Immunization against nucleus pulposus antigens to accelerate degenerative disc disease in a rabbit model.

Low back pain poses a significant societal burden, with progressive intervertebral disc degeneration (IDD) emerging as a pivotal contributor to chronic pain. Improved animal models of progressive IDD are needed to comprehensively investigate new diagnostic and therapeutic approaches to managing IDD. Recent studies underscore the immune system's involvement in IDD, particularly with regards to the role of immune privileged tissues such as the nucleus pulposus (NP) becoming an immune targeting following initial disc injury. We therefore hypothesized that generating an active immune response against NP antigens with an NP vaccine could significantly accelerate and refine an IDD animal model triggered by mechanical puncture of the disc. To address this question, rabbits were immunized against NP antigens following disc puncture, and the impact on development of progressive IDD was assessed radiographically, functionally, and histologically compared between vaccinated and non-vaccinated animals over a 12-week period. Immune responses to NP antigens were assessed by ELISA and Western blot. We found that the vaccine elicited strong immune responses against NP antigens, including a dominant ~37 kD antigen. Histologic evaluation revealed increases IDD in animals that received the NP vaccine plus disc puncture, compared to disc puncture and vaccine only animals. Imaging evaluation evidenced a decrease in disc height index and higher scores of disc degeneration in animals after disc punctures and in those animals that received the NP vaccine in addition to disc puncture. These findings therefore indicate that it is possible to elicit immune responses against NP antigens in adult animals, and that these immune responses may contribute to accelerated development of IDD in a novel immune-induced and accelerated IDD model.

Evaluation of stem cell therapies in companion animal disease models: A concise review (2015-2023).

Companion animals in veterinary medicine develop multiple naturally occurring diseases analogous to human conditions. We previously reported a comprehensive review on the feasibility, safety, and biologic activity of using novel stem cell therapies to treat a variety of inflammatory conditions in dogs and cats (2008-2015) [1]. The purpose of this review is to provide an updated summary of current studies in companion animal disease models that have evaluated stem cell therapeutics that are relevant to human disease. Here we have reviewed the literature from 2015 to 2023 for publications on stem cell therapies that have been evaluated in companion animals, including dogs, cats, and horses. The review excluded case reports or studies performed in experimentally induced models of disease, studies involving cancer, or studies in purpose-bred laboratory species such as rodents. We identified 45 manuscripts meeting these criteria, an increase from 19 that were described in the previous review [1]. The majority of studies were performed in dogs (n=28), with additional studies in horses (n=9) and cats (n=8). Disease models included those related to musculoskeletal disease (osteoarthritis, tendon/ligament injury), neurologic disease (canine cognitive dysfunction, intervertebral disc disease, spinal cord injury) gingival/dental disease (gingivostomatitis), dermatologic disease (atopic dermatitis), chronic multi-drug resistant infections, ophthalmic disease (keratoconjunctivitis sicca, eosinophilic keratitis, immune mediated keratitis), cardiopulmonary disease (asthma, degenerative valve disease, dilated cardiomyopathy), gastrointestinal disease (inflammatory bowel disease, chronic enteropathy) and renal disease (chronic kidney disease). The majority of studies reported beneficial responses to stem cell treatment, with the exception of those related to more chronic processes such as spinal cord injury and chronic kidney disease. However, it should also be noted that 22 studies were open-label, baseline-controlled trials and only 12 studies were randomized and controlled, making overall study interpretation difficult. As noted in the previous review, improved regulatory oversight and consistency in manufacturing of stem cell therapies is needed. Enhanced understanding of the temporal course of disease processes using advanced -omics approaches may further inform mechanisms of action and help define appropriate timing of interventions. Future directions of stem cell-based therapies could include use of stem-cell derived extracellular vesicles, or cell conditioning approaches to direct cells to specific pathways that are tailored to individual disease processes and stages of illness.

Immunohistochemical and transcriptomic characterization of T and myeloid cell infiltrates in canine malignant melanoma.

