Kathleen L. Graham

Supplementary Table S1. Primer sequences for transcript analysis. Supplementary Figure S1. Gating strategy for identification of monocyte subpopulations. Supplementary Figure S2. CD40 agonist induces accumulation of resident macrophages in spleen. Supplementary Figure S3. Impact of anti-CD40 treatment on Ly6G+ cells within PDAC tumors of KPC mice. Supplementary Figure S4. Strategy for depletion of resident and inflammatory monocyte populations in vivo. Supplementary Figure S5. Effect of clodronate encapsulated liposome (CEL) treatment on peripheral blood myeloid cells. Supplementary Figure S6. Specificity of Ly6C depleting antibody, Monts-1. Supplementary Figure S7. CCL2 regulates CD40-dependent Ly6C+ myeloid cell recruitment to PDAC tumors in KPC mice. Supplementary Figure S8. Anti-CD40 treatment induces systemic cytokine release in mice and humans. Supplementary Figure S9. STAT1 signaling in human monocytes induced by plasma from PDAC patient treated with CP-870,893. Supplementary Figure S10. Anti-fibrotic activity induced with a CD40 agonist is dependent on IFN-gamma. Supplementary Figure S11. CD40 activation does not alter extracellular matrix production or FAP+ fibroblast proliferation. Supplementary Figure S12. Anti-CD40 treatment selectively depletes extracellular matrix proteins within the tumor microenvironment. Supplementary Figure S13. Implantable tumor model of PDAC that reproduces tumor microenvironment of KPC model of spontaneous PDAC. Supplementary Figure S14. Development of an implantable model of PDAC for evaluating monocyte-dependent degradation of cancer fibrosis. Supplementary Figure S15. Gene expression of extracellular matrix and adhesion molecules within the tumor microenvironment in response to anti-CD40 treatment. Supplementary Figure S16. IFN-gamma induces Mmp13 expression in myeloid cells. Supplementary Figure S17. RNA in situ hybridization (ISH) for MMP13 in PDAC tumors. Supplementary Figure S18. Detection of MMP activity in cellular subsets within the tumor microenvironment. Supplementary Figure S19. Protein expression of MMP13 and MMP14 within the tumor microenvironment of spontaneously arising pancreatic tumors in the KPC model. Supplementary Figure S20. Gene expression of Mmp2 and Mmp14 within the tumor microenvironment of pancreatic tumors. Supplementary Figure S21. Anti-fibrotic activity of a CD40 agonist is dependent on MMPs. Supplementary Figure S22. Impact of MMP13 specific inhibitors on CD40 agonist induced collagen degradation. Supplementary Figure S23. Kinetics of anti-CD40 activity on tumor microenvironment. Supplementary Figure S24. Impact of anti-CD40 on CD31+ blood vessel density and vascular patency. Supplementary Figure 25. Anti-CD40 treatment improves gemcitabine efficacy in vivo. Supplementary Figure 26. Anti-CD40 treatment improves gemcitabine efficacy in KPC tumor-bearing mice. Supplementary Figure S27. Impact of MMPs and Ly6C+ cells on anti-tumor activity induced by gemcitabine administered two days after an agonist CD40 antibody. Supplementary Figure S28. Treatment tolerability is dependent on timing of gemcitabine administration after anti-CD40 treatment.

