Canine colostrum exosomes: characterization and influence on the canine mesenchymal stem cell secretory profile and fibroblast anti-oxidative capacity.

BACKGROUND: Canine colostrum milk (CCM) is a specific secretion of the mammary gland that is fundamental for the survival of the newborn. CCM has many described components (immunoglobulins, proteins or fat), but its small vesicles, named exosomes, are largely unknown. RESULTS: A characterization of CCM exosomes was performed. Exosomes were abundant in CCM and appeared with the characteristic cup-shaped morphology and well-defined round vesicles. The size distribution of exosomes was between 37 and 140 nm, and western blot analysis showed positive expression of specific exosomal markers. Proteomic analysis revealed a total of 826 proteins in exosome cargo. We also found that exosomes modified the proliferation and secretory profiles in canine mesenchymal stem cells derived from bone marrow (cBM-MSCs) and adipose tissue (cAd-MSCs). Additionally, CCM exosomes demonstrated a potent antioxidant effect on canine fibroblasts in culture. CONCLUSIONS: Our findings highlight, for the first time, the abundant presence of exosomes in CCM and their ability to interact with mesenchymal stem cells (MSCs). The addition of exosomes to two types of MSCs in culture resulted in specific secretory profiles with functions related to angiogenesis, migration and chemotaxis of immune cells. In particular, the cAd-MSCs secretory profile showed higher potential in adipose tissue development and neurogenesis, while cBM-MSC production was associated with immunity, cell mobilization and haematopoiesis. Finally, exosomes also presented antioxidant capacity on fibroblasts against reactive oxygen species activity within the cell, demonstrating their fundamental role in the development and maturation of dogs in the early stages of their life.

Safety and efficacy of the mesenchymal stem cell in feline eosinophilic keratitis treatment.

BACKGROUND: Feline eosinophilic keratitis (FEK) is a chronic keratopathy caused by a suspected immune mediated response to an unknown antigenic stimulus. The purpose of this study was to investigate the safety and therapeutic effects of allogeneic feline adipose-derived mesenchymal stromal cells (fAd-MSCs) implanted subconjunctival around the ocular surface lesion in five cats with FEK refractory to current available treatments. RESULTS: FEK was diagnosed by clinical appearance and evidence of eosinophil and/or mast cells in corneal cytology. Each animal was treated with two applications of 2 × 106 million of fAd-MSCs 2 months apart. Ocular surface integrity was assessed before treatment and at 1, 3, 6 and 11 months after treatment. Clinical signs showed a significant change during the follow-up with resolution of the corneal and conjunctiva lesions and there were no signs of regression or worsening. CONCLUSIONS: Implanted cells were well-tolerated and effective reducing clinical signs of FEK with a sustained effect during the study period. None of the animals showed systemic or local complications during the study. To our knowledge, this is the first time in literature that local implantation of allogeneic fAd-MSCs has been found as an effective therapeutic alternative to treat cats with FEK.

Regenerative Therapies in Dry Eye Disease: From Growth Factors to Cell Therapy.

Dry eye syndrome is a complex and insidious pathology with a high level of prevalence among the human population and with a consequently high impact on quality of life and economic cost. Currently, its treatment is symptomatic, mainly based on the control of lubrication and inflammation, with significant limitations. Therefore, the latest research is focused on the development of new biological strategies, with the aim of regenerating affected tissues, or at least restricting the progression of the disease, reducing scar tissue, and maintaining corneal transparency. Therapies range from growth factors and cytokines to the use of different cell sources, in particular mesenchymal stem cells, due to their multipotentiality, trophic, and immunomodulatory properties. We will review the state of the art and the latest advances and results of these promising treatments in this pathology.

Secretory Profile of Adipose-Tissue-Derived Mesenchymal Stem Cells from Cats with Calicivirus-Positive Severe Chronic Gingivostomatitis.

