Publications by authors named "Lyndah Chow"

22 Publications

  • Page 1 of 1

Intra-articular administration of antibiotics in horses: Justifications, risks, reconsideration of use and outcomes.

Equine Vet J 2021 Aug 30. Epub 2021 Aug 30.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.

Antibiotics have been injected intra-articularly by equine veterinarians for decades, either prophylactically when other drugs are administered for osteoarthritis or therapeutically to treat septic arthritis. This route of administration has also more recently gained attention in human orthopaedic clinical practice, particularly as an alternative to systemic antibiotic administration to treat infections following prosthetic arthroplasty. While the rationale for injecting antibiotics intra-articularly has been largely focused on achieving high local drug concentrations, there has been relatively little focus on pharmacokinetic parameters of antibiotics administered by this route, or on the potential for local toxicity. The increasing incidence of antibiotic resistance in veterinary and human medicine prompts reconsideration of off-label antibiotic usage and evaluation of evidence-based dosing strategies. The purpose of this review was to summarise the current literature describing intra-articular antibiotic usage, including specific studies where pharmacokinetics, potential safety and toxicity have been evaluated. This review will advance practitioners' understanding of the use of intra-articularly administered antibiotics, including the overall pros and cons of the approach.
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http://dx.doi.org/10.1111/evj.13502DOI Listing
August 2021

Enthesis trauma as a means for the development of translatable chronic rotator cuff degeneration in an ovine model.

Ann Transl Med 2021 May;9(9):741

Orthopaedic Bioengineering Research Laboratory, Colorado State University, Fort Collins, CO, USA.

Background: Untreated rotator cuff tears lead to irreversible tendon degeneration, resulting in unacceptable repair prognosis. The inability of current animal models of degenerated rotator cuff tendons to more fully emulate the manifestation and degree of pathology seen in humans with a previously torn rotator cuff tendon (s) significantly impairs the development of novel therapeutics. Therefore, the objective of this study was to develop a large-animal translational model of enthesis damage to the rotator cuff tendons to mimic the chronic degenerative changes that occur in patients that demonstrate clinical manifestations of tendinopathy.

Methods: A partial enthesis tear model (i.e., sharp transection) in adult sheep was created by cutting the tendon fibers perpendicularly through the enthesis midpoint, while leaving the other portion of the tendon in-tact. To assess tendon integrity, non-destructive biomechanical tests were performed, followed by histopathological, histomorphological, and gene expression analysis. Samples of degenerated human rotator cuff tendons obtained from patients undergoing reverse total shoulder arthroplasty to use for comparative pathological analysis.

Results: In the sheep model, transected tendons at all timepoints had significantly decreased mechanical properties. Histopathologic evaluation and Bonar scoring revealed that the tendons in sheep underwent degenerative changes similar in magnitude and manifestation as the degenerated human tendon samples. Furthermore, similar levels of collagen disorganization were noted between the 6 and 12-week ovine samples and the degenerated human samples.

Conclusions: These findings indicate that the new sheep model of rotator cuff injury reliably recapitulates the structural and cellular changes that occur clinically in humans with chronic rotator cuff tendon injuries and suggest that this new model is well suited to evaluation of new therapeutic interventions.
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http://dx.doi.org/10.21037/atm-21-354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8246224PMC
May 2021

Evaluation of Intra-Articular Amikacin Administration in an Equine Non-inflammatory Joint Model to Identify Effective Bactericidal Concentrations While Minimizing Cytotoxicity.

Front Vet Sci 2021 21;8:676774. Epub 2021 May 21.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, United States.

Septic arthritis causes significant morbidity and mortality in veterinary and human clinical practice and is increasingly complicated by multidrug-resistant infections. Intra-articular (IA) antibiotic administration achieves high local drug concentrations but is considered off-label usage, and appropriate doses have not been defined. Using an equine joint model, we investigated the effects of amikacin injected at three different doses (500, 125, and 31.25 mg) on the immune and cartilage responses in tibiotarsal joints. Synovial fluid (SF) was sampled at multiple time points over 24 h, the cell counts determined, and amikacin concentrations measured by liquid chromatography-mass spectrometry. Cytokine concentrations and collagen degradation products in SF were measured by ELISA and multiplex immunoassays. The mean amikacin concentrations in SF were greater than or equal to the minimum inhibitory concentration (MIC) (0.004 mg/ml) for most common equine joint pathogens at all time points tested to 24 h for all three amikacin doses evaluated. The inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) increased significantly in SF in the highest amikacin dose group, despite the fact that increases in SF cell counts were not observed. Similarly, the biomarkers of cartilage type II collagen cleavage (C2C and C12C) were increased in SF following amikacin injection. Mechanistically, we further demonstrated using studies that chondrocytes and synoviocytes killed by exposure to amikacin underwent apoptotic cell death and were phagocytosed by macrophages in a non-inflammatory process resembling efferocytosis. Neutrophils and T cells were susceptible to amikacin cytotoxicity at clinically relevant doses, which may result in blunting of cellular inflammatory responses in SF and account for the lack of increase in total nucleated cell counts following amikacin injection. In summary, decisions on whether to inject cytotoxic antibiotics such as aminoglycosides intra-articularly and what doses to use should take into account the potential harm that antibiotics may cause and consider lower doses than those previously reported in equine practice.
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http://dx.doi.org/10.3389/fvets.2021.676774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8175670PMC
May 2021

Impact of Three Different Serum Sources on Functional Properties of Equine Mesenchymal Stromal Cells.

