Investigation of Gastrointestinal Toxicities Associated with Concurrent Abdominal Radiation Therapy and the Tyrosine Kinase Inhibitor Sunitinib in a Mouse Model.

Tyrosine kinase inhibitors (TKIs) may be combined with radiation therapy (RT) to enhance tumor control; however, increased incidences of gastrointestinal (GI) toxicity have been reported with this combination. We hypothesize that toxicity is due to compromised intestinal healing caused by inhibition of vascular repair and proliferation pathways. This study explores underlying tissue toxicity associated with abdominal RT and concurrent sunitinib in a mouse model. Four groups of CD-1 mice were treated with 12 Gy abdominal RT, oral sunitinib, abdominal RT + sunitinib, or sham treatment. Mice received oral sunitinib or the vehicle via gavage for 14 days. On day 7, mice were irradiated with 12 Gy abdominal RT or sham treated. Mice were euthanized on day 14 and intestinal tract was harvested for semiquantitative histopathologic evaluation and immunohistochemical quantification of proliferation (Ki67) and vascular density (CD31). Non-irradiated groups had stable weights while abdominal irradiation resulted in weight loss, with mice receiving RT + SUN having greater weight loss than mice receiving RT alone. Semiquantitative analysis showed significant increases in inflammation in irradiated groups. The difference in the density of CD31+ cells was significantly increased in RT alone compared to SUN alone. Ki67+ density was not significant. In summary, we identify a lack of angiogenic response in irradiated GI tissues when abdominal RT is combined with a TKI, which may correlate with clinical toxicities seen in canine and human patients receiving combined treatment.

Biphasic Interpositional Allograft for Rotator Cuff Repair Augmentation Is Safe in an Ovine Model.

PURPOSE: To perform a preclinical histologic assessment of a biphasic acellular interpositional cancellous allograft in an ovine model of rotator cuff repair (RCR) designed to better understand its safety profile and effects on tendon healing after RCR. METHODS: Thirty skeletally mature sheep with clinically normal shoulders with an artificially created degenerative infraspinatus tendon tear were randomized to control and treatment groups. Animals were euthanized at 3 weeks, 6 weeks, and 12 weeks. After gross dissection, rotator cuff specimens were fixed with formalin and polymerized for sectioning and staining. Blinded histologic scores evaluated inflammatory cell infiltrates, signs of degradation, particulate debris, collagen arrangement, neovascularization, and enthesis qualitative measures. RESULTS: There were no treatment specimens that exhibited histologic signs of a significant infection, inflammatory infiltrate, or foreign body reaction such as granuloma or fibrous capsule formation. Histologic scores in all categories were not significantly different at all time points, including the primary end point mean cumulative inflammatory score (control: 3.66 ± 1.21 vs treated: 4.33 ± 1.51, P = .42), when comparing the treatment and control RCR groups. In general, the degree of tendon healing and host tissue response was essentially equivalent between the 2 groups with observation of low overall levels of inflammation and progressive improvements in collagen organization, reduced tenocyte activity, and fibrocartilaginous enthesis reformation. CONCLUSIONS: This histologic study demonstrated the use of a biphasic interpositional allograft for RCR augmentation in an ovine model does not generate an inflammatory response or foreign body reaction. Use of the biphasic interpositional allograft resulted in a histological profile that was essentially equivalent to that of a standard RCR at 3-, 6-, and 12-week postoperative timepoints. These findings suggest that a biphasic interpositional allograft is safe for further clinical investigation in humans before broader clinical application. CLINICAL RELEVANCE: Patch augmentation of RCR is a popular technique that has shown clinical success in improving the likelihood of a successful repair in patients at elevated risk for retear. Newer augmentation technologies are being developed to address the biology at the interface between the bone and soft tissue where failure typically occurs.

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 influence of estrogen receptor α signaling independent of the estrogen response element on avoidance behavior, social interactions, and palatable ingestive behavior in female mice.