The feline calicivirus (FCV) causes infections in cats all over the world and seems to be related to a broad variety of clinical presentations, such as feline chronic gingivostomatitis (FCGS), a severe oral pathology in cats. Although its etiopathogeny is largely unknown, FCV infection is likely to be a main predisposing factor for developing this pathology. During recent years, new strategies for treating FCGS have been proposed, based on the use of mesenchymal stem cells (MSC) and their regenerative and immunomodulatory properties. The main mechanism of action of MSC seems to be paracrine, due to the secretion of many biomolecules with different biological functions (secretome). Currently, several pathologies in humans have been shown to be related to functional alterations of the patient's MSCs. However, the possible roles that altered MSCs might have in different diseases, including virus-mediated diseases, remain unknown. We have recently demonstrated that the exosomes produced by the adipose-tissue-derived MSCs (fAd-MSCs) from cats suffering from FCV-positive severe and refractory FCGS showed altered protein contents. Based on these findings, the goal of this work was to analyze the proteomic profile of the secretome produced by feline adipose-tissue-derived MSCs (fAd-MSCs) from FCV-positive patients with FCGS, in order to identify differences between them and to increase our knowledge of the etiopathogenesis of this disease. We used high-resolution mass spectrometry and functional enrichment analysis with Gene Ontology to compare the secretomes produced by the fAd-MSCs of healthy and calicivirus-positive FCGS cats. We found that the fAd-MSCs from cats with FCGS had an increased expression of pro-inflammatory cytokines and an altered proteomic profile compared to the secretome produced by cells from healthy cats. These findings help us gain insight on the roles of MSCs and their possible relation to FCGS, and may be useful for selecting specific biomarkers and for identifying new therapeutic targets.

Suicide gene therapy by canine mesenchymal stem cell transduced with thymidine kinase in a u-87 glioblastoma murine model: Secretory profile and antitumor activity.

The role played by certain domestic species such as dogs as a translational model in comparative oncology shows great interest to develop new therapeutic strategies in brain tumors. Gliomas are a therapeutic challenge that represents the most common form of malignant primary brain tumors in humans and the second most common form in dogs. Gene-directed enzyme/prodrug therapy using adipose mesenchymal stem cells (Ad-MSCs) expressing the herpes simplex virus thymidine kinase (TK) has proven to be a promising alternative in glioblastoma therapy, through its capacity to migrate and home to the tumor and delivering local cytotoxicity avoiding other systemic administration. In this study, we demonstrate the possibility for canine Ad-MSCs (cAd-MSCs) to be genetically engineered efficiently with a lentiviral vector to express TK (TK-cAd-MSCs) and in combination with ganciclovir (GCV) prodrug demonstrated its potential antitumor efficacy in vitro and in vivo in a mice model with the human glioblastoma cell line U87. TK-cAd-MSCs maintained cell proliferation, karyotype stability, and MSCs phenotype. Genetic modification significantly affects its secretory profile, both the analyzed soluble factors and exosomes. TK-cAd-MSCs showed a high secretory profile of some active antitumor immune response cytokines and a threefold increase in the amount of secreted exosomes, with changes in their protein cargo. We also found that the prodrug protein is not released directly into the culture medium by TK-cAd-MSCs. We believe that our work provides new perspectives for glioblastoma gene therapy in dogs and a better understanding of this therapy in view of its possible implantation in humans.

Effect of Canine Adipose Mesenchymal Stem Cell Secretome on a Model of Second-Intention Wound Healing in the Red-Eared Slider Turtle (Trachemys scripta).