Immune checkpoint inhibitor therapy can provide significant clinical benefit in patients with certain cancer types including melanoma; however, objective responses are only observed for a subset of patients. Mucosal melanoma is a rare melanoma subtype associated with a poor prognosis and, compared with cutaneous melanoma, is significantly less responsive to immune checkpoint inhibitors. Spontaneous canine tumours have emerged as valuable models to inform human cancer studies. In contrast to human melanoma, most canine melanomas are mucosal-an incidence that may be leveraged to better understand the subtype in humans. However, a more comprehensive understanding of the immune landscape of the canine disease is required. Here, we quantify tumour infiltrative T and myeloid cells in canine mucosal (n = 13) and cutaneous (n = 5) melanomas using immunohistochemical analysis of CD3 and MAC387 expression, respectively. Gene expression analysis using the Canine IO NanoString panel was also performed to identify genes and pathways associated with immune cell infiltration. T and myeloid cell densities were variable with geometric means of 158.7 cells/mm2 and 166.7 cells/mm2, respectively. Elevated T cell infiltration was associated with increased expression of cytolytic genes as well as genes encoding the coinhibitory checkpoint molecules PD-1, CTLA-4, TIM-3 and TIGIT; whereas increased myeloid cell infiltration was associated with elevated expression of protumourigenic cytokines. These data provide a basic characterization of the tumour microenvironment of canine malignant melanoma and suggest that, like human melanoma, inherent variability in anti-tumour T cell responses exists and that a subset of canine melanomas may respond better to immunomodulation.

Impact of Equine Ocular Surface Squamous Neoplasia on Interactions between Ocular Transcriptome and Microbiome.

Ocular surface squamous neoplasia (OSSN) represents the most common conjunctival tumor in horses and frequently results in vision loss and surgical removal of the affected globe. Multiple etiologic factors have been identified as contributing to OSSN progression, including solar radiation exposure, genetic mutations, and a lack of periocular pigmentation. Response to conventional treatments has been highly variable, though our recent work indicates that these tumors are highly responsive to local immunotherapy. In the present study, we extended our investigation of OSSN in horses to better understand how the ocular transcriptome responds to the presence of the tumor and how the ocular surface microbiome may also be altered by the presence of cancer. Therefore, we collected swabs from the ventral conjunctival fornix from 22 eyes in this study (11 with cytologically or histologically confirmed OSSN and 11 healthy eyes from the same horses) and performed RNA sequencing and 16S microbial sequencing using the same samples. Microbial 16s DNA sequencing and bulk RNA sequencing were both conducted using an Illumina-based platform. In eyes with OSSN, we observed significantly upregulated expression of genes and pathways associated with inflammation, particularly interferon. Microbial diversity was significantly reduced in conjunctival swabs from horses with OSSN. We also performed interactome analysis and found that three bacterial taxa (Actinobacillus, Helcococcus and Parvimona) had significant correlations with more than 100 upregulated genes in samples from animals with OSSN. These findings highlight the inflammatory nature of OSSN in horses and provide important new insights into how the host ocular surface interacts with certain microbial populations. These findings suggest new strategies for the management of OSSN in horses, which may entail immunotherapy in combination with ocular surface probiotics or prebiotics to help normalize ocular cell and microbe interactions.

Single-cell RNA sequencing reveals the cellular and molecular heterogeneity of treatment-naïve primary osteosarcoma in dogs.

Osteosarcoma (OS) is a heterogeneous, aggressive malignancy of the bone that disproportionally affects children and adolescents. Therapeutic interventions for OS are limited, which is in part due to the complex tumor microenvironment (TME). As such, we used single-cell RNA sequencing (scRNA-seq) to describe the cellular and molecular composition of the TME in 6 treatment-naïve dogs with spontaneously occurring primary OS. Through analysis of 35,310 cells, we identified 41 transcriptomically distinct cell types including the characterization of follicular helper T cells, mature regulatory dendritic cells (mregDCs), and 8 tumor-associated macrophage (TAM) populations. Cell-cell interaction analysis predicted that mregDCs and TAMs play key roles in modulating T cell mediated immunity. Furthermore, we completed cross-species cell type gene signature homology analysis and found a high degree of similarity between human and canine OS. The data presented here act as a roadmap of canine OS which can be applied to advance translational immuno-oncology research.

Review of cellular therapies provides new insights into the potential treatment of diverse neurologic diseases in horses and dogs.