Women are vulnerable to developing mental disorders that are associated with circulating estrogens. Estrogens, especially 17ß-estradiol (E2), have a wide array of effects on the brain, affecting many behavioral endpoints associated with mental illness. By using a total estrogen receptor (ER) α knockout (KO), an ERα knock in/knock out (KIKO) that lacks a functional DNA-binding domain, and wild type (WT) controls treated with either oil or E2, we evaluated ERα signaling, dependent and independent of the estrogen response element (ERE), on avoidance behavior, social interactions and memory, and palatable ingestive behavior using the open field test, the elevated plus maze, the light dark box, the 3-chamber test, and palatable feeding. We found that ERα does not mediate control of anxiety-like behaviors but rather yielded differences in locomotor activity. In evaluating social preference and social recognition memory, we observed that E2 may modulate these measures in KIKO females but not KO females, suggesting that ERE-independent signaling is likely involved in sociability. Lastly, observations of palatable (high-fat) food intake suggested an increase in palatable eating behavior in oil-treated KIKO females. Oil-treated KO females had a longer latency to food intake, indicative of an anhedonic phenotype compared to oil-treated WT and KIKO females. We have observed that social-related behaviors are potentially influenced by ERE-independent ERα signaling and hedonic food intake requires signaling of ERα.

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.

Evaluation of Myogenin and MyoD1 as Immunohistochemical Markers of Canine Rhabdomyosarcoma.

Canine rhabdomyosarcoma (RMS) presents a diagnostic challenge due to its overlapping histologic features with other soft tissue sarcomas. The diagnosis of RMS currently relies on positive immunohistochemical (IHC) labeling for desmin; however, desmin expression is also observed in non-RMS tumors. Myogenin and MyoD1 are transcription factors reported to be sensitive and specific IHC markers for human RMS, but they are not widely used in veterinary oncology. The goals of this study were to develop an IHC protocol for myogenin and MyoD1, evaluate myogenin and MyoD1 labeling in canine RMS, and report clinical outcomes. Sixteen cases of possible RMS were retrospectively evaluated. A diagnosis of RMS was confirmed in 13 cases based on histological features and immunolabeling for myogenin and MyoD1, with the aid of electron microscopy in 2 cases. Desmin was negative in 3 cases of RMS. Two cases were of the sclerosing variant. The median age of dogs with RMS was 7.2 years. Anatomic tumor locations included previously reported sites such as bladder, larynx, heart, and orbit, as well as other locations typical of soft tissue sarcomas. Survival ranged from 47 to 1480 days for 5 dogs with available data. This study demonstrated that MyoD1 and myogenin should be included with desmin as part of a diagnostic IHC panel for canine RMS. Utilization of these antibodies to improve the accuracy of canine RMS diagnosis will ultimately allow for better characterization of the biological behavior and clinical outcomes of this disease, providing the groundwork for future comparative investigations in canine RMS.

Role of Periostin Expression in Canine Osteosarcoma Biology and Clinical Outcome.

Periostin is a matricellular protein important in regulating bone, tooth, and cardiac development. In pathologic conditions, periostin drives allergic and fibrotic inflammatory diseases and is also overexpressed in certain cancers. Periostin signaling in tumors has been shown to promote angiogenesis, metastasis, and cancer stem cell survival in rodent models, and its overexpression is associated with poor prognosis in human glioblastoma. However, the role of periostin in regulating tumorigenesis of canine cancers has not been evaluated. Given its role in bone development, we sought to evaluate mRNA and protein expression of periostin in canine osteosarcoma (OS) and assess its association with patient outcome. We validated an anti-human periostin antibody cross-reactive to canine periostin via western blot and immunohistochemistry and evaluated periostin expression in microarray data from 49 primary canine OS tumors and 8 normal bone samples. Periostin mRNA was upregulated greater than 40-fold in canine OS tumors compared to normal bone and was significantly correlated with periostin protein expression based on quantitative image analysis. However, neither periostin mRNA nor protein expression were associated with time to metastasis in this cohort. Gene Set Enrichment Analysis demonstrated significant enhancement of pro-tumorigenic pathways including canonical WNT signaling, epithelial-mesenchymal transition, and angiogenesis in periostin-high tumors, while periostin-low tumors demonstrated evidence of heightened antitumor immune responses. Overall, these data identify a novel antibody that can be used as a tool for evaluation of periostin expression in dogs and suggest that investigation of Wnt pathway-targeted drugs in periostin overexpressing canine OS may be a potential therapeutic target.

