Dori L. Borjesson

Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue

OBJECTIVE To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. ANIMALS 10 adult Quarter Horses and 8 newborn Thoroughbred foals. PROCEDURES eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. RESULTS BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP activity and calcium deposition. There was no significant difference in BM-eMSC yield from the tuber coxae or sternum, but yield was significantly greater from the first 60-mL aspirate than from subsequent aspirates. The BM-eMSC expansion rate was significantly higher when cells were cultured in fetal bovine serum instead of autologous serum (AS). CONCLUSIONS AND CLINICAL RELEVANCE eMSCs from BM possessed the highest in vitro osteogenic potential; eMSCs from adipose tissue also had robust osteogenic potential. The tuber coxae and the sternum were viable sources of BM-eMSCs in yearlings, and 60 mL of BM aspirate was sufficient for culture and expansion. Expanding BM-eMSCs in AS to avoid potential immunologic reactions decreased the total yield because BM-eMSCs grew significantly slower in AS than in fetal bovine serum. Additional studies are needed to determine optimal ex vivo eMSC culture and expansion conditions, including the timing and use of growth factor—supplemented AS.

Insights into Pathogen Immune Evasion Mechanisms: Anaplasma phagocytophilum Fails to Induce an Apoptosis Differentiation Program in Human Neutrophils

Polymorphonuclear leukocytes (PMNs or neutrophils) are essential to human innate host defense. However, some bacterial pathogens circumvent destruction by PMNs and thereby cause disease. Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, survives within PMNs in part by altering normal host cell processes, such as production of reactive oxygen species (ROS) and apoptosis. To investigate the molecular basis of A. phagocytophilum survival within neutrophils, we used Affymetrix microarrays to measure global changes in human PMN gene expression following infection with A. phagocytophilum. Notably, A. phagocytophilum uptake induced fewer perturbations in host cell gene regulation compared with phagocytosis of Staphylococcus aureus. Although ingestion of A. phagocytophilum did not elicit significant PMN ROS, proinflammatory genes were gradually up-regulated, indicating delayed PMN activation rather than loss of proinflammatory capacity normally observed during phagocytosis-induced apoptosis. Importantly, ingestion of A. phagocytophilum failed to trigger the neutrophil apoptosis differentiation program that typically follows phagocytosis and ROS production. Heat-killed A. phagocytophilum caused some similar initial alterations in neutrophil gene expression and function, which included delaying normal PMN apoptosis and blocking Fas-induced programmed cell death. However, at 24 h, down-regulation of PMN gene transcription may be more reliant on active infection. Taken together, these findings suggest two separate antiapoptotic processes may work concomitantly to promote bacterial survival: 1) uptake of A. phagocytophilum fails to trigger the apoptosis differentiation program usually induced by bacteria, and 2) a protein or molecule on the pathogen surface can mediate an early delay in spontaneous neutrophil apoptosis.

Evaluation of Senescence in Mesenchymal Stem Cells Isolated from Equine Bone Marrow, Adipose Tissue, and Umbilical Cord Tissue

Mesenchymal stem cells (MSCs) from adult and neonatal tissues are intensively investigated for their use in regenerative medicine. The purpose of this study was to compare the onset of replicative senescence in MSCs isolated from equine bone marrow (BMSC), adipose tissue (ASC), and umbilical cord tissue (UCMSC). MSC proliferation (cell doubling), senescence-associated β-galactosidase staining, telomere length, Sox-2, and lineage-specific marker expression were assessed for MSCs harvested from tissues of 4 different donors. The results show that before senescence ensued, all cell types proliferated at ∼1 day/cell doubling. BMSCs significantly increased population doubling rate by passage 10 and ceased proliferation after a little >30 total population doublings, whereas UCMSCs and ASCs achieved about 60 to 80 total population doublings. UCMSC and ASCs showed marked β-galactosidase staining after ∼70 population doublings, whereas BMSCs stained positive by ∼30 population doublings. The onset of senescence was associated with a significant reduction in telomere length averaging 10.2 kbp at passage 3 and 4.5 kbp in senescent cultures. MSCs stained intensively for osteonectin at senescence compared with earlier passages, whereas vimentin and low levels of smooth muscle actin were consistently expressed. Sox-2 gene expression was consistently noted in all 3 MSC types. In conclusion, equine BMSCs appear to senesce much earlier than ASCs and UCMSCs. These results demonstrate the limited passage numbers of subcultured BMSCs available for use in research and tissue engineering and suggest that adipose tissue and umbilical cord tissue may be preferable for tissue banking purposes.