Neurological diseases and injuries in veterinary patients (horses, dogs, and cats) are complex, and effective treatment options are limited. Neuronal loss, damage to nerve conduction pathways, and inflammation and scarring associated with spinal cord injury pose major challenges in managing many neurological diseases. Furthermore, most of these neuropathologies lack definitive pharmacological treatments, driving interest and research into novel interventions. Our objective is to provide a narrative review of the current literature surrounding cellular therapies including neuronal and glial stem cells, neurotrophic factors, mesenchymal stem or stromal cells, and cells derived from induced pluripotent stem cells for the treatment of diverse neurological pathologies. Cellular therapies have the potential for cellular replacement, immune modulation, and paracrine signaling and the flexibility of being used alone or alongside surgical intervention. Mesenchymal stem or stromal cells are arguably the most researched cellular therapy and have been administered intrathecally, IV, intra-arterially, intranasally, and intraspinally with few adverse reactions. Limited clinical and experimental studies have suggested efficacy in diseases including acute spinal cord injury and intervertebral disc disease. Little is currently known about the safety and efficacy of neural stem cells, precursor cell administration, and induced pluripotent stem cell-derived treatments. Further research is necessary to determine the efficacy and long-term safety of cellular therapies. Future aims should include larger controlled clinical trials in companion animals for common neurologic conditions including acute spinal cord injury, intervertebral disc disease, peripheral nerve injury, degenerative neuropathies, and age-associated cognitive decline.

Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity.

The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. The goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed B7-H3; whereas, levels were undetectable on normal dog tissues. Both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little anti-tumor activity was generated by B7-H3 CAR T cells; whereas, B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept.

Intralesional interferon alpha-2b as a novel treatment for periocular squamous cell carcinoma in horses.

OBJECTIVE: To determine the safety and efficacy of perilesional human recombinant interferon alpha-2b (IFNα2b) for treatment of periocular squamous cell carcinoma (PSCC) in horses. ANIMALS STUDIED: Eleven horses (12 eyes) with PSCC were enrolled in this prospective clinical study with owner consent. PROCEDURES: Systemically healthy horses were included in the study following confirmation of PSCC via biopsy. Every two weeks for a maximum of six treatments, horses were sedated and perilesional injection of IFNα2b (10 million IU) was performed. Tumors were measured prior to each injection and at one, three, and 12 months after treatment completion. A greater than 50% reduction in tumor size was considered positive response to treatment (i.e., partial or complete response). Development of anti-IFNα2b antibodies was assessed using serum samples obtained after treatment initiation and compared with treatment responses. Antibody concentrations were analyzed using a mixed model. Statistical significance was considered p < 0.05. RESULTS: Each horse received four to six perilesional injections of IFNα2b. Five of 12 eyes (4/11 horses) responded to treatment. Two of five eyes showed complete resolution of gross PSCC. No systemic adverse effects were seen. Local swelling occurred during treatment protocol in 6/11 horses but resolved without intervention. All horses developed serum anti-IFNα2b antibodies. There was no evidence of statistical difference in antibody concentration between responders and non-responders. CONCLUSIONS: Perilesional administration of IFNα2b was found to be well-tolerated in horses with PSCC, and induced tumor regression in 42% of treated eyes. Treatment failure appears unrelated to the development of IFNα2b antibodies.

Direct comparison of canine and human immune responses using transcriptomic and functional analyses.

The canine spontaneous cancer model is increasingly utilized to evaluate new combined cancer immunotherapy approaches. While the major leukocyte subsets and phenotypes are closely related in dogs and humans, the functionality of T cells and antigen presenting cells in the two species has not been previously compared in detail. Such information would be important in interpreting immune response data and evaluating the potential toxicities of new cancer immunotherapies in dogs. To address this question, we used in vitro assays to compare the transcriptomic, cytokine, and proliferative responses of activated canine and human T cells, and also compared responses in activated macrophages. Transcriptomic analysis following T cell activation revealed shared expression of 515 significantly upregulated genes and 360 significantly downregulated immune genes. Pathway analysis identified 33 immune pathways shared between canine and human activated T cells, along with 34 immune pathways that were unique to each species. Activated human T cells exhibited a marked Th1 bias, whereas canine T cells were transcriptionally less active overall. Despite similar proliferative responses to activation, canine T cells produced significantly less IFN-γ than human T cells. Moreover, canine macrophages were significantly more responsive to activation by IFN-γ than human macrophages, as reflected by co-stimulatory molecule expression and TNF-α production. Thus, these studies revealed overall broad similarity in responses to immune activation between dogs and humans, but also uncovered important key quantitative and qualitative differences, particularly with respect to T cell responses, that should be considered in designing and evaluating cancer immunotherapy studies in dogs.

