Sandra M. Axiak-Bechtel

Gum arabic-coated radioactive gold nanoparticles cause no short-term local or systemic toxicity in the clinically relevant canine model of prostate cancer

Introduction Gum arabic-coated radioactive gold nanoparticles (GA-198AuNPs) offer several advantages over traditional brachytherapy in the treatment of prostate cancer, including homogenous dose distribution and higher dose-rate irradiation. Our objective was to determine the short-term safety profile of GA-198AuNPs injected intralesionally. We proposed that a single treatment of GA-198AuNPs would be safe with minimal-to-no evidence of systemic or local toxicity. Methods Nine dogs with spontaneously occurring prostatic cancer were treated. Injections were performed with ultrasound or computerized tomography guidance. Complete blood counts, chemistry panels, and urinalyses were performed at weekly intervals for 1 month and imaging was repeated 4 weeks postinjection. Planar scintigraphic images were obtained within 30 minutes of injection. Results No statistically significant difference was found in any hematologic or biochemical parameter studied, nor was any evidence of tumor swelling or abscessation found in eight dogs with repeat imaging; one dog died secondary to urethral obstruction 12 days following injection. At 30 minutes postinjection, an average of 53% of injected dose in seven dogs was retained in the prostate, with loss of remaining activity in the bladder and urethra; no systemic uptake was detected. Conclusion GA-198AuNP therapy had no short-term toxicity in the treatment of prostatic cancer. While therapeutic agent was found in the prostate immediately following injection, some loss of agent was detected in the bladder and urethra. Localization of radioactivity within the prostate was lower than anticipated and likely due to normal vestigial prostatic ducts. Therefore, further study of retention, dosimetry, long-term toxicity, and efficacy of this treatment is warranted prior to Phase I trials in men.

Effects of opioids on phagocytic function, oxidative burst capacity, cytokine production and apoptosis in canine leukocytes.

Opioids alter immune and apoptotic pathways in several species. They are commonly used in companion animals affected with infectious and/or inflammatory disease, but the immunomodulatory and apoptotic effects of these drugs in dogs are relatively unknown. The aim of the present study was to evaluate the effects of morphine, buprenorphine and fentanyl on canine phagocyte function, oxidative burst capacity, leukocyte cytokine production, and lymphocyte apoptosis. Blood from six healthy adult dogs was incubated in the presence or absence of morphine (200 ng/mL), buprenorphine (10 ng/mL) or fentanyl (10 ng/mL) for 3 h (phagocytic function; cytokine production) or 8 h (apoptosis). Neutrophil phagocytosis of opsonized Escherichia coli, respiratory burst capacity after stimulation with opsonized E. coli or phorbol 12-myristate 13-acetate (PMA), and Annexin V-FITC staining of apoptotic lymphocytes were evaluated using flow cytometry. Leukocyte production of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 was assessed after incubation with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or peptidoglycan. Morphine promoted a more intense respiratory burst. Morphine, buprenorphine and fentanyl all promoted LPS- or LTA-induced TNF-α and IL-10 production. However, the opioids tested did not alter TNF-α:IL-10 ratios. Morphine, buprenorphine and fentanyl all inhibited neutrophil apoptosis, an effect that was not concentration dependent in nature. These data indicate that opioids alter immune and apoptotic pathways in dogs. The possible effects of opioids on immune and cellular responses should be considered when designing studies and interpreting outcomes of studies involving administration of opioids in dogs.

Chemotherapy and remission status do not alter pre-existing innate immune dysfunction in dogs with lymphoma

Dogs with lymphoma have altered innate immunity and little is known about the effects of chemotherapy on innate immune function in dogs. Lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PG) - induced leukocyte cytokine production capacity, and phagocytosis and respiratory burst were evaluated in dogs prior to and following 6 weeks of chemotherapy. Dogs had decreased TNF production following LPS stimulation and increased IL-10 production following PG stimulation, which did not improve following remission of lymphoma. Dogs also had reduced E. coli-induced respiratory burst function after chemotherapy induced complete or partial remission. Dogs with lymphoma have an imbalance in pro-and anti-inflammatory cytokine production which did not improve with remission, and, following treatment, a decrease in respiratory burst function. Altered immune responses following exposure to bacterial pathogen associated molecular pattern motifs and bacteria may have many implications in the management of canine lymphoma.