Mesenchymal stem cell (MSC) secretome refers to a variety of bioactive compounds that represents the more important pathway by which MSCs participate in tissue regeneration. Many of these compounds have shown variable functional activity even across nonmammalian vertebrate species, although MSCs in turtles have not yet been described. Canine adipose MSC secretome has been successfully used experimentally in skin healing. Our aim was to conduct a blinded controlled study to evaluate the effect of canine adipose MSC secretome (cS-MSC) as an alternative for the healing of soft skin, second intention wounds of red-eared slider turtles (Trachemys scripta). Under general anesthesia, one circular, 6-mm full thickness wound was made in each rear leg of 14 females. After randomization, cS-MSC was injected subcutaneously around one wound at days 1, 7, and 14, whereas the other wound acted as control. Biopsies from three animals' wounds were obtained at days 21, 28, 42, and 63. Differences in mean wound retraction at days 21 (n=14) and 28 (n=11) were statistically nonsignificant. The clinical and histopathologic scores performed blind by two different investigators were similar for treated and control wounds. In conclusion, we could not detect a significant functional activity of cS-MSC on wound healing of Trachemys scripta.

Proteomic Analysis of the Secretome and Exosomes of Feline Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been shown to have therapeutic efficacy in different complex pathologies in feline species. This effect is attributed to the secretion of a wide variety of bioactive molecules and extracellular vesicles, such as exosomes, with significant paracrine activity, encompassed under the concept of the secretome. However, at present, the exosomes from feline MSCs have not yet been studied in detail. The objective of this study is to analyze and compare the protein profiles of the secretome as a whole and its exosomal fraction from feline adipose-derived MSCs (fAd-MSCs). For this, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Protein-Protein Interaction Networks Functional Enrichment Analysis (STRING) were utilized. A total of 239 proteins were identified in the secretome, and 228 proteins specific to exosomes were identified, with a total of 133 common proteins. The proteins identified in the secretome were located in the extracellular regions and in the cytoplasm, while the exosomal proteins were located mainly in the membrane, cytoplasm and cytosol. Regarding function, in the secretome, proteins involved in different metabolic pathways, in pathways related to the immune system and the endocrine system and in the processing of proteins in the endoplasmic reticulum predominated. In contrast, proteins specific to exosomes were predominantly associated with endocytosis, cell junctions, platelet activation and other cell signaling pathways. The possible future use of the secretome, or some of its components, such as exosomes, would provide a non-cell-based therapeutic strategy for the treatment of different diseases that would avoid the drawbacks of cell therapy.

Altered Proteomic Profile of Adipose Tissue-Derived Mesenchymal Stem Cell Exosomes from Cats with Severe Chronic Gingivostomatitis.

Feline chronic gingivostomatitis (FCGS) is a pathology with a complicated therapeutic approach and with a prevalence between 0.7 and 12%. Although the etiology of the disease is diverse, feline calicivirus infection is known to be a predisposing factor. To date, the available treatment helps in controlling the disease, but cannot always provide a cure, which leads to a high percentage of refractory animals. Mesenchymal stem cells (MSCs) play a pivotal role in the homeostasis and reparation of different tissues and have the ability to modulate the immune system responses. This ability is, in part, due to the capacity of exosomes to play a part in intercellular cell communication. However, the precise role of MSC-derived exosomes and their alterations in immunocompromised pathologies remains unknown, especially in veterinary patients. The goal of this work was to analyze the proteomic profile of feline adipose tissue-derived MSCs (fAd-MSCs) from calicivirus-positive FCGS patients, and to detect possible modifications of the exosomal cargo, to gain better knowledge of the disease's etiopathogenesis. Using high-resolution mass spectrometry and functional enrichment analysis with Gene Ontology, exosomes isolated from the fAd-MSCs of five healthy cats and five calicivirus-positive FCGS patients, were pooled and compared. The results showed that the fAd-MSCs from cats suffering from FCGS not only had a higher exosome production, but also their exosomes showed significant alterations in their proteomic profile. Eight proteins were exclusively found in the exosomes from the FCGS group, and five proteins could only be found in the exosomes from the healthy cats. When comparing the exosomal cargo between the two groups, significant upregulation of 17 and downregulation of 13 proteins were detected in the FCGS group compared to the control group. These findings shed light on new perspectives on the roles of MSCs and their relation to this disease, which may help in identifying new therapeutic targets and selecting specific biomarkers.