Targeted transcriptomic analysis of synovial tissues from horses with septic arthritis treated with immune-activated mesenchymal stromal cells reveals induction of T-cell response pathways.

OBJECTIVE: To investigate mechanistically the reported beneficial effects of immune-activated mesenchymal stromal cell (MSC) therapy to treat equine septic arthritis, leveraging Nanostring technology. ANIMALS: 8 Quarter Horses with induced tibiotarsal Staphylococcus aureus septic arthritis treated IA with either Toll-like receptor-3 agonist polyinosinic:polycytidylic acid-activated MSCs + vancomycin antimicrobials (TLR-MSC-VAN; n = 4) or antimicrobials (VAN; 4). METHODS: Synovial tissues were collected and fixed in neutral-buffered 10% formalin, and formalin-fixed paraffin-embedded synovial and osteochondral tissues were sequenced using a custom-designed 200-gene equine Nanostring nCounter immune panel to directly quantify expression of key immune and cartilage-related genes. Immunohistochemistry to detect CD3+ T cells was performed on synovial tissues to further quantify T-cell infiltration in TLR-MSC-VAN- versus VAN-treated joints. RESULTS: Comparison of synovial transcriptomes between groups revealed moderate changes in differential gene expression, with upregulated expression of 9 genes and downregulated expression of 17 genes with fold change ≥ 2 or ≤ -2 and a significant false discovery rate-adjusted P value of ≤ .05. The most upregulated genes in TLR-MSC-VAN-treated horses included those related to T-lymphocyte recruitment and function, while pathways related to innate immune activation and inflammation were significantly downregulated. Immunohistochemistry and quantitation of CD3+ T-cell infiltrates revealed a numerically greater infiltrate in synovial tissues of TLR-MSC-VAN-treated horses, which did not reach statistical significance in this small sample set (P = .20). CLINICAL RELEVANCE: Targeted transcriptomic analyses using an equine Nanostring immune and cartilage health panel provided new mechanistic insights into how innate and adaptive immune cells within synovial tissues respond to TLR-activated MSC treatment when used to treat septic arthritis.

Modulation of In Vitro Macrophage Responses via Primary and Secondary Bile Acids in Dogs.

Bile acids (BA) are important metabolites secreted into the intestinal lumen and impacted by luminal microbes and dietary intake. Prior studies in humans and rodents have shown that BAs are immunologically active and that primary and secondary BAs have distinct immune properties. Therefore, the composition of the gut BA pool may influence GI inflammatory responses. The current study investigated the relative immune modulatory properties of primary (cholic acid, CA) and secondary BAs (lithocholic acid, LCA) by assessing their effects on canine macrophage cytokine secretion and BA receptor (TGR5) expression. In addition, RNA sequencing was used to further interrogate how CA and LCA differentially modulated macrophage responses to LPS (lipopolysaccharide). We found that exposure to either CA or LCA influenced LPS-induced cytokine production via macrophages similarly, with suppression of TNF-α secretion and enhancement of IL-10 secretion. Neither BA altered the expression of the BA receptor TGR5. Transcriptomic analysis revealed that CA activated inflammatory signaling pathways in macrophages involving type II interferon signaling and the aryl hydrocarbon receptor, whereas LCA activated pathways related to nitric oxide signaling and cell cycle regulation. Thus, we concluded that both primary and secondary BAs are active modulators of macrophage responses in dogs, with differential and shared effects evident with sequencing analysis.

Efficacy of an elemental diet in achieving clinical remission in dogs with chronic enteropathy.