Canine granulocytic anaplasmosis: a review.

Anaplasma phagocytophilum is an emerging pathogen of humans, horses, and dogs worldwide that is transmitted by Ixodid ticks and maintained in a variety of small wild mammal species. Recent studies suggest that multiple strains of A. phagocytophilum may be circulating in wild and domestic animal populations, and these strains may have differential host tropisms and pathogenicity. The organism infects and survives within neutrophils by disabling key neutrophil functions, including neutrophil motility, phagocytosis, the oxidative burst mechanism, and neutrophil-endothelial cell interactions, as well as interfering with neutrophil apoptosis. Coinfections with other tick-borne pathogens may occur, especially Borrelia burgdorferi. A. phagocytophilum causes an acute febrile illness in dogs with lethargy and inappetence. Less frequent signs include lameness, coughing, polydipsia, intermittent vomiting, and hemorrhages. Diagnosis is based on finding morulae within granulocytes in the peripheral blood, the combination of acute and convalescent serology using immunofluorescent antibody techniques, and detection of the DNA of A. phagocytophilum using specific polymerase chain reaction assays. Whether persistent infection or reinfection with A. phagocytophilum occurs after natural infection requires additional study, with most reports suggesting that anaplasmosis is a self-limiting disease in dogs that responds well to a 2-week course of doxycycline therapy.

Dynamics of Neutrophil Infiltration during Cutaneous Wound Healing and Infection Using Fluorescence Imaging

Neutrophil influx is an early inflammatory response that is essential for the clearance of bacteria and cellular debris during cutaneous wounding. A non-invasive real-time fluorescence imaging technique was developed to examine the kinetics of enhanced green fluorescence protein-polymorphonuclear leukocyte (EGFP-PMN) influx within a wound. We hypothesized that infection or systemic availability would directly regulate the dynamics of EGFP-PMN recruitment and the efficiency of wound closure. Neutrophil recruitment increased dramatically over the first 24 hours from 10(6) at 4 hours up to a maximum of 5 x 10(6) EGFP-PMNs at 18 hours. A high rate of EGFP-PMN turnover was evidenced by approximately 80% decrease in EGFP signal within 6 hours. In response to wound colonization by Staphylococcus aureus or injection of GM-CSF, systemic PMNs increased twofold above saline control. This correlated with an increase in EGFP-PMN recruitment up to approximately 10(7) within the wound. Despite this effect by these distinct inflammatory drivers, wound closure occurred at a rate similar to the saline-treated control group. In summary, a non-invasive fluorescence-based imaging approach combined with genetic labeling of neutrophils provides a dynamic inner view of inflammation and the kinetics of neutrophil infiltration into the wounded skin over extended durations.

Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses.

BACKGROUND AIMS The development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horses. METHODS Sixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injection. RESULTS MSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSC. CONCLUSIONS The healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.

Borrelia burgdorferi Population Kinetics and Selected Gene Expression at the Host-Vector Interface

ABSTRACT By using real-time quantitative PCR, the population dynamics and gene transcription of Borrelia burgdorferi were examined in ticks and skin of mice during acquisition of the infection from mice by ticks and during transmission of the infection from ticks to mice. Population dynamics were determined by using a flaB DNA target. A quantitative analysis of flaB, ospA, ospC, dbpA, and arp transcription was also performed. The results revealed that both uninfected larval and nymphal Ixodes scapularis ticks acquired B. burgdorferi as early as 1 day after attachment and that the sizes of spirochete populations within ticks increased during feeding. In addition, all gene targets revealed that there was RNA transcription during feeding. Similar events occurred within infected nymphal ticks feeding on uninfected hosts. Transmission from infected nymphal ticks to mice could be detected within 1 day after attachment. Analysis of skin during the first 3 days after attachment of infected ticks revealed rising numbers of spirochetes but minimal gene transcription. In contrast, the skin of mice with established infections revealed static populations of spirochetes and active but stable transcription of flaB, ospC, dbpA, and arp. There were consistent reductions in the number of spirochetes in the skin at the tick attachment sites compared to the number of spirochetes in the skin at nontick sites, but there were no differences in gene expression between tick and nontick skin sites. Evidence of ospA transcription in skin could be found 1 day after tick attachment but not thereafter.

