspected by the caretaker and underwent a physical examination with hematological and biochemical analysis at day 0 (T0), week 2 (T1), week 4 (T2) and week 6 (T3) by a veterinarian. Furthermore, a modified mixed lymphocyte reaction (MLR) was performed at T0 and T3 for each cat in order to evaluate immunogenic and immunomodulatory properties of the ePB-MSCs RESULTS: No adverse clinical effects could be detected following repeated i.v. administration of ePB-MSCs in all cats. Significant lower protein (T1: P-value = 0.002; T2: P-value > 0.001; T3: P-value = 0.004) and albumin levels (T1: P-value = 0.003; T2: P-value = 0.001) were seen after repeated administration of ePB-MSCs, compared to T0. However, all biochemical and hematological parameters stayed within clinical acceptance level. In addition, the repeated injections did not induce a cellular immune response before and after repeated ePB-MSCs administration. Furthermore, convincing immunomodulatory properties of ePB-MSCs on feline peripheral blood mononuclear cells were confirmed in the MLR-assay CONCLUSION: This preliminary study demonstrates that ePB-MSCs can safely be administered in healthy cats and provide a promising alternative for the treatment of various inflammatory diseases in cats.

Blood-brain barrier transport kinetics of NOTA-modified proteins: the somatropin case.

BACKGROUND: Chemical modifications such as PEG, polyamine and radiolabeling on proteins can alter their pharmacokinetic behavior and their blood-brain barrier (BBB) transport characteristics. NOTA, i.e. 1,4,7-triazacyclononane-1,4,7-triacetic acid, is a bifunctional chelating agent that has attracted the interest of the scientific community for its high complexation constant with metals like gallium. Until now, the comparative BBB transport characteristics of NOTA-modified proteins versus unmodified proteins are not yet described. METHODS: Somatropin (i.e. recombinant human growth hormone), NOTA-conjugated somatropin and gallium-labelled NOTA-conjugated somatropin were investigated for their brain penetration characteristics (multiple time regression and capillary depletion [CD]) in an in vivo mice model to determine the blood-brain transfer properties. RESULTS: The three compounds showed comparable initial brain influx, with Kin=0.38±0.14 µL/(g×min), 0.36±0.16 µL/(g×min) and 0.28±0.18 µL/(g×min), respectively. CD indicated that more than 80% of the influxed compounds reached the brain parenchyma. All three compounds were in vivo stable in serum and brain during the time frame of the experiments. CONCLUSIONS: Our results show that modification of NOTA as well as gallium chelation onto proteins, in casu somatropin, does not lead to a significantly changed pharmacokinetic profile at the blood-brain barrier.

Detection and quantification of Enterococcus faecalis RNPP-type quorum sensing peptides in bacterial culture media by UHPLC-MS.

Bacteria communicate with each other using quorum sensing; i.e. the production and sensing of signalling molecules. Enterococcus faecalis, a Gram-positive bacterium, employs peptides as quorum sensing molecules. These peptides have previously been isolated from culture media by elaborate, time and medium-consuming sample preparation approaches and specific bacteria-based bio-sensors. Here, a method for the detection and quantification of all nine currently reported E. faecalis quorum sensing peptides belonging to the RNPP family in bacterial cell culture medium was developed. The approach developed consists of solid-phase extraction (SPE) sample preparation followed by a UHPLC-triple quadrupole mass spectroscopic method, operated in Multiple Reaction Monitoring (MRM) mode. All nine peptides were quantified with a total analysis time below 90 min per sample and limited cell culture medium volumes of only 1 ml per sample. A method verification, performed in uniplicate, was carried out to obtain an idea of the method performance. The recovery varied between 19.9 and 119.0%, and the limit of detection is in the low nM range. Analytical stability, carry-over and dilution integrity were investigated and were acceptable. This method will be a useful tool in the investigation of the roles of the RNPP-type quorum sensing peptides in microbial processes.