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.

High-fat diet induced central adiposity (visceral fat) is associated with increased fibrosis and decreased immune cellularity of the mesenteric lymph node in mice.

PURPOSE: Accumulation of visceral, but not subcutaneous, adipose tissue is highly associated with metabolic disease. Inflammation inciting from adipose tissue is commonly associated with metabolic disease risk and comorbidities. However, constituents of the immune system, lymph nodes, embedded within these adipose depots remain under-investigated. We hypothesize that, lymph nodes are inherently distinct and differentially respond to diet-induced obesity much like the adipose depots they reside in. METHODS: Adipose tissue and lymph nodes were collected from the visceral and inguinal depots of male mice fed 13 weeks of standard CHOW or high fat diet (HFD). Immune cells were isolated from tissues, counted and characterized by flow cytometry or plated for proliferative capacity following Concanavalin A stimulation. Lymph node size and fibrosis area were also characterized. RESULTS: In HFD fed mice visceral adipose tissue accumulation was associated with significant enlargement of the lymph node encased within. The subcutaneous lymph node did not change. Compared with mice fed CHOW for 13 weeks, mice fed HFD had a decline in immune cell populations and immune cell proliferative ability, as well as, exacerbated fibrosis accumulation, within the visceral, but not subcutaneous, lymph node. CONCLUSIONS: Obesity-induced chronic low-grade inflammation is associated with impaired immunity and increased susceptibility to disease. Excessive visceral adiposity and associated inflammation driven by diet likely leads to obesity-induced immune suppression by way of lymph node/lymphatic system pathophysiology.

Iliac vein stent migration with extensive cardiac damage in a patient with May-Thurner syndrome.

In May-Thurner syndrome, or iliac vein compression syndrome, the left common iliac vein is compressed between the right common iliac artery and corresponding vertebral body, increasing patient risk for deep vein thrombosis. Iliac vein stenting for patients with symptomatic May-Thurner syndrome has become standard practice in many centers. This article describes a patient whose stent embolized completely to the right ventricle, destroying most structures in its path.

Characterization of an Inducible Alcoholic Liver Fibrosis Model for Hepatocellular Carcinoma Investigation in a Transgenic Porcine Tumorigenic Platform.

PURPOSE: This study used the Oncopig Cancer Model (OCM) to develop alcohol-induced fibrosis in a porcine model capable of developing hepatocellular carcinoma. MATERIALS AND METHODS: Liver injury was induced in 8-week-old Oncopigs (n = 10) via hepatic transarterial infusion of 0.75 mL/kg ethanol-ethiodized oil (1:3 v/v). Feasibility was assessed in an initial Oncopig cohort (n = 5) by histologic analysis at 8 weeks after induction, and METAVIR results were compared to age- and sex-matched healthy controls (n = 5). Liver injury was then induced in a second OCM cohort (n = 5) for a time-course study, with post-induction disease surveillance via biweekly physical exam, lab analysis, and liver biopsies until 20 weeks after induction. RESULTS: In Cohort 1, 8-week post-induction liver histologic analysis revealed median METAVIR F3 (range, F3-F4) fibrosis, A2 (range, A2-A3) inflammation, and 15.3% (range, 5.0%-22.9%) fibrosis. METAVIR and inflammation scores were generally elevated compared to healthy controls (F0-F1, P = 0.0013; A0-A1, P = .0013; median percent fibrosis 8.7%, range, 5.8%-12.1%, P = .064). In Cohort 2, histologic analysis revealed peak fibrosis severity of median METAVIR F3 (range, F2-F3). However, lack of persistent alcohol exposure resulted in liver recovery, with median METAVIR F2 (range, F1-F2) fibrosis at 20 weeks after induction. No behavioral or biochemical abnormalities were observed to indicate liver decompensation. CONCLUSIONS: This study successfully validated a protocol to develop METAVIR F3-F4 fibrosis within 8 weeks in the OCM, supporting its potential to serve as a model for hepatocellular carcinoma in a fibrotic liver background. Further investigation is required to determine if repeated alcohol liver injury is required to develop an irreversible METAVIR grade F4 porcine cirrhosis model.