Front Vet Sci 2021 30;8:634064. Epub 2021 Apr 30.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, United States.

Culture and expansion of equine mesenchymal stromal cells (MSCs) are routinely performed using fetal bovine serum (FBS) as a source of growth factors, nutrients, and extracellular matrix proteins. However, the desire to minimize introduction of xenogeneic bovine proteins or pathogens and to standardize cellular products intended for clinical application has driven evaluation of alternatives to FBS. Replacement of FBS in culture for several days before administration has been proposed to reduce antigenicity and potentially prolong survival after injection. However, the functional consequences of MSC culture in different serum types have not been fully evaluated. The objective of this study was to compare the immunomodulatory and antibacterial properties of MSCs cultured in three serum sources: FBS or autologous or allogeneic equine serum. We hypothesized that continuous culture in FBS would generate MSCs with improved functionality compared to equine serum and that there would not be important differences between MSCs cultured in autologous vs. allogeneic equine serum. To address these questions, MSCs from three healthy donor horses were expanded in medium with FBS and then switched to culture in FBS or autologous or allogeneic equine serum for 72 h. The impact of this 72-h culture period in different sera on cell viability, cell doubling time, cell morphology, bactericidal capability, chondrogenic differentiation, and production of cytokines and antimicrobial peptides was assessed. Altering serum source did not affect cell viability or morphology. However, cells cultured in FBS had shorter cell doubling times and secreted more interleukin 4 (IL-4), IL-5, IL-17, RANTES, granulocyte-macrophage colony-stimulating factor, fibroblast growth factor 2, eotaxin, and antimicrobial peptide cathelicidin/LL-37 than cells cultured in either source of equine serum. Cells cultured in FBS also exhibited greater spontaneous bactericidal activity. Notably, significant differences in any of these parameters were not observed when autologous vs. allogeneic equine serum was used for cell culture. Chondrogenic differentiation was not different between different serum sources. These results indicate that MSC culture in FBS will generate more functional cells based on a number of parameters and that the theoretical risks of FBS use in MSC culture should be weighed against the loss of MSC function likely to be incurred from culture in equine serum.
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http://dx.doi.org/10.3389/fvets.2021.634064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8119767PMC
April 2021

Toll-like receptor activation of equine mesenchymal stromal cells to enhance antibacterial activity and immunomodulatory cytokine secretion.

Vet Surg 2021 May 2;50(4):858-871. Epub 2021 Apr 2.

Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Colorado, USA.

Objective: To evaluate effects of Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor (TLR, NLR) ligand stimulation of equine mesenchymal stromal cells (MSCs) on antibacterial and immunomodulatory properties in vitro.

Study Design: Controlled laboratory study.

Sample Population: Equine bone-marrow-derived MSCs (three horses).

Methods: MSCs were stimulated with TLR (polyinosinic:polycytidylic acid [pIC] and lipopolysaccharide [LPS]) and NLR agonists (γ-d-Glu-mDAP [IE-DAP]) for 2 h, and plated at 1 × 10 cells/well 24 h. MSC-conditioned media (MSC-CM) were collected and assessed for antimicrobial peptide cathelicidin/LL-37 production, bactericidal action against multidrug-resistant planktonic and biofilm Staphylococcus aureus and neutrophil phagocytosis. Bacterial growth was measured by plating bacteria and counting viable colonies, reading culture absorbance, and live-dead staining with confocal microscopy imaging. Following initial comparison of activating stimuli, TLR3-agonist pIC protocols (cell density during activation and plating, culture time, %serum) were further optimized for bactericidal activity and secretion of interleukin-8 (IL-8), monocyte-chemoattractant-protein (MCP-1), and cathelicidin/LL37.

Results: MSCs stimulation with pIC (p = .004) and IE-DAP (p = .03) promoted increased bactericidal activity, evidenced by reduced viable planktonic colony counts. PIC stimulation (2 × 10 cells/ml, 2 h, 10 μg/ml) further suppressed biofilm formation (p = .001), enhanced neutrophil bacterial phagocytosis (p = .009), increased MCP-1 secretion (p < .0001), and enhanced cathelicidin/LL-37 production, which was apparent when serum concentration in media was reduced to 1% (p = .01) and 2.5% (p = .05).

Conclusion: TLR-3 pIC MSCs activation was most effective to enhance antibacterial and cytokine responses, which were affected by serum reduction.

Clinical Significance: In vitro TLR-3 activation of equine MSCs tested here may be a strategy to improve antibacterial properties of MSCs to treat antibiotic-resistant infections.
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http://dx.doi.org/10.1111/vsu.13628DOI Listing
May 2021

Susceptibility of canine chondrocytes and synoviocytes to antibiotic cytotoxicity in vitro.

Vet Surg 2021 Apr 19;50(3):650-658. Epub 2021 Feb 19.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Colorado.

Objective: To evaluate relative cytotoxicity of antibiotics to normal canine joint tissues in vitro.

Study Design: Experimental in vitro study.

Sample Population: Chondrocytes and synoviocytes (three dogs); cartilage explants (three dogs); six dogs total.