Design, Synthesis, and Biological Evaluation of Hybrid Glypromate Analogues Using 2-Azanorbornane as a Prolyl and Pipecolyl Surrogate.

Neurodegenerative disorders of the central nervous system are a class of heterogeneous pathologies affecting millions of people worldwide and represent a global health burden in developed and developing countries. Without restorative treatments currently available, research on neuroprotective drugs is considered a health priority. In this study, new analogues of the glycyl-l-prolyl-l-glutamic acid (Glypromate) neuropeptide were designed, synthesized, and biologically evaluated using (1R,3S,4S)-2-azanorbornane-3-carboxylic acid as a hybrid construct of l-proline and l-pipecolic acid. Neuroprotection assays carried out in human neuroblastoma SH-SY5Y cells using 6-hydroxydopamine as a stress inducer showed great percentage of recovery (29.7-40.0%) at 100 µM. Among this series, [(1R,3S,4S)-2-glycyl-2-azanorbornane-3-carbonyl]-l-aspartic acid (2a) stands out with a remarkable percentage of recovery (40.0%, at 100 µM) and safe toxicological profile in SH-SY5Y and human adipose mesenchymal stem cells.

Discovery of New Potent Positive Allosteric Modulators of Dopamine D2 Receptors: Insights into the Bioisosteric Replacement of Proline to 3-Furoic Acid in the Melanostatin Neuropeptide.

The control of Parkinson's disease (PD) is challenged by the motor and non-motor fluctuations as well as dyskinesias associated with levodopa long-term therapy. As such, pharmacological alternatives to reduce the reliance on this drug are needed. Melanostatin (MIF-1), a positive allosteric modulator (PAM) of D2 receptors (D2R), is being explored as a novel pharmacological approach focused on D2R potentiation. In this work, 3-furoic acid (3-Fu) was successfully employed as an l-proline (Pro) surrogate, affording two potent MIF-1 analogues, methyl 3-furoyl-l-leucylglycinate (4a) and 3-furoyl-l-leucylglycinamide (6a). In this series, the C-terminal carboxamide moiety was found crucial to enhancing the potency and toxicological profile, yet it is not considered a requisite for the PAM activity. Conformational analysis excludes 4a from adopting the claimed type II ß-turn. The discovery and validation of 6a as a lead compound open a new avenue for the development of a novel class of anti-Parkinson therapeutics targeting the D2R.

Characterization of the secretory profile and exosomes of limbal stem cells in the canine species.

Limbal stem cells (LSCs) are a quiescent cell population responsible for the renewal of the corneal epithelium. Their deficiency is responsible for the conjunctivization of the cornea that is seen in different ocular pathologies, both in humans and in the canine species. The canine species represents an interesting preclinical animal model in ocular surface pathologies. However, the role of LSCs in physiological and pathological conditions in canine species is not well understood. Our objective was to characterize for the first time the soluble factors and the proteomic profile of the secretome and exosomes of canine LSCs (cLSCs). In addition, given the important role that fibroblasts play in the repair of the ocular surface, we evaluated the influence of the secretome and exosomes of cLSCs on their proliferation in vitro. Our results demonstrated a secretory profile of cLSCs with high concentrations of MCP-1, IL-8, VEGF-A, and IL-10, as well as significant production of exosomes. Regarding the proteomic profile, 646 total proteins in the secretome and 356 in exosomes were involved in different biological processes. Functionally, the cLSC secretome showed an inhibitory effect on the proliferation of fibroblasts in vitro, which the exosomes did not. These results open the door to new studies on the possible use of the cLSC secretome or some of its components to treat certain pathologies of the ocular surface in canine species.

Allogeneic adipose-derived mesenchymal stem cell therapy in dogs with refractory atopic dermatitis: clinical efficacy and safety.