Comparative Analysis of the Immunomodulatory Properties of Equine Adult-Derived Mesenchymal Stem Cells().

Mesenchymal stem cells (MSCs) derived from bone marrow (BM), adipose tissue (AT), umbilical cord blood (CB), and umbilical cord tissue (CT) are increasingly being used to treat equine inflammatory and degenerative lesions. MSCs modulate the immune system in part through mediator secretion. Animal species and MSC tissue of origin are both important determinants of MSC function. In spite of widespread clinical use, how equine MSCs function to heal tissues is fully unknown. In this study, MSCs derived from BM, AT, CB, and CT were compared for their ability to inhibit lymphocyte proliferation and secrete mediators in response to activation. Five MSC lines from each tissue were isolated. Lymphocyte proliferation was assessed in a mixed leukocyte reaction, and mediator secretion was determined by ELISA. Regardless of tissue of origin, quiescent MSCs did not alter lymphocyte proliferation or secrete mediators, except for transforming growth factor-β (TGF-β1). When stimulated, MSCs of all tissue types decreased lymphocyte proliferation, increased prostaglandin (PGE(2)) and interleukin-6 (IL-6) secretion, and decreased production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). BM-MSCs and CB-MSCs also produced nitric oxide (NO), while AT-MSCs and CT-MSCs did not. Equine MSCs did not produce indoleamine 2,3-dioxygenase (IDO). These data suggest that activated equine MSCs derived from BM, AT, CT, and CB secrete high concentration of mediators and are similar to MSCs from rodents and humans in their immunomodulatory profiles. These findings have implication for the treatment of inflammatory lesions dominated by activated lymphocytes and TNF-α and IFN-γ in vivo.

Intradermal injections of equine allogeneic umbilical cord-derived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions

BACKGROUND AIMS. The use of allogeneic mesenchymal stem cells (MSC) to treat acute equine lesions would greatly expand equine cellular therapy options; however, the safety and antigenicity of these cells have not been well-studied. We hypothesized that equine allogeneic umbilical cord tissue (UCT)-derived MSC would not elicit acute graft rejection or a delayed-type hypersensitivity response when injected intradermally. METHODS. Six Quarterhorse yearlings received 12 intradermal injections (autologous MSC, allogeneic MSC, positive control and negative control, in triplicate) followed by the same series of 12 injections, 3-4 weeks later, at another site. Wheals were measured and palpated at 0.25, 4, 24, 48, 72 h and 7 days post-injection. Biopsies were obtained at 48 and 72 h and 7 days post-injection. Mixed leukocyte reactions were performed 1 week prior to the first injections and 3 weeks after the second injections. RESULTS. There were no adverse local or systemic responses to two intradermal injections of allogeneic MSC. MSC injection resulted in minor wheal formation, characterized by mild dermatitis, dermal edema and endothelial hyperplasia, that fully resolved by 48-72 h. No differences were noted between allogeneic and autologous MSC. The second injection of MSC did not elicit more significant physical or histomorphologic alterations compared with the first MSC injection. Neither allogeneic nor autologous UCT-derived MSC stimulated or suppressed baseline T-cell proliferation in vitro prior to or after two MSC administrations. CONCLUSIONS. Equine allogeneic UCT MSC may be safely administered intradermally on multiple occasions without eliciting a measurable cellular immune response.

Application of thrombelastography/thromboelastometry to veterinary medicine

Thrombelastograph analyzers are point-of-care hemostatic analyzers that provide global assessment of the hemostatic process. Thrombelastography (TEG) detects and provides a continuous recording of the changes in the viscoelastic properties of whole blood from initial clot formation through fibinolysis. TEG has been validated for use in dogs, horses, and cats. Hemostasis research using TEG has focused on test validation, alterations of TEG tracings in animals with naturally occurring diseases, and the use of TEG for monitoring various therapeutic modalities. This article reviews TEG methodology and terminology, including potential sources of preanalytical and analytical errors, the correlation between TEG and other routine hemostatic assays, and current clinical applications of TEG, with emphasis on veterinary medical practice. Data suggest that TEG may be a sensitive and useful adjunctive tool for evaluating an animal with an underlying coagulopathy, including hypercoagulability and hypocoagulability. Additional prospective studies are needed to (1) correlate TEG tracing patterns with a clinical predisposition for bleeding or thrombosis in various disease states and (2) determine whether monitoring and treating hemostatic disorders based on TEG tracings improve clinical outcome.