Methods: Chondrocytes and synoviocytes from normal femoropatellar joints of three dogs were plated on 24-well plates (50 000 cells/cm , triplicate, 48 hours) and exposed to antibiotics (ampicillin sulbactam, vancomycin, cefazolin, ceftazidime, amikacin, enrofloxacin; 0.39-25 mg/mL, 24 hours). Viability was assessed by using trypan blue dye exclusion. Antibiotic concentrations at which 50% cell death occurred (half-maximal inhibitory concentration) were determined to rank antibiotics for relative cytotoxicity. Occurrence of caspase-3 expression after antibiotic exposure was assessed as an indication of apoptosis induction. Cartilage explants from three different dogs were minced and exposed to antibiotics (amikacin, ceftazidime, cefazolin, enrofloxacin; 5 mg/mL, 72 hours). Live/dead staining was performed, and fluorescence was visualized by using confocal microscopy. Percentage of live vs dead cells was quantitated.

Results: Viability of chondrocytes and synoviocytes decreased with increasing antibiotic concentrations. Half-maximal inhibitory concentrations were determined for synoviocytes (vancomycin 13.77, ampicillin sulbactam 3.07, amikacin 2.26, ceftazidime 1.62, cefazolin 1.48, enrofloxacin 1.25 mg/mL) and chondrocytes (vancomycin 8.65, ampicillin sulbactam 8.63, ceftazidime 3.16, amikacin 2.74, cefazolin 1.67, enrofloxacin 0.78 mg/mL). Caspase-3 expression was upregulated, providing evidence that apoptotic pathways were active in cell death.

Conclusion: Half-maximal inhibitory concentration data provided evidence of lower toxicity of vancomycin and ampicillin sulbactam to joint tissues in vitro.

Clinical Significance: These results provide evidence to justify future in vitro work with osteoarthritic joint tissues and in vivo clinical trials to evaluate safety and efficacy of intra-articular antibiotics to treat dogs with septic arthritis.
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http://dx.doi.org/10.1111/vsu.13591DOI Listing
April 2021

Optimizing In Vitro Osteogenesis in Canine Autologous and Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells with Dexamethasone and BMP-2.

Stem Cells Dev 2021 02 8;30(4):214-226. Epub 2021 Feb 8.

Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA.

A growing body of work suggests that canine mesenchymal stromal cells (cMSCs) require additional agonists such as bone morphogenic protein-2 (BMP-2) for consistent in vitro osteogenic differentiation. BMP-2 is costly and may challenge the translational relevance of the canine model. Dexamethasone enhances osteogenic differentiation of human MSCs (hMSCs) and is widely utilized in osteogenic protocols. The aim of this study was to determine the effect of BMP-2 and dexamethasone on early- and late-stage osteogenesis of autologous and induced pluripotent stem cell (iPS)-derived cMSCs. Two preparations of marrow-derived cMSCs were selected to represent exceptionally or marginally osteogenic autologous cMSCs. iPS-derived cMSCs were generated from canine fibroblasts. All preparations were evaluated using alkaline phosphatase (ALP) activity, Alizarin Red staining of osteogenic monolayers, and quantitative polymerase chain reaction. Data were reported as mean ± standard deviation and compared using one- or two-way analysis of variance and Tukey or Sidak post hoc tests. Significance was established at  < 0.05. In early-stage assays, dexamethasone decreased ALP activity for all cMSCs in the presence of BMP-2. In late-stage assays, inclusion of dexamethasone and BMP-2 at Day 1 of culture produced robust monolayer mineralization for autologous cMSCs. Delivering 100 nM dexamethasone at Day 1 improved mineralization and reduced the BMP-2 concentrations required to achieve mineralization of the marginal cMSCs. For iPS-cMSCs, dexamethasone was inhibitory to both ALP activity and monolayer mineralization. There was increased expression of osteocalcin and osterix with BMP-2 in autologous cMSCs but a more modest expression occurred in iPS cMSCs. While autologous and iPS-derived cMSCs respond similarly in early-stage osteogenic assays, they exhibit unique responses to dexamethasone and BMP-2 in late-stage mineralization assays. This study demonstrates that dexamethasone and BMP-2 can be titrated in a time- and concentration-dependent manner to enhance osteogenesis of autologous cMSC preparations. These results will prove useful for investigators performing translational studies with cMSCs while providing insight into iPS-derived cMSC osteogenesis.
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http://dx.doi.org/10.1089/scd.2020.0144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891305PMC
February 2021

Generation of Neural Progenitor Cells From Canine Induced Pluripotent Stem Cells and Preliminary Safety Test in Dogs With Spontaneous Spinal Cord Injuries.

Front Vet Sci 2020 5;7:575938. Epub 2020 Nov 5.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Center for Immune and Regenerative Medicine, Colorado State University, Ft. Collins, CO, United States.

Advances in stem cell technology, including the use of induced pluripotent stem cells (iPSC) to produce neurons and glial cells, offer new hope for patients with neurological disease and injuries. Pet dogs with spinal cord injuries provide an important spontaneous animal model for evaluating new approaches to stem cell therapy. Therefore, studies were conducted to identify optimal conditions for generating neural progenitor cells (NPC) from canine induced pluripotent stem cells (iPSC) for preliminary evaluation in animals with spinal cord injury. We found that canine NPC could be induced to differentiate into mature neural cells, including glia and neurons. In addition, canine NPC did not form teratomas when injected in NOD/SCID mice. In a pilot study, two dogs with chronic spinal cord injury underwent fluoroscopically guided intrathecal injections of canine NPC. In follow-up MRI evaluations, tumor formation was not observed at the injection sites. However, none of the animals experienced meaningful clinical or electrophysiological improvement following NPC injections. These studies provide evidence that canine iPSC can be used to generate NPC for evaluation in cellular therapy of chronic spinal cord injury in the dog spontaneous injury model. Further refinements in the cell implantation procedure are likely required to enhance stem cell treatment efficacy.
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http://dx.doi.org/10.3389/fvets.2020.575938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674778PMC
November 2020

Non-specific protection from respiratory tract infections in cattle generated by intranasal administration of an innate immune stimulant.