BACKGROUND: Diet may induce clinical remission in dogs with chronic enteropathy (CE). Elemental diets (EDs), providing protein as amino acids, modulate intestinal immunity and microbiome in rodents and humans. HYPOTHESIS: Evaluate the impact of an amino acid-based kibble (EL) on CE clinical activity and gastrointestinal (GI)-relevant variables. ANIMALS: Client-owned dogs (n = 23) with inadequately controlled CE. METHODS: Prospective, uncontrolled clinical trial. Diagnostic evaluation including upper and lower GI endoscopy was performed before study entry. Canine chronic enteropathy clinical activity index (CCECAI), serum biomarkers, and fecal microbiome were evaluated before and after 2 weeks of EL. Dogs with stable or improved CE remained in the study for another 6 weeks. Pre- and post-EL clinical and microbiological variables were compared statistically using a mixed model. RESULTS: After 2 weeks of EL, 15 of 22 dogs (68%; 95% confidence interval [CI], 47%-84%) consuming the diet were classified as responders with a median (range) decrease in CCECAI from 6 (3-12) to 2 (0-9; P < .001). Fourteen of 15 responders and 2/7 nonresponders at 2 weeks completed the trial; all 16 were experiencing adequate control at week 8 with a median CCECAI of 2 (0-3). In total, 16/23 dogs (70%; 95% CI, 49%-84%) were responders. Feeding EL caused shifts in fecal bacterial communities, which differed between responders and nonresponders. Serum biomarker concentrations were unchanged throughout the study apart from serum alkaline phosphatase activity. CONCLUSIONS: Exclusive feeding of EL improved clinical signs in 16 of 23 dogs with uncontrolled CE. Fecal microbiome shifts were associated with response to diet and may represent a mechanism for clinical improvement.

Single-cell RNA sequencing reveals the cellular and molecular heterogeneity of treatment-naïve primary osteosarcoma in dogs.

Osteosarcoma (OS) is a heterogeneous, aggressive malignancy of the bone that disproportionally affects children and adolescents. Therapeutic interventions for OS are limited, which is in part due to the complex tumor microenvironment (TME) that has proven to be refractory to immunotherapies. Thus, there is a need to better define the complexity of the OS TME. To address this need, we used single-cell RNA sequencing (scRNA-seq) to describe the cellular and molecular composition of the TME in 6 treatment-naïve dogs with spontaneously occurring primary OS. Through analysis of 35,310 cells, we identified 30 distinct immune cell types, 9 unique tumor populations, 1 cluster of fibroblasts, and 1 cluster of endothelial cells. Independent reclustering of major cell types revealed the presence of follicular helper T cells, mature regulatory dendritic cells (mregDCs), and 8 transcriptomically distinct macrophage/monocyte populations. Cell-cell interaction inference analysis predicted that mregDCs and tumor-associated macrophages (TAMs) play key roles in modulating T cell mediate immunity. Furthermore, we used publicly available human OS scRNA-seq data to complete a cross-species cell type gene signature homology analysis. The analysis revealed a high degree of cell type gene signature homology between species, suggesting the cellular composition of OS is largely conserved between humans and dogs. Our findings provide key new insights into the biology of canine OS and highlight the conserved features of OS across species. Generally, the data presented here acts as a cellular and molecular roadmap of canine OS which can be applied to advance the translational immuno-oncology research field.

Erythrocyte removal from bone marrow aspirate concentrate improves efficacy as intra-articular cellular therapy in a rodent osteoarthritis model.

Background: Despite the high prevalence of osteoarthritis (OA), there remains a need for additional therapeutic options. Cellular therapies with minimally manipulated cells such as bone marrow aspirate concentrates (BMAC) are increasingly popular in the U.S. but clear-cut evidence of efficacy has not been established. In theory, BMAC injections provide a source of stromal cells to stimulate healing in OA and ligamentous injuries; however, BMAC injections are also often associated with inflammation, short-term pain, and mobility impairment. Given that blood is known to trigger inflammation in joints, we hypothesized that removing erythrocytes [red blood cells (RBCs)] from BMAC preparations prior to intra-articular injection would improve efficacy for OA treatment. Methods: To test this hypothesis, BMAC was collected from the bone marrow of mice. Three treatment groups were pursued: (I) untreated; (II) BMAC; or (III) BMAC depleted of RBCs by lysis. Product was injected into the femorotibial joint of mice 7 days after OA had been induced by destabilization of the medial meniscus (DMM). To assess the impact of treatment on joint function, individual cage monitoring (ANY-mazeTM) and Digigait treadmill-based analyses were performed over 4 weeks. At study completion, joint histopathology was assessed and immune transcriptomes within joint tissues were compared using a species-specific NanoString panel. Results: Significant improvements in activity, gait parameters, and histology scores were seen in animals receiving RBC-depleted BMAC compared to untreated mice; animals treated with non-depleted BMAC did not demonstrate this same extent of consistent significant improvement. Transcriptomic analysis of joint tissues revealed significant upregulation of key anti-inflammatory genes, including interleukin-1 receptor antagonist (IRAP), in mice treated with RBC-depleted BMAC compared to animals treated with non-RBC depleted BMAC. Conclusions: These findings indicate that RBC depletion in BMAC prior to intra-articular injection improves treatment efficacy and reduces joint inflammation compared to BMAC.