Canine atopic dermatitis (AD) is a common skin disease with a 10-15 per cent prevalence. Current treatments vary in their efficacy and safety. The immunomodulatory properties of mesenchymal stem cells (MSCs) make them a promising alternative treatment. The aim of this study was to evaluate the therapeutic efficacy and safety of allogeneic canine adipose MSCs (cAd-MSCs) in dogs with refractory AD. Twenty-six dogs, suffering from AD for at least 12 months, not responding to conventional therapy, received an intravenous dose of 1.5×106 cAd-MSCs/kg bodyweight. Clinical signs, haematological and biochemistry profiles, and AD severity were assessed in a six-month follow-up using a validated scoring system (Canine Atopic Dermatitis Extent and Severity Index, version 4 (CADESI-04)). The degree of pruritus was quantified using a validated visual analogue scale, and also owner's global assessment of treatment efficacy. Twenty-two animals completed the study. Pruritus and CADESI-04 scores decreased significantly after one week or month of treatment, respectively, and remained stable for six months. Owner's global assessment score was 2.15±1.15 for all the animals in the study. In conclusion, systemic administration of allogeneic cAd-MSCs appeared to be a simple therapy with positive outcome in the remission of clinical signs for AD refractory to conventional medications, for at least six months and with no adverse events.

Carbon Quantum Dot Surface-Chemistry-Dependent Ag Release Governs the High Antibacterial Activity of Ag-Metal-Organic Framework Composites.

Nanocomposites and hybrid materials of Ag-1,3,5-benzenetricarboxylic acid metal-organic frameworks (MOFs) with S- and N-carbon quantum dots (CQDs) were synthesized and evaluated for their antibacterial activity against representative Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial strains using the qualitative disk-diffusion approach and the quantitative minimum inhibitory concentration test. The composites and hybrids were found to be nontoxic to living cells. The composite formation fostered a synergistic effect that enhanced their antibacterial activity compared with those of their pristine components. Charge transfer from AgMOF to CQDs facilitated the electrostatic interactions of the composites and hybrids with the bacterial cell membranes. Enhanced bactericidal activity was linked to morphological features (a nanorod-like morphology) and specific surface chemistry. The latter affected the release of silver. Silver on the surface of the MOFs rather than silver in the bulk was found to be important. The destruction of the MOF component in the extracellular environment led to the release of silver ions, which have a high affinity to S compounds of the cell physiology. The formation of metallic silver (Ag°) and silver sulfides (Ag2S) was suggested as essential for the ability of the composites and hybrids to inhibit bacterial growth. To the best of our knowledge, this is the first study that introduces the bactericidal effect of AgMOF-CQDs composites and hybrids.

Glioblastoma Bystander Cell Therapy: Improvements in Treatment and Insights into the Therapy Mechanisms.

A preclinical model of glioblastoma (GB) bystander cell therapy using human adipose mesenchymal stromal cells (hAMSCs) is used to address the issues of cell availability, quality, and feasibility of tumor cure. We show that a fast proliferating variety of hAMSCs expressing thymidine kinase (TK) has therapeutic capacity equivalent to that of TK-expressing hAMSCs and can be used in a multiple-inoculation procedure to reduce GB tumors to a chronically inhibited state. We also show that up to 25% of unmodified hAMSCs can be tolerated in the therapeutic procedure without reducing efficacy. Moreover, mimicking a clinical situation, tumor debulking previous to cell therapy inhibits GB tumor growth. To understand these striking results at a cellular level, we used a bioluminescence imaging strategy and showed that tumor-implanted therapeutic cells do not proliferate, are unaffected by GCV, and spontaneously decrease to a stable level. Moreover, using the CLARITY procedure for tridimensional visualization of fluorescent cells in transparent brains, we find therapeutic cells forming vascular-like structures that often associate with tumor cells. In vitro experiments show that therapeutic cells exposed to GCV produce cytotoxic extracellular vesicles and suggest that a similar mechanism may be responsible for the in vivo therapeutic effectiveness of TK-expressing hAMSCs.