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.

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.

Novel ocular immunotherapy induces tumor regression in an equine model of ocular surface squamous neoplasia.

Ocular surface squamous neoplasia (OSSN) is the major cause of corneal cancer in man and horses worldwide, and the prevalence of OSSN is increasing due to greater UVB exposure globally. Currently, there are no approved treatments for OSSN in either species, and most patients are managed with surgical excision or off-label treatment with locally injected interferon alpha, or topically applied cytotoxic drugs such as mitomycin C. A more broadly effective and readily applied immunotherapy could exert a significant impact on management of OSSN worldwide. We therefore evaluated the effectiveness of a liposomal TLR complex (LTC) immunotherapy, which previously demonstrated strong antiviral activity in multiple animal models following mucosal application, for ocular antitumor activity in a horse spontaneous OSSN model. In vitro studies demonstrated strong activation of interferon responses in horse leukocytes by LTC and suppression of OSSN cell growth and migration. In a trial of 8 horses (9 eyes), treatment with topical or perilesional LTC resulted in an overall tumor response rate of 67%, including durable regression of large OSSN tumors. Repeated treatment with LTC ocular immunotherapy was also very well tolerated clinically. We conclude therefore that ocular immunotherapy with LTC warrants further investigation as a novel approach to management of OSSN in humans.

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.

Immunologic Effects of Stereotactic Body Radiotherapy in Dogs with Spontaneous Tumors and the Impact of Intratumoral OX40/TLR Agonist Immunotherapy.

Stereotactic body radiotherapy (SBRT) is known to induce important immunologic changes within the tumor microenvironment (TME). However, little is known regarding the early immune responses within the TME in the first few weeks following SBRT. Therefore, we used the canine spontaneous tumor model to investigate TME responses to SBRT, and how local injection of immune modulatory antibodies to OX40 and TLR 3/9 agonists might modify those responses. Pet dogs with spontaneous cancers (melanoma, carcinoma, sarcoma, n = 6 per group) were randomized to treatment with either SBRT or SBRT combined with local immunotherapy. Serial tumor biopsies and serum samples were analyzed for immunologic responses. SBRT alone resulted at two weeks after treatment in increased tumor densities of CD3+ T cells, FoxP3+ Tregs, and CD204+ macrophages, and increased expression of genes associated with immunosuppression. The addition of OX40/TLR3/9 immunotherapy to SBRT resulted in local depletion of Tregs and tumor macrophages and reduced Treg-associated gene expression (FoxP3), suppressed macrophage-associated gene expression (IL-8), and suppressed exhausted T cell-associated gene expression (CTLA4). Increased concentrations of IL-7, IL-15, and IL-18 were observed in serum of animals treated with SBRT and immunotherapy, compared to animals treated with SBRT. A paradoxical decrease in the density of effector CD3+ T cells was observed in tumor tissues that received combined SBRT and immunotherapy as compared to animals treated with SBRT only. In summary, these results obtained in a spontaneous large animal cancer model indicate that addition of OX40/TLR immunotherapy to SBRT modifies important immunological effects both locally and systemically.

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

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.

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

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 × 105 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 × 106 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.

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

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/cm2 , 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.

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

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.

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

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.

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.

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.

The potential for senotherapy as a novel approach to extend life quality in veterinary medicine.

Cellular senescence, a condition where cells undergo arrest and can assume an inflammatory phenotype, has been associated with initiation and perpetuation of inflammation driving multiple disease processes in rodent models and humans. Senescent cells secrete inflammatory cytokines, proteins, and matrix metalloproteinases, termed the senescence associated secretory phenotype (SASP), which accelerates the aging processes. In preclinical models, drug interventions termed "senotherapeutics" selectively clear senescent cells and represent a promising strategy to prevent or treat multiple age-related conditions in humans and veterinary species. In this review, we summarize the current available literature describing in vitro evidence for senotheraputic activity, preclinical models of disease, ongoing human clinical trials, and potential clinical applications in veterinary medicine. These promising data to date provide further justification for future studies identifying the most active senotherapeutic combinations, dosages, and routes of administration for use in veterinary medicine.

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.

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.

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.

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.

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.

Nanoparticle ocular immunotherapy for herpesvirus surface eye infections evaluated in cat infection model.

Ocular herpes simplex type 1 (HSV-1) infections can trigger conjunctivitis, keratitis, uveitis, and occasionally retinitis, and is a major cause of blindness worldwide. The infections are lifelong and can often recrudesce during periods of stress or immune suppression. Currently HSV-1 infections of the eye are managed primarily with anti-viral eye drops, which require frequent administration, can cause irritation, and may take weeks for full resolution of symptoms. We therefore evaluated the effectiveness of an ocular immune activating nanoparticle eye drop as a novel approach to treating HSV-1 infection, using a cat feline herpesvirus -1 (FHV-1) ocular infection model. In vitro studies demonstrated significant induction of both type I and II interferon responses by the liposome-dual TLR 3/9 agonist nanoparticles, along with suppression of FHV-1 replication. In cats with naturally occurring eye infections either proven or suspected to involve FHV-1, ocular nanoparticle treated animals experienced resolution of signs within several days of treatment, including resolution of keratitis and corneal ulcers. In a cat model of recrudescent FHV-1 infection, cats treated twice daily with immune nanoparticle eye drops experienced significant lessening of ocular signs of infection and significantly fewer episodes of viral shedding compared to control cats. Treatment was well-tolerated by all cats, without signs of drug-induced ocular irritation. We concluded therefore that non-specific ocular immunotherapy offers significant promise as a novel approach to treatment of HSV-1 and FHV-1 ocular infections.

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.