PLoS One 2020 25;15(6):e0235422. Epub 2020 Jun 25.

Department of Clinical Sciences, From the Center for Immune and Regenerative Medicine, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft. Collins, Colorado, United States of America.

Alternatives to antibiotics for prevention of respiratory tract infections in cattle are urgently needed given the increasing public and regulatory pressure to reduce overall antibiotic usage. Activation of local innate immune defenses in the upper respiratory tract is one strategy to induce non-specific protection against infection with the diverse array of viral and bacterial pathogens associated with bovine respiratory disease complex (BRDC), while avoiding the use of antibiotics. Our prior studies in rodent models demonstrated that intranasal administration of liposome-TLR complexes (LTC) as a non-specific immune stimulant generated high levels of protection against lethal bacterial and viral pathogens. Therefore, we conducted studies to assess LTC induction of local immune responses and protective immunity to BRDC in cattle. In vitro, LTC were shown to activate peripheral blood mononuclear cells in cattle, which was associated with secretion of INFγ and IL-6. Macrophage activation with LTC triggered intracellular killing of Mannheimia hemolytica and several other bacterial pathogens. In studies in cattle, intranasal administration of LTC demonstrated dose-dependent activation of local innate immune responses in the nasopharynx, including recruitment of monocytes and prolonged upregulation (at least 2 weeks) of innate immune cytokine gene expression by nasopharyngeal mucosal cells. In a BRDC challenge study, intranasal administration of LTC prior to pathogen exposure resulted in significant reduction in both clinical signs of infection and disease-associated euthanasia rates. These findings indicate that intranasal administration of a non-specific innate immune stimulant can be an effective method of rapidly generating generalized protection from mixed viral and bacterial respiratory tract infections in cattle.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235422PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316291PMC
September 2020

Use of in vitro assays to identify antibiotics that are cytotoxic to normal equine chondrocytes and synovial cells.

Equine Vet J 2021 May 3;53(3):579-589. Epub 2020 Jul 3.

Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.

Background: Intra-articular (IA) antibiotic usage is prevalent in equine practice. However, recent emergence of antimicrobial resistance prompts re-evaluation of antibiotic selection, particularly when used prophylactically. Furthermore, many commonly used antibiotics exert direct cytotoxicity to equine cells, and appropriate IA doses have not been defined.

Objectives: To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues.

Study Design: In vitro experimental study.

Methods: Chondrocytes and synovial cells were harvested from three horses and plated on 24-well plates (100 000 cells/wells in triplicate) for 48 hours prior to addition of antibiotics. Joint cells were exposed to antibiotics (n = 15) at various doses (25-0.39 mg/mL in complete DMEM media) for 24 hours and viability was assessed by trypan blue dye exclusion. The half maximal inhibitory concentration (IC50) was determined for each antibiotic. Cartilage explants were obtained from 3 horses, minced and exposed to antibiotics (n = 5) for 72 hours. Live/dead staining was performed, and fluorescence was visualised using Olympus IX83 spinning disk confocal microscope. Percentage of live vs dead cells was quantified.

Results: Antibiotics from different antimicrobial classes expressed dose-dependent but variable cytotoxicity to equine joint cells in vitro. Aminoglycosides and doxycycline had the lowest IC50 (most toxic). Ampicillin sulbactam, imipenem, tobramycin, ceftiofur sodium and amoxicillin had IC50 > 25 mg/mL for at least one cell line, representing potentially less cytotoxic alternatives.

Main Limitations: Further studies are necessary to extrapolate these in vitro data results to the in vivo joint environment.

Conclusions: Targeted IA antibiotic therapy would involve selection of the safest antibiotics (highest IC50) with efficacy based on bacterial culture/sensitivity. Antimicrobial selection and evidence-based dosing may minimise damage to native articular cartilage and synovial cells and development of antimicrobial resistance when IA antibiotics are used in equine practice.
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http://dx.doi.org/10.1111/evj.13314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738387PMC
May 2021

Amikacin induces rapid dose-dependent apoptotic cell death in equine chondrocytes and synovial cells in vitro.

Equine Vet J 2020 Sep 21;52(5):715-724. Epub 2020 Feb 21.

Translational Medicine Institute, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.

Background: Equine veterinarians frequently inject aminoglycoside antibiotics intra-articularly, either to treat septic arthritis or for prophylaxis with other medications when injecting joints for osteoarthritis. Although aminoglycosides have been demonstrated to be toxic to equine mesenchymal stem cells (MSC), their effects on resident joint cells have not been previously investigated. Moreover, safe and effective intra-articular doses have not been defined.

Objectives: To determine effects of concentration, duration of exposure, pH and the presence of synovial fluid on the cytotoxic effects of amikacin on equine chondrocytes, synoviocytes and bone marrow- and adipose-derived MSC.

Study Design: In vitro experimental study.