Antimicrobial Properties of Equine Stromal Cells and Platelets and Future Directions.

Increasing antimicrobial resistance in veterinary practice has driven the investigation of novel therapeutic strategies including regenerative and biologic therapies to treat bacterial infection. Integration of biological approaches such as platelet lysate and mesenchymal stromal cell (MSC) therapy may represent adjunctive treatment strategies for bacterial infections that minimize systemic side effects and local tissue toxicity associated with traditional antibiotics and that are not subject to antibiotic resistance. In this review, we will discuss mechanisms by which biological therapies exert antimicrobial effects, as well as potential applications and challenges in clinical implementation in equine practice.

A single-cell RNA sequencing atlas of circulating leukocytes from healthy and osteosarcoma affected dogs.

Translationally relevant animal models are essential for the successful translation of basic science findings into clinical medicine. While rodent models are widely accessible, there are numerous limitations that prevent the extrapolation of findings to human medicine. One approach to overcome these limitations is to use animal models that are genetically diverse and naturally develop disease. For example, pet dogs spontaneously develop diseases that recapitulate the natural progression seen in humans and live in similar environments alongside humans. Thus, dogs represent a useful animal model for many areas of research. Despite the value of the canine model, species specific reagent limitations have hampered in depth characterization of canine immune cells, which constrains the conclusions that can be drawn from canine immunotherapy studies. To address this need, we used single-cell RNA sequencing to characterize the heterogeneity of circulating leukocytes in healthy dogs (n = 7) and osteosarcoma (OS) affected dogs (n = 10). We present a cellular atlas of leukocytes in healthy dogs, then employ the dataset to investigate the impact of primary OS tumors on the transcriptome of circulating leukocytes. We identified 36 unique cell populations amongst dog circulating leukocytes, with a remarkable amount of heterogeneity in CD4 T cell subtypes. In our comparison of healthy dogs and dogs with OS, we identified relative increases in the abundances of polymorphonuclear (PMN-) and monocytic (M-) myeloid-derived suppressor cells (MDSCs), as well as aberrations in gene expression within myeloid cells. Overall, this study provides a detailed atlas of canine leukocytes and investigates how the presence of osteosarcoma alters the transcriptional profiles of circulating immune cells.

Evaluation of Immune Nanoparticles for Rapid and Non-Specific Activation of Antiviral and Antibacterial Immune Responses in Cattle, Swine, and Poultry.

Given the rapid potential spread of agricultural pathogens, and the lack of vaccines for many, there is an important unmet need for strategies to induce rapid and non-specific immunity against these viral and bacterial threats. One approach to the problem is to generate non-specific immune responses at mucosal surfaces to rapidly protect from entry and replication of both viral and bacterial pathogens. Using complexes of charged nanoparticle liposomes with both antiviral and antibacterial toll-like receptor (TLR) nucleic acid ligands (termed liposome-TLR complexes or LTC), we have previously demonstrated considerable induction of innate immune responses in nasal and oropharyngeal tissues and protection from viral and bacterial pathogens in mixed challenge studies in rodents, cattle, and companion animals. Therefore, in the present study, we used in vitro assays to evaluate the ability of the LTC immune stimulant to activate key innate immune pathways, particularly interferon pathways, in cattle, swine, and poultry. We found that LTC complexes induced strong production of type I interferons (IFNα and IFNß) in both macrophages and leukocyte cultures from all three species. In addition, the LTC complexes induced the production of additional key protective cytokines (IL-6, IFNγ, and TNFα) in macrophages and leukocytes in cattle and poultry. These findings indicate that the LTC mucosal immunotherapeutic has the capability to activate key innate immune defenses in three major agricultural species and potentially induce broad protective immunity against both viral and bacterial pathogens. Additional animal challenge studies are warranted to evaluate the protective potential of LTC immunotherapy in cattle, swine, and poultry.

Strategies to overcome myeloid cell induced immune suppression in the tumor microenvironment.