Objective To describe measurements of in vivo structures of the visual pathway beyond the retina and optic nerve head associated with canine primary angle‐closure glaucoma (PACG). Methods A prospective pilot study was conducted using magnetic resonance diffusion tensor imaging (DTI) to obtain quantitative measures of the optic nerve, chiasm, tract, and lateral geniculate nucleus (LGN) in dogs with and without PACG. 3‐Tesla DTI was performed on six affected dogs and five breed, age‐ and sex‐matched controls. DTI indices of the optic nerve, optic chiasm, optic tracts, and LGN were compared between normal, unilateral PACG, and bilateral PACG groups. Intra‐class correlation coefficient (ICC) was calculated to assess intra‐observer reliability. Results Quantitative measurements of the optic nerve, optic tract, optic chiasm, and LGN were obtained in all dogs. There was a trend for reduced fractional anisotropy (FA) associated with disease for all structures assessed. Compared to the same structure in normal dogs, FA, and radial diffusivity (RD) of the optic nerve was consistently higher in the unaffected eye in dogs with unilateral PACG. Intra‐observer reliability was excellent for measurements of the optic nerve (ICC: 0.92), good for measurements of the optic tract (ICC: 0.89) and acceptable for measures of the optic chiasm (ICC: 0.71) and lateral geniculate nuclei (ICC: 0.76). Conclusion Diffusivity and anisotropy measures provide a quantifiable means to evaluate the visual pathway in dogs. DTI has potential to provide in vivo measures of axonal and myelin injury and transsynaptic degeneration in canine PACG.

Objective: To explore the use of a modified pupillometry technique in dogs without chemical restraint.Materials and Methods: Following dark adaptation, pupillary light reflexes were assessed in six dogs with sudden acquired retinal degeneration syndrome, in the unaffected eye of eight dogs with unilateral primary glaucoma (“predisposed”), and in 11 healthy dogs. Responses to red, blue and white lights were recorded and relative pupil sizes subsequently determined based on video recordings of each test.Results: Mean testing time was 2.3 minutes (range 1.8 to 3.1 minutes), excluding time for dark adaptation. Baseline pupil size in dogs with sudden acquired retinal degeneration syndrome was greater than in normal and predisposed eyes. Pupil constriction was reduced in predisposed compared to normal and sudden acquired retinal degeneration syndrome eyes when stimulated with high‐intensity blue light. Compared to normal eyes, those with sudden acquired retinal degeneration syndrome had reduced pupil constriction when stimulated with low‐ and high‐intensity red light, low‐intensity blue light and white light.Clinical Significance: Quantitative measures of pupil function were obtained from healthy and diseased eyes without the need for chemical restraint. Further investigations are warranted to validate the technique and evaluate its use in the management of canine glaucoma.

Objective: To introduce a protocol for the characterization of protein patterns in tears of dogs with primary angle closure glaucoma (PACG) and primary open‐angle glaucoma (POAG).Animals: Nineteen dogs (25 eyes).Methods: Tear samples were collected using a Schirmer tear strip, from dogs with PACG (PACG‐affected eyes, n = 8; unaffected eyes predisposed to PACG, n = 7), POAG (n = 4), and healthy controls (n = 6). Protein precipitation and trypsin digestion were performed for analyses via liquid chromatography‐tandem mass spectrometry. Proteins were identified using the SwissProt protein sequence database. Relative protein expression in 17 eyes (15 dogs) was evaluated using Proteome Discoverer 2.0. Pathway analyses were performed to investigate molecular mechanisms associated with primary glaucoma.Results: Unique peptides were identified in 505 proteins, with Major allergen Can f 1 and albumin identified with high confidence. Proteins unique to tears from diseased eyes (PACG: n = 7; POAG: n = 14) were identified. Nucleoside diphosphate was unique to tears in PACG eyes naïve to therapy, while retinal binding protein and NSFL1 cofactor p47 were unique to medicated PACG eyes. Relative expression of 34 proteins differed between disease states. Pathway analyses identified that the ‘inflammatory response’ was among the top disease/disorders in dogs with primary glaucoma (PACG and POAG) but not in healthy controls.Conclusion: Tear samples suitable for mass spectrometry were readily obtained from pet dogs without needing specialized equipment. Further studies to validate the findings and explore potential candidate biomarkers for early disease detection and potential therapeutic targets are indicated.