Regulation of PD-L1 expression on murine tumor-associated monocytes and macrophages by locally produced TNF-α.

PD-L1 is an immune checkpoint protein that has emerged as a major signaling molecule involved with tumor escape from T cell immune responses. Studies have shown that intra-tumoral expression of PD-L1 can inhibit antitumor immune responses. However, it has recently been shown that expression of PD-L1 on myeloid cells from the tumor is a stronger indicator of prognosis than tumor cell PD-L1 expression. Therefore, it is important to understand the factors that govern the regulation of PD-L1 expression on tumor-infiltrating myeloid cells. We found that immature bone marrow monocytes in tumor-bearing mice had low levels of PD-L1 expression, while higher levels of expression were observed on monocytes in circulation. In contrast, macrophages found in tumor tissues expressed much higher levels of PD-L1 than circulating monocytes, implying upregulation by the tumor microenvironment. We demonstrated that tumor-conditioned media strongly induced increased PD-L1 expression by bone marrow-derived monocytes and TNF-α to be a cytokine that causes an upregulation of PD-L1 expression by the monocytes. Furthermore, we found production of TNF-α by the monocytes themselves to be a TLR2-dependent response to versican secreted by tumor cells. Thus, PD-L1 expression by tumor macrophages appears to be regulated in a different manner than by tumor cells themselves.

Autophagy Inhibition Delays Early but Not Late-Stage Metastatic Disease.

The autophagy pathway has been recognized as a mechanism of survival and therapy resistance in cancer, yet the extent of autophagy's function in metastatic progression is still unclear. Therefore, we used murine models of metastatic cancer to investigate the effect of autophagy modulation on metastasis development. Pharmacologic and genetic autophagy inhibition were able to impede cell proliferation in culture, but did not impact the development of experimentally induced 4T1 and B16-F10 metastases. Similarly, autophagy inhibition by adjuvant chloroquine (CQ) treatment did not delay metastasis in an orthotopic 4T1, tumor-resection model. However, neoadjuvant CQ treatment or genetic autophagy inhibition resulted in delayed metastasis development, whereas stimulation of autophagy by trehalose hastened development. Cisplatin was also administered either as a single agent or in combination with CQ. The combination of cisplatin and CQ was antagonistic. The effects of autophagy modulation on metastasis did not appear to be due to alterations in the intrinsic metastatic capability of the cells, as modulating autophagy had no impact on migration, invasion, or anchorage-independent growth in vitro. To explore the possibility of autophagy's influence on the metastatic microenvironment, bone marrow-derived cells (BMDCs), which mediate the establishment of the premetastatic niche, were measured in the lung and in circulation. Trehalose-treated mice had significantly more BMDCs than either vehicle- or CQ-treated mice. Autophagy inhibition may be most useful as a treatment to impede early metastatic development. However, modulating autophagy may also alter the efficacy of platinum-based therapies, requiring caution when considering combination therapies.

Do Mesenchymal Stromal Cells Influence Microscopic Residual or Metastatic Osteosarcoma in a Murine Model?