Methods: Four cell types were harvested from three donor horses and plated in triplicate wells for 48 hours prior to the addition of amikacin. The effects of amikacin on cell viability were assessed for different exposure times, concentrations and with pH buffered or unbuffered in media, as well as in the presence of synovial fluid. Cell metabolism/viability was assessed by colorimetric MTT assay. Cell proliferation was assessed by live cell imaging. Cell viability was assessed using trypan blue and dimeric cyanine nucleic acid stain (yoyo-1). To determine the mechanism of cell death, apoptosis was evaluated using Annexin V and 7AAD staining with flow cytometric quantification. Induction of apoptotic cell death pathways was assessed using caspase-3 expression.

Results: Amikacin is cytotoxic to equine joint cells and MSC in a rapid, dose-dependent, pH-independent manner, which occurs primarily by apoptosis. Amikacin cytotoxicity was not mitigated by the addition of synovial fluid in vitro.

Main Limitations: Further studies are necessary to determine whether these in vitro results predict joint injury in live animal models.

Conclusions: Amikacin at clinically applied doses induces rapid, pronounced cell death of equine joint cells. These findings suggest that amikacin doses currently used intra-articularly should be reconsidered pending in vivo joint titration studies.
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http://dx.doi.org/10.1111/evj.13243DOI Listing
September 2020

Antibacterial activity of human mesenchymal stem cells mediated directly by constitutively secreted factors and indirectly by activation of innate immune effector cells.

Stem Cells Transl Med 2020 02 8;9(2):235-249. Epub 2019 Nov 8.

Center for Immune and Regenerative Medicine, Department of Clinical Sciences, Colorado State University, Ft. Collins, Colorado.

Mesenchymal stem cells (MSC) have been shown to improve wound healing and suppress inflammatory immune responses. Newer research also indicates that MSC exhibit antimicrobial activity, although the mechanisms underlying this activity have not been fully elucidated. Therefore, we conducted in vitro and in vivo studies to examine the ability of resting and activated MSC to kill bacteria, including multidrug resistant strains. We investigated direct bacterial killing mechanisms and the interaction of MSC with host innate immune responses to infection. In addition, the activity of MSC against chronic bacterial infections was investigated in a mouse biofilm infection model. We found that MSC exhibited high levels of spontaneous direct bactericidal activity in vitro. Moreover, soluble factors secreted by MSC inhibited Staphylococcus aureus biofilm formation in vitro and disrupted the growth of established biofilms. Secreted factors from MSC also elicited synergistic killing of drug-resistant bacteria when combined with several major classes of antibiotics. Other studies demonstrated interactions of activated MSC with host innate immune responses, including triggering of neutrophil extracellular trap formation and increased phagocytosis of bacteria. Finally, activated MSC administered systemically to mice with established S. aureus biofilm infections significantly reduced bacterial numbers at the wound site and improved wound healing when combined with antibiotic therapy. These results indicate that MSC generate multiple direct and indirect, immunologically mediated antimicrobial activities that combine to help eliminate chronic bacterial infections when the cells are administered therapeutically.
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http://dx.doi.org/10.1002/sctm.19-0092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988770PMC
February 2020

Local immune and microbiological responses to mucosal administration of a Liposome-TLR agonist immunotherapeutic in dogs.

BMC Vet Res 2019 Sep 13;15(1):330. Epub 2019 Sep 13.

From the Center for Immune and Regenerative Medicine and the Center for Companion Animal Studies, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 80523, Ft. Collins, Colorado, CO, USA.

Background: Non-specific immunotherapeutics have been evaluated previously in dogs, primarily for cancer treatment. However, there remains a need for a more broadly targeted, general purpose immunotherapeutic capable of activating innate immune defenses for non-specific protection or early treatment of viral and bacterial infections. To address need, our group has developed a liposomal immune stimulant (liposome-TLR complexes, LTC) containing TLR 3 and 9 agonists specifically designed to activate mucosal immune defenses in sites such as nasal cavity and oropharynx, following topical delivery. In this study, we evaluated the local immune stimulatory properties of LTC in vitro and in healthy purpose-bred dogs, including activation of cellular recruitment and cytokine production. The ability of LTC treatment to elicit effective antiviral immunity was assessed in dogs following a canine herpesvirus outbreak, and the impact of LTC treatment on the local microbiome of the oropharynx was also investigated.

Results: These studies revealed that LTC potently activated innate immune responses in vitro and triggered significant recruitment of inflammatory monocytes and T cells into the nasal cavity and oropharynx of healthy dogs. Administration of LTC to dogs shortly after an outbreak of canine herpesvirus infection resulted in significant reduction in clinical signs of infection. Interestingly, administration of LTC to healthy dogs did not disrupt the microbiome in the oropharynx, suggesting resiliency of the microflora to transient immune activation.

Conclusions: Taken together, these results indicate that LTC administration mucosally to dogs can trigger local innate immune activation and activation of antiviral immunity, without significantly disrupting the composition of the local microbiome. Thus, the LTC immune stimulant has potential for use as a non-specific immunotherapy for prevention or early treatment of viral and bacterial infections in dogs.
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http://dx.doi.org/10.1186/s12917-019-2073-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6743184PMC
September 2019

Humoral immune responses against gut bacteria in dogs with inflammatory bowel disease.

PLoS One 2019 1;14(8):e0220522. Epub 2019 Aug 1.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America.