Y-chromosome DNA Is Present in the Blood of Female Dogs Suggesting the Presence of Fetal Microchimerism

Fetal microchimerism has been suggested to play contradictory roles in women’s health, with factors including age of the recipient, time elapsed since microchimerism occurred, and microchimeric cell type modulating disease. Both beneficial and harmful effects have been identified in wound healing and tissue regeneration, immune mediated disease, and cancer. This area of research is relatively new, and hindered by the time course from occurrence of fetal microchimerism to the multi-factorial development of disease. Dogs represent an excellent model for study of fetal microchimerism, as they share our environment, have a naturally condensed lifespan, and spontaneously develop immune-mediated diseases and cancers similar to their human counterparts. However, fetal microchimerism has not been described in dogs. These experiments sought preliminary evidence that dogs develop fetal microchimerism following pregnancy. We hypothesized that Y chromosomal DNA would be detected in the peripheral blood mononuclear cells of female dogs collected within two months of parturition. We further hypothesized that Y chromosomal DNA would be detected in banked whole blood DNA samples from parous female Golden Retrievers with at least one male puppy in a prior litter. Amplification of DNA extracted from five female Golden Retrievers that had whelped within the two months prior to collection revealed strong positive bands for the Y chromosome. Of banked, parous samples, 36% yielded positive bands for the Y chromosome. This is the first report of persistent Y chromosomal DNA in post-partum female dogs and these results suggest that fetal microchimerism occurs in the canine species. Evaluation of the contributions of fetal microchimeric cells to disease processes in dogs as a model for human disease is warranted.

Resveratrol decreases oxidative burst capacity and alters stimulated leukocyte cytokine production in vitro.

INTRODUCTION Resveratrol, a naturally-occurring phytophenol, has been shown to bolster immune surveillance and reverse immunosenescence in a dose dependent manner in rodents and humans. Although safety and pharmacokinetic studies have been completed in dogs, the immunomodulatory effects of resveratrol in dogs has not yet been investigated. The objective of this study was to determine the effect of resveratrol on canine innate immune system function in vitro. The hypothesis was that similar to other species, low concentrations of resveratrol would stimulate while high concentrations would depress innate immune system function. METHODS Whole blood was collected from six healthy, adult, client-owned dogs and was incubated with resveratrol at final concentrations of 6000 ng ml(-1), 3000 ng ml(-1), 1000 ng ml(-1), or control solution for 4h. Following incubation, phagocytosis and oxidative burst were evaluated using flow cytometry, and LPS-, lipoteichoic acid (LTA) - and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay. RESULTS Phagocytosis was not altered by resveratrol at any concentration compared to control. However, while the number of PMNs capable of performing oxidative burst did not change, the robustness of the reaction following stimulation with Escherichia coli and PMA was reduced in a dose dependent manner. In addition, LPS-, LTA-, PG, and PBS-stimulated TNF production was increased following incubation with all concentrations of resveratrol compared to control, and this effect was dose dependent. LTA-stimulated IL-6 was increased with resveratrol compared to control. Furthermore, LTA-stimulated IL-10 was decreased with 6000 ng ml(-1) and 3000 ng ml(-1) concentrations of resveratrol and PG-stimulated IL-10 production was decreased with all concentrations of resveratrol compared to control. The LPS-, LTA-, and PG-stimulated TNF:IL-10 ratio was increased with 6000 ng ml(-1) of resveratrol compared to control and lower resveratrol concentrations. CONCLUSION While resveratrol was sparing to PMN phagocytosis, it reduced the robustness of PMN oxidative burst. Resveratrol also increased pro-inflammatory and decreased anti-inflammatory leukocyte cytokine production capacity in vitro. These data suggest that resveratrol supplementation may depress oxidative burst reactions while promoting pro-inflammatory leukocyte cytokine production and decreasing anti-inflammatory cytokine production. Based on these findings, further in vivo study regarding the effects of resveratrol on PMN oxidative burst capability and leukocyte cytokine production capacity are indicated prior to routine supplementation.