Objective: To evaluate the retina and optic nerve head (ONH) in canine eyes predisposed to glaucoma using optical coherence tomography (OCT).Animals: Twenty‐five eyes (24 dogs).Methods: Measures of peripapillary retinal, retinal nerve fiber layer (RNFL), and ganglion cell complex (GCC) thickness and ONH parameters were obtained in vivo by OCT of the unaffected eye in dogs diagnosed with unilateral primary glaucoma (predisposed; n = 12) and compared with measures of healthy control eyes (normal; n = 13). Repeatability and intrarater reliability were explored using intraclass correlation coefficients (ICC).Results: Compared to normal eyes, predisposed eyes had a thinner retina in the temporal (P = 0.005), inferior quadrants (P = 0.003), and decreased inner retinal thickness (superior: P = 0.003, temporal: P = 0.001, inferior: P < 0.001, nasal: P = 0.001). Predisposed eyes had a thinner RNFL compared to normal eyes (P = 0.005), and when analyzed in quadrants, it was thinner in the superior (P < 0.001), temporal (P = 0.034), and nasal quadrants (P = 0.001). Repeatability (ICC 0.763‐0.835) and intrarater reliability (ICC 0.824‐0.942) were good to excellent for measures of retinal thickness and adequate for RNFL measurements (ICC 0.701‐0.798). Reliable measurements of optic disk area were obtained and were similar between groups (P = 0.597). Measurements of parameters relying on automated software detection (GCC, optic cup, optic rim) had inadequate repeatability and reliability.Conclusion: Statistically significant differences in retinal and RNFL thicknesses were identified in normal and predisposed eyes. Reliable and consistent measurements of variables with manual adjustment of software detected parameters were obtained. Validation of OCT as a diagnostic tool for clinical assessment in canine glaucoma is warranted.

Aim: To describe the development and initial validation of a questionnaire measuring functional vision in dogs.Methods: A 17‐item survey was designed to quantify functional vision in dogs. The Vision Impairment Score (VIS) was determined by summing responses to each question. Questions were assigned to one of five subcategories: overall vision, daily activities, peripheral vision, near vision, and distance vision. Content validity was established during development phases, and construct validity via comparing results of known groups (blind vs sighted; normal vs impaired vision; surgery to improve vision vs nonrestorative surgery), and through factor analysis. Concurrent criterion validity was determined with use of a validated health‐related quality‐of‐life (HRQL) assessment tool. Reliability and responsiveness assessments were investigated using intraclass correlation coefficient (ICC) and effect size (ES), respectively.Results: Responses (221) from 201 dog owners were included. Compared to sighted dogs (n = 153), blind dogs (n = 48) had a higher VIS and greater impairment in all subcategories. Among sighted dogs, a higher VIS was obtained in dogs with low vision compared to those with normal vision (P < 0.001). A higher VIS was associated with poorer HRQL (P < 0.001). Perfect reliability was obtained for 6/17 questions, and excellent reliability for 11/17 questions (intraclass correlation 1.0 and >0.9, respectively), and the VIS was highly responsive to therapeutic intervention (effect size 1.46).Conclusion: Results suggest the VIS may be clinically useful in assessing and obtaining a quantifiable measure of functional vision in dogs. Ongoing validation of the tool for clinical use is needed.

Objectives To describe preliminary use of a forced‐choice preferential looking task for the clinical assessment of vision in dogs. Materials and Methods The vision of 18 pet dogs was investigated in two separate studies using a forced‐choice preferential looking task: multiple observers watched eye, head and body movements on video recordings to identify cues suggesting when a dog had seen the feature of interest. Human observer reliability was determined using eight dogs and computer‐generated stimuli. Visual acuity was assessed using computer‐generated grating stimuli: in real‐time, an observer watched each dog's eye movement patterns and behaviour to decide whether each grating was seen. Stimuli were presented in a step‐wise manner and were controlled by the observer. Acuity was estimated as the highest spatial frequency the dog was determined to have seen. Results Median estimated visual acuity was better at 1 m compared to that at 3 m. Average test time was longer at a 3‐m distance than at 1 m. Inter‐ and intra‐observer reliability was better from 1 m than from 3 m. Clinical Significance Preliminary use of a forced‐choice preferential looking task for measurement of visual acuity in dogs has potential use as a clinical tool for the assessment of vision in dogs.