Inflammatory bowel disease (IBD) in dogs is associated with clinical signs of intestinal dysfunction, as well as abnormal lymphocytic and myeloid cell infiltrates in the small and/or large intestine. Thus, in many respects IBD in dogs resembles IBD in humans. However, the factors that trigger intestinal inflammation in dogs with IBD are not well understood and have been variously attributed to immune responses against dietary antigens or intestinal antigens. Previous studies in humans with IBD have documented increased production of IgG and IgA antibodies specific to intestinal bacteria, and this abnormal immune response has been linked to disease pathogenesis. Therefore, we investigated the humoral immune response against gut bacteria in dogs with IBD, using flow cytometry to quantitate IgG and IgA binding. Studies were also done to investigate the source of these antibodies (locally produced versus systemic production) and whether greater antibody binding to bacteria is associated with increased inflammatory responses. We found that dogs with IBD had significantly higher percentages and overall amounts of IgG bound to their intestinal bacteria compared to healthy dogs. Similarly, significantly higher percentages of bacteria were IgA+ bacteria were also found in dogs with IBD. Serum antibody recognition of gut bacteria was not different between healthy dogs and dogs with IBD, suggesting that anti-bacterial antibodies were primarily produced locally in the gut rather than systemically. Importantly, bacteria in the Actinobacteria phylum and in particular the genus Collinsella had significantly greater levels of antibody binding in dogs with IBD. Based on these findings, we concluded that antibody binding to commensal gut bacteria was significantly increased in dogs with IBD, that particular phyla were preferential targets for gut antibodies, and that anti-bacterial antibody responses may play an important role in regulating gut inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220522PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675102PMC
March 2020

The Angiotensin Receptor Blocker Losartan Suppresses Growth of Pulmonary Metastases via AT1R-Independent Inhibition of CCR2 Signaling and Monocyte Recruitment.

J Immunol 2019 05 10;202(10):3087-3102. Epub 2019 Apr 10.

Flint Animal Cancer Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523;

Inflammatory monocytes have been shown to play key roles in cancer metastasis through promotion of tumor cell extravasation, growth, and angiogenesis. Monocyte recruitment to metastases is mediated primarily via the CCL2-CCR2 chemotactic axis. Thus, disruption of this axis represents an attractive therapeutic target for the treatment of metastatic disease. Losartan, a type I angiotensin II receptor (AT1R) antagonist, has been previously shown to have immunomodulatory actions involving monocyte and macrophage activity. However, the exact mechanisms accounting for these effects have not been fully elucidated. Therefore, we investigated the effects of losartan and its primary metabolite on CCL2-mediated monocyte recruitment and CCR2 receptor function using mouse tumor models and in vitro human monocyte cultures. We show, in this study, that losartan and its metabolite potently inhibit monocyte recruitment through the noncompetitive inhibition of CCL2-induced ERK1/2 activation, independent of AT1R activity. Studies in experimental metastasis models demonstrated that losartan treatment significantly reduced the metastatic burden in mice, an effect associated with a significant decrease in CD11b/Ly6C-recruited monocytes in the lungs. Collectively, these results indicate that losartan can exert antimetastatic activity by inhibiting CCR2 signaling and suppressing monocyte recruitment and therefore suggest that losartan (and potentially other AT1R blocker drugs) could be repurposed for use in cancer immunotherapy.
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http://dx.doi.org/10.4049/jimmunol.1800619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504574PMC
May 2019

Activation of upper respiratory tract mucosal innate immune responses in cats by liposomal toll-like receptor ligand complexes delivered topically.

J Vet Intern Med 2019 Mar 15;33(2):838-845. Epub 2019 Feb 15.

Center for Immune and Regenerative Medicine and the Center for Companion Animal Studies, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado.

Background: Nonspecific induction of local innate immune responses by mucosally administered immunotherapy is a new approach to protection from upper respiratory tract infections. Therefore, a new liposome-toll-like receptor complex (LTC) immune stimulant was developed and investigated for its ability to activate innate immune responses in cats, both in vitro and in vivo, as part of an initial evaluation of LTC for use as an immunotherapeutic agent in cats.

Objectives: We hypothesized that LTC could activate innate immune responses in cats after topical application to nasal and oropharyngeal mucosal surfaces.

Animals: Mucosal immune responses to topical administration of LTC were assessed in 7 healthy, purpose-bred cats, and in vitro responses were assessed using blood samples from healthy cats.

Methods: Cytokine and cellular immune responses to LTC were evaluated in blood samples, nasal lavage specimens, and pharyngeal swabs from cats, using reverse transcriptase polymerase chain reaction assays, ELISA assays, and flow cytometry.

Results: Liposome-TLR complexes rapidly activated leukocytes in vitro, including upregulation of costimulatory molecule expression and cytokine production. Topical administration of LTC in healthy cats triggered rapid recruitment of monocytes to the nasal and oropharyngeal mucosa.

Conclusions And Clinical Importance: Liposome-TLR complexes were found to effectively activate innate immune responses in cats after mucosal administration. These findings suggest that LTC have potential for use as a new mucosally administered immunotherapy for nonspecific protection from viral and bacterial respiratory tract infections.
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http://dx.doi.org/10.1111/jvim.15426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430862PMC
March 2019

Programmed Cell Death Ligand 1 (PD-L1) Signaling Regulates Macrophage Proliferation and Activation.