BACKGROUND: Mesenchymal stromal cells (MSCs) have been shown in rodent models to promote primary and pulmonary metastatic sarcoma growth when injected in the presence of gross tumor. In theory, this would limit their use in a clinical setting after limb salvage treatment for osteosarcoma. Although concerning, these models do not translate to the clinical setting wherein MSCs could be used after primary tumor resection to aid in bone healing and incorporation of tumor endoprostheses. If we can determine whether the use of MSCs in this setting is safe, it might improve our ability to augment bone healing in patients undergoing limb salvage. QUESTIONS/PURPOSES: The purpose of this study was to determine (1) whether MSCs promote pulmonary metastatic disease progression in a murine osteosarcoma model; and/or (2) whether they affect local disease recurrence in the presence of microscopic residual osteosarcoma. METHODS: An orthotopic model of luciferase-expressing osteosarcoma was developed. At 10 days, resection of the primary tumor was performed. One hundred fourteen female C3H mice were inoculated with DLM8-luc osteosarcoma in the proximal tibia. Ninety-four mice developed orthotopic osteosarcoma with luciferase expression. Mice with bioluminescent evidence of a primary tumor received either a microscopically "clean" amputation at a time when residual microscopic metastatic disease was present in the lungs (pulmonary metastasis group; n = 65) or a "dirty" amputation (local recurrence group; n = 29). Mice were randomized to receive intravenous MSCs, MSCs at the surgical site, or no MSCs. Mice were monitored for development and progression of pulmonary metastasis and local recurrence by bioluminescence imaging and daily measurements at the surgical site. The number of pulmonary nodules, time to first evidence of metastasis, and size of recurrent tumor were compared using Kruskal-Wallis, analysis of variance, Welch's, t-tests, or Mann-Whitney tests as appropriate for the specific data sets with p < 0.05 considered significant. RESULTS: Mice receiving intravenous MSCs had a faster time to first detection of pulmonary metastasis (2.93 ± 1.90 days) compared with mice with local injection of MSCs (6.94 ± 6.78 days) or no MSCs (5.93 ± 4.55 days) (p = 0.022). MSC treatment did not influence whether mice developed local recurrence (p = 0.749) or size of recurrent tumors (p = 0.221). CONCLUSIONS: MSCs delivered to the surgical site did not promote local recurrence or size of recurrent tumors, but intravenous injection of MSCs did hasten onset of detection of pulmonary metastatic disease. Although local administration of MSCs into a surgical site does not appear to promote either pulmonary metastatic disease or local recurrence, large variation within groups and small numbers diminished statistical power such that a Type II error cannot be ruled out. CLINICAL RELEVANCE: If MSCs are to be used to augment bone healing in the postlimb salvage setting in patients with osteosarcoma, it will be important to understand their influence, if any, on pulmonary micrometastsis or residual microscopic local disease. Although murine models do not completely recapitulate the clinical scenario, these results suggest that intravenous delivery of MSCs may promote micrometastatic pulmonary disease. Local administration into a surgical wound, even in the presence of residual microscopic disease, may be safe, at least in this murine model, but further investigation is warranted before considering the use of MSCs for clinical use in patients with osteosarcoma.

Effects of recent cannabis consumption on eye-tracking and pupillometry.

Introduction: Cannabis consumption is known to immediately affect ocular and oculomotor function, however, cannabis consumption is also known to affect it for a prolonged period of time. The purpose of this study is to identify an eye tracking or pupillometry metric which is affected after recent cannabis consumption but is not confounded by cannabis consumption history or demographic variables. Methods: Quasi-experimental design. Participants who would consume inhalable cannabis (n = 159, mean age 31.0 years, 54% male) performed baseline neurobehavioral testing and eye-function assessments when they were sober. Eye function assessments included eye-tracking [gaze (point of visual focus), saccades (smooth movement)] and pupillometry. Participants then inhaled cannabis until they self-reported to be high and performed the same assessment again. Controls who were cannabis naïve or infrequent users (n = 30, mean age 32.6 years, 57% male) performed the same assessments without consuming cannabis in between. Results: Cannabis significantly affected several metrics of pupil dynamics and gaze. Pupil size variability was the most discriminant variable after cannabis consumption. This variable did not change in controls on repeat assessment (i.e., no learning effect), did not correlate with age, gender, race/ethnicity, or self-reported level of euphoria, but did correlate with THC concentration of cannabis inhaled. Discussion: A novel eye-tracking metric was identified that is affected by recent cannabis consumption and is not different from non-users at baseline. A future study that assesses pupil size variability at multiple intervals over several hours and quantifies cannabis metabolites in biofluids should be performed to identify when this variable normalizes after consumption and if it correlates with blood THC levels.

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.

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.

Characterization of an intermediate in the ammonia-forming reaction of Fe(DMeOPrPE)2N2 with acid (DMeOPrPE = 1,2-[bis(dimethoxypropyl)phosphino]ethane).