Cancer progression and metastasis due to tumor immune evasion and drug resistance is strongly associated with immune suppressive cellular responses, particularly in the case of metastatic tumors. The myeloid cell component plays a key role within the tumor microenvironment (TME) and disrupts both adaptive and innate immune cell responses leading to loss of tumor control. Therefore, strategies to eliminate or modulate the myeloid cell compartment of the TME are increasingly attractive to non-specifically increase anti-tumoral immunity and enhance existing immunotherapies. This review covers current strategies targeting myeloid suppressor cells in the TME to enhance anti-tumoral immunity, including strategies that target chemokine receptors to deplete selected immune suppressive myeloid cells and relieve the inhibition imposed on the effector arms of adaptive immunity. Remodeling the TME can in turn improve the activity of other immunotherapies such as checkpoint blockade and adoptive T cell therapies in immunologically "cold" tumors. When possible, in this review, we have provided evidence and outcomes from recent or current clinical trials evaluating the effectiveness of the specific strategies used to target myeloid cells in the TME. The review seeks to provide a broad overview of how myeloid cell targeting can become a key foundational approach to an overall strategy for improving tumor responses to immunotherapy.

Distinct differences in immunological properties of equine orthobiologics revealed by functional and transcriptomic analysis using an activated macrophage readout system.

Introduction: Multiple biological therapies for orthopedic injuries are marketed to veterinarians, despite a lack of rigorous comparative biological activity data to guide informed decisions in selecting a most effective compound. Therefore, the goal of this study was to use relevant bioassay systems to directly compare the anti-inflammatory and immunomodulatory activity of three commonly used orthobiological therapies (OTs): mesenchymal stromal cells (MSC), autologous conditioned serum (ACS), and platelet rich plasma (PRP). Methods: Equine monocyte-derived macrophages were used as the readout system to compare therapies, including cytokine production and transcriptomic responses. Macrophages were stimulated with IL-1ß and treated 24 h with OTs, washed and cultured an additional 24 h to generate supernatants. Secreted cytokines were measured by multiplex immunoassay and ELISA. To assess global transcriptomic responses to treatments, RNA was extracted from macrophages and subjected to full RNA sequencing, using an Illumina-based platform. Data analysis included comparison of differentially expressed genes and pathway analysis in treated vs. untreated macrophages. Results: All treatments reduced production of IL-1ß by macrophages. Secretion of IL-10 was highest in MSC-CM treated macrophages, while PRP lysate and ACS resulted in greater downregulation of IL-6 and IP-10. Transcriptomic analysis revealed that ACS triggered multiple inflammatory response pathways in macrophages based on GSEA, while MSC generated significant downregulation of inflammatory pathways, and PRP lysate induced a mixed immune response profile. Key downregulated genes in MSC-treated cultures included type 1 and type 2 interferon response, TNF-α and IL-6. PRP lysate cultures demonstrated downregulation of inflammation-related genes IL-1RA, SLAMF9, ENSECAG00000022247 but concurrent upregulation of TNF-α, IL-2 signaling, and Myc targets. ACS induced upregulation of inflammatory IL-2 signaling, TNFα and KRAS signaling and hypoxia, but downregulation of MTOR signaling and type 1 interferon signaling. Discussion: These findings, representing the first comprehensive look at immune response pathways for popular equine OTs, reveal distinct differences between therapies. These studies address a critical gap in our understanding of the relative immunomodulatory properties of regenerative therapies commonly used in equine practice to treat musculoskeletal disease and will serve as a platform from which further in vivo comparisons may build.

Chronic adrenergic stress and generation of myeloid-derived suppressor cells: Implications for cancer immunotherapy in dogs.

Recent studies have highlighted a key role played by the sympathetic nervous system (SNS) and adrenergic stress in mediating immune suppression associated with chronic inflammation in cancer and other diseases. The connection between chronic SNS activation, adrenergic stress and immune suppression is linked in part to the ability of catecholamines to stimulate the bone marrow release and differentiation of myeloid-derived suppressor cells (MDSC). Rodent model studies have revealed an important role for ß-adrenergic receptor signalling in suppression of cancer immunity in mice subjected to chronic stresses, including thermal stress. Importantly, therapeutic blockade of beta-adrenergic responses by drugs such as propranolol can partially reverse the generation and differentiation of MDSC, and partly restore tumour immunity. Clinical trials in both humans and dogs with cancer have demonstrated that propranolol blockade can improve responses to radiation therapy, cancer vaccines and immune checkpoint inhibitors. Thus, the SNS stress response has become an important new target to relieve immune suppression in cancer and other chronic inflammatory conditions.