Cancer Immunol Res 2018 10 16;6(10):1260-1273. Epub 2018 Jul 16.

Animal Cancer Center, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Ft. Collins, Colorado.

Tumor-associated macrophages (TAMs) express programmed cell death ligand 1 (PD-L1) and contribute to the immune-suppressive tumor microenvironment. Although the role of the PD-L1 and PD-1 interaction to regulate T-cell suppression is established, less is known about PD-L1 signaling in macrophages and how these signals may affect the function of TAMs. We used and models to investigate PD-L1 signaling in macrophages and the effects of PD-L1 antibody treatment on TAM responses. Treatment of mouse and human macrophages with PD-L1 antibodies increased spontaneous macrophage proliferation, survival, and activation (costimulatory molecule expression, cytokine production). Similar changes were observed in macrophages incubated with soluble CD80 and soluble PD-1, and in PD-L1 macrophages. Macrophage treatment with PD-L1 antibodies upregulated mTOR pathway activity, and RNAseq analysis revealed upregulation of multiple macrophage inflammatory pathways. treatment with PD-L1 antibody resulted in increased tumor infiltration with activated macrophages. In tumor-bearing RAG mice, upregulated costimulatory molecule expression by TAMs and reduced tumor growth were observed. Combined PD-1/ PD-L1 antibody treatment of animals with established B16 melanomas cured half of the treated mice, whereas treatment with single antibodies had little therapeutic effect. These findings indicate that PD-L1 delivers a constitutive negative signal to macrophages, resulting in an immune-suppressive cell phenotype. Treatment with PD-L1 antibodies reverses this phenotype and triggers macrophage-mediated antitumor activity, suggesting a distinct effect of PD-L1, but not PD-1, antibody treatment. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0537DOI Listing
October 2018

Mesenchymal Stem Cells (MSC) Derived from Induced Pluripotent Stem Cells (iPSC) Equivalent to Adipose-Derived MSC in Promoting Intestinal Healing and Microbiome Normalization in Mouse Inflammatory Bowel Disease Model.

Stem Cells Transl Med 2018 06 10;7(6):456-467. Epub 2018 Apr 10.

Center for Immune and Regenerative Medicine, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA.

Cellular therapy with allogeneic or autologous mesenchymal stem cells (MSC) has emerged as a promising new therapeutic strategy for managing inflammatory bowel disease (IBD). However, MSC therapy ideally requires a convenient and relatively homogenous cell source (typically bone marrow or adipose tissues) and the ability to generate cells with stable phenotype and function. An alternative means of generating allogeneic MSC is to derive them from induced pluripotent stem cells (iPSC), which could in theory provide an indefinite supply of MSC with well-defined phenotype and function. Therefore, we compared the effectiveness of iPSC-derived MSC (iMSC) and adipose-derived MSC (adMSC) in a mouse model of IBD (dextran sodium sulfate-induced colitis), and investigated mechanisms of intestinal protection. We found that iMSC were equivalent to adMSC in terms of significantly improving clinical abnormalities in treated mice and reducing lesion scores and inflammation in the gut. Administration of iMSC also stimulated significant intestinal epithelial cell proliferation, increased in the numbers of Lgr5+ intestinal stem cells, and increased intestinal angiogenesis. In addition, the microbiome alterations present in mice with colitis were partially restored to resemble those of healthy mice following treatment with iMSC or adMSC. Thus, iMSC administration improved overall intestinal health and healing with equivalent potency to treatment with adMSC. This therefore is the first report of the effectiveness of iMSC in the treatment of IBD, along with a description of unique mechanisms of action with respect to intestinal healing and microbiome restoration. Stem Cells Translational Medicine 2018;7:456-467.
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http://dx.doi.org/10.1002/sctm.17-0305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980202PMC
June 2018

Mesenchymal Stem Cells Recruit CCR2 Monocytes To Suppress Allergic Airway Inflammation.

J Immunol 2018 02 19;200(4):1261-1269. Epub 2018 Jan 19.

Center for Immune and Regenerative Medicine, Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523.

Mesenchymal stem cells (MSC) exert immune modulatory properties and previous studies demonstrated suppressive effects of MSC treatment in animal models of allergic airway inflammation. However, the underlying mechanisms have not been fully elucidated. We studied the role of MSC in immune activation and subsequent recruitment of monocytes in suppressing airway hyperresponsiveness and airway inflammation using a mouse model of allergic airway inflammation. MSC administration prior to or after allergen challenge inhibited the development of airway inflammation in allergen-sensitized mice. This was accompanied by an influx of CCR2-positive monocytes, which were localized around injected MSC in the lungs. Notably, IL-10-producing monocytes and/or macrophages were also increased in the lungs. Systemic administration of liposomal clodronate or a CCR2 antagonist significantly prevented the suppressive effects of MSC. Activation of MSC by IFN-γ leading to the upregulation of CCL2 expression was essential for the suppressive effects, as administration of wild-type MSC into IFN-γ-deficient recipients, or IFN-γ receptor-deficient or CCL2-deficient MSC into wild-type mice failed to suppress airway inflammation. These results suggest that MSC activation by IFN-γ, followed by increased expression of CCL2 and recruitment of monocytes to the lungs, is essential for suppression by MSC in allergen-induced airway hyperresponsiveness and airway inflammation.
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http://dx.doi.org/10.4049/jimmunol.1700562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809262PMC
February 2018

Safety and immune regulatory properties of canine induced pluripotent stem cell-derived mesenchymal stem cells.