The reactivity of Fe(DMeOPrPE)2N2 with water and acid was explored. (DMeOPrPE is the bidentate phosphine 1,2-[bis(dimethoxypropyl)phosphino]ethane.) The complex reacts with acid to form trans-[Fe(DMeOPrPE)2(N2)H](+) and small amounts of ammonia and hydrazine. When reacted with H2O, cis-Fe(DMeOPrPE)2(H)2 is formed. To increase the yields of ammonia and hydrazine, we investigated the effect of anion, solvent, and acid addition rate on the yields of ammonia. Of these parameters, only the properties of the anion (i.e., of the acid) had a significant impact on the yields of ammonia. The highest yields of NH3 occurred with the largest/least-coordinating anion (triflate). A short-lived purple intermediate (τ1/2 < 5 s at 23 °C) was observed in the reaction of Fe(DMeOPrPE)2N2 with triflic acid. Because the structure of this purple species could potentially provide valuable insights into the mechanism of ammonia formation, a method was developed for independently synthesizing and stabilizing the complex. Spectroscopic characterization of the purple species identified it as the paramagnetic [((DMeOPrPE)2Fe)2(µ-N2)](2+) complex (1). This purple dimer (1) exists in equilibrium with yellow, monomeric, paramagnetic [Fe(DMeOPrPE)2N2](+) (2). The role of 1 in the formation of hydrazine and ammonia was probed by reacting 1 with acid.

To what extent does the Health Professions Admission Test-Ireland predict performance in early undergraduate tests of communication and clinical skills? An observational cohort study.

BACKGROUND: Internationally, tests of general mental ability are used in the selection of medical students. Examples include the Medical College Admission Test, Undergraduate Medicine and Health Sciences Admission Test and the UK Clinical Aptitude Test. The most widely used measure of their efficacy is predictive validity.A new tool, the Health Professions Admission Test- Ireland (HPAT-Ireland), was introduced in 2009. Traditionally, selection to Irish undergraduate medical schools relied on academic achievement. Since 2009, Irish and EU applicants are selected on a combination of their secondary school academic record (measured predominately by the Leaving Certificate Examination) and HPAT-Ireland score. This is the first study to report on the predictive validity of the HPAT-Ireland for early undergraduate assessments of communication and clinical skills. METHOD: Students enrolled at two Irish medical schools in 2009 were followed up for two years. Data collected were gender, HPAT-Ireland total and subsection scores; Leaving Certificate Examination plus HPAT-Ireland combined score, Year 1 Objective Structured Clinical Examination (OSCE) scores (Total score, communication and clinical subtest scores), Year 1 Multiple Choice Questions and Year 2 OSCE and subset scores. We report descriptive statistics, Pearson correlation coefficients and Multiple linear regression models. RESULTS: Data were available for 312 students. In Year 1 none of the selection criteria were significantly related to student OSCE performance. The Leaving Certificate Examination and Leaving Certificate plus HPAT-Ireland combined scores correlated with MCQ marks.In Year 2 a series of significant correlations emerged between the HPAT-Ireland and subsections thereof with OSCE Communication Z-scores; OSCE Clinical Z-scores; and Total OSCE Z-scores. However on multiple regression only the relationship between Total OSCE Score and the Total HPAT-Ireland score remained significant; albeit the predictive power was modest. CONCLUSION: We found that none of our selection criteria strongly predict clinical and communication skills. The HPAT- Ireland appears to measures ability in domains different to those assessed by the Leaving Certificate Examination. While some significant associations did emerge in Year 2 between HPAT Ireland and total OSCE scores further evaluation is required to establish if this pattern continues during the senior years of the medical course.

Primary primitive neuroectodermal tumors of the retina and ciliary body in dogs.

We describe the clinical, histological, and immunohistochemical features of primary intraocular primitive neuroectodermal tumors in eight dogs. Four of eight tumors exhibited histological features similar to human retinoblastomas characterized by Flexner-Wintersteiner rosettes, and fleurettes, and demonstrated variable immunoreactivity for retinal markers opsin, S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). All dogs with tumors displaying histological and immunohistochemical features of retinal differentiation were ≤2 years of age. All tumors diagnosed as medulloepitheliomas (n = 4) did not display histological and immunohistochemical features of retinal differentiation and were present in dogs 7 years or older. Age of onset, in conjunction with immunohistochemistry for opsin, S-Ag, and IRBP, is an important aid in the differentiation of primary, primitive neuroectodermal tumors arising within the canine ciliary body, retina, and optic papilla.