Stem Cell Res 2017 12 14;25:221-232. Epub 2017 Nov 14.

Center for Immune and Regenerative Medicine, Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Ft. Collins, CO, United States. Electronic address:

Mesenchymal stem cells (MSCs) exhibit broad immune modulatory activity in vivo and can suppress T cell proliferation and dendritic cell activation in vitro. Currently, most MSC for clinical usage are derived from younger donors, due to ease of procurement and to the superior immune modulatory activity. However, the use of MSC from multiple unrelated donors makes it difficult to standardize study results and compare outcomes between different clinical trials. One solution is the use of MSC derived from induced pluripotent stem cells (iPSC); as iPSC-derived MSC have nearly unlimited proliferative potential and exhibit in vitro phenotypic stability. Given the value of dogs as a spontaneous disease model for pre-clinical evaluation of stem cell therapeutics, we investigated the functional properties of canine iPSC-derived MSC (iMSC), including immune modulatory properties and potential for teratoma formation. We found that canine iMSC downregulated expression of pluripotency genes and appeared morphologically similar to conventional MSC. Importantly, iMSC retained a stable phenotype after multiple passages, did not form teratomas in immune deficient mice, and did not induce tumor formation in dogs following systemic injection. We concluded therefore that iMSC were phenotypically stable, immunologically potent, safe with respect to tumor formation, and represented an important new source of cells for therapeutic modulation of inflammatory disorders.
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http://dx.doi.org/10.1016/j.scr.2017.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457437PMC
December 2017

Mechanisms of Immune Suppression Utilized by Canine Adipose and Bone Marrow-Derived Mesenchymal Stem Cells.

Stem Cells Dev 2017 03 24;26(5):374-389. Epub 2017 Jan 24.

1 Center for Immune and Regenerative Medicine, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University , Ft. Collins, Colorado.

Mesenchymal stem cells (MSCs) from rodents and humans have been shown to suppress T cells by distinct primary pathways, with nitric oxide (NO)-dependent pathways dominating in rodents and indoleamine 2,3-deoxygenase (IDO)-dependent pathways dominating in humans. However, the immune suppressive pathways utilized by canine MSC have not been thoroughly studied, nor have bone marrow-derived MSC (BM-MSC) and adipose-derived MSC (Ad-MSC) been directly compared for their immune modulatory potency or pathway utilization. Therefore, canine BM-MSC and Ad-MSC were generated in vitro and their potency in suppressing T cell proliferation and cytokine production was compared, and differential gene expression. Mechanisms of T cells suppression were also investigated for both MSC types. We found that BM-MSC and Ad-MSC were roughly equivalent in terms of their ability to suppress T cell activation. However, the two MSC types used both shared and distinct biochemical pathways to suppress T cell activation. Ad-MSC utilized TGF-β signaling pathways and adenosine signaling to suppress T cell activation, whereas BM-MSC used cyclooxygenase, TGF-β and adenosine signaling pathways to suppress T cell activation. These results indicate that canine MSC are distinct from human and rodent MSC terms of their immune suppressive pathways, relying primarily on cyclooxygenase and TGF-β pathways for T cell suppression, rather than on NO or IDO-mediated pathways.
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http://dx.doi.org/10.1089/scd.2016.0207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5327053PMC
March 2017

Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways.

Stem Cells Dev 2017 02 22;26(4):249-262. Epub 2016 Dec 22.

Department of Clinical Sciences, Center for Immune and Regenerative Medicine, College of Veterinary Medicine and Biomedical Sciences, Colorado State University , Fort Collins, Colorado.

Mesenchymal stem cells (MSC) represent a readily accessible source of cells with potent immune modulatory activity. MSC can suppress ongoing inflammatory responses by suppressing T cell function, while fewer studies have examined the impact of MSC on dendritic cell (DC) function. The dog spontaneous disease model represents an important animal model with which to evaluate the safety and effectiveness of cellular therapy with MSC. This study evaluated the effects of canine MSC on the activation and maturation of canine monocyte-derived DC, as well as mechanisms underlying these effects. Adipose-derived canine MSC were cocultured with canine DC, and the MSC effects on DC maturation and activation were assessed by flow cytometry, cytokine ELISA, and confocal microscopy. We found that canine MSC significantly suppressed lipopolysaccharide (LPS)-stimulated upregulation of DC activation markers such as major histocompatibility class II (MHCII), CD86, and CD40. Furthermore, pretreatment of MSC with interferon gamma (IFNγ) augmented this suppressive activity. IFNγ-activated MSC also significantly reduced LPS-elicited DC secretion of tumor necrosis factor alpha without reducing secretion of interleukin-10. The suppressive effect of IFNγ-treated MSC on LPS-induced DC activation was mediated by soluble factors secreted by both MSC and DC. Pathways of DC functional suppression included programmed death ligand-1 expression and secretion of nitrous oxide, prostaglandin E2, and adenosine by activated MSC. Coculture of DC with IFNγ-treated MSC maintained DC in an immature state and prolonged DC antigen uptake during LPS maturation stimulus. Taken together, canine MSC are capable of potently suppressing DC function in a potentially inflammatory microenvironment through several separate immunological pathways and confirm the potential for immune therapy with MSC in canine immune-mediated disease models.
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http://dx.doi.org/10.1089/scd.2016.0199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913781PMC
February 2017
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