Evaluation of immunomodulatory effect of recombinant human granulocyte-macrophage colony-stimulating factor on polymorphonuclear cell from dogs with cancer in vitro.

The objective of this in vitro study was to evaluate the immunomodulatory effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on polymorphonuclear cell (PMN) function in dogs with cancer. PMNs were harvested from dogs with naturally developing cancer as a pre-clinical model to evaluate the immunomodulatory effects of rhGM-CSF on PMN phagocytic and cytotoxic functions, cytokine production and receptor expression. Some aspects of cancer-related PMN dysfunction in dogs with cancer were restored following incubation with rhGM-CSF including PMN phagocytosis, respiratory burst and LPS-induced TNF-α production. In addition, rhGM-CSF increased surface HLA-DR expression on the PMNs of dogs with cancer. These data suggests that dysfunction of innate immune response in dogs with cancer may be improved by rhGM-CSF. The results of this study provided a pathophysiologic rationale for the initiation of clinical trials to continue evaluating rhGM-CSF as an immunomodulatory therapy in dogs with cancer.

Phase I-II clinical trial of hyaluronan-cisplatin nanoconjugate in dogs with naturally occurring malignant tumors

OBJECTIVE To conduct a phase I-II clinical trial of hyaluronan-cisplatin nanoconjugate (HA-Pt) in dogs with naturally occurring malignant tumors. ANIMALS 18 healthy rats, 9 healthy mice, and 16 dogs with cancer. PROCEDURES HA-Pt was prepared and tested by inductively coupled plasma mass spectrometry; DNA-platinum adduct formation and antiproliferation effects of cisplatin and HA-Pt were compared in vitro. Effects of cisplatin (IV) and HA-Pt (SC) in rodents were tested by clinicopathologic assays. In the clinical trial, dogs with cancer received 1 to 4 injections of HA-Pt (10 to 30 mg/m(2), intratumoral or peritumoral, q 3 wk). Blood samples were collected for pharmacokinetic analysis; CBC, serum BUN and creatinine concentration measurement, and urinalysis were conducted before and 1 week after each treatment. Some dogs underwent hepatic enzyme testing. Tumors were measured before the first treatment and 3 weeks after each treatment to assess response. RESULTS No adverse drug effects were detected in pretrial assessments in rodents. Seven of 16 dogs completed the study; 3 had complete tumor responses, 3 had stable disease, and 1 had progressive disease. Three of 7 dogs with oral and nasal squamous cell carcinoma (SCC) that completed the study had complete responses. Myelosuppression and cardiotoxicosis were identified in 6 and 2 dogs, respectively; none had nephrotoxicosis. Four of 5 dogs with hepatic enzymes assessed had increased ALT activities, attributed to diaquated cisplatin products in the HA-Pt. Pharmacokinetic data fit a 3-compartment model. CONCLUSIONS AND CLINICAL RELEVANCE HA-Pt treatment resulted in positive tumor responses in some dogs, primarily those with SCC. The adverse effect rate was high. IMPACT FOR HUMAN MEDICINE Oral SCC in dogs has characteristics similar to human head and neck SCC; these results could be useful in developing human treatments.

Morphine and buprenorphine do not alter leukocyte cytokine production capacity, early apoptosis, or neutrophil phagocytic function in healthy dogs.

Opioids have immunomodulatory properties in many species, but there is little information pertaining to these properties in dogs. Our objective was to compare the in vivo effects of morphine, buprenorphine, and control solution on innate immune system function and apoptosis in healthy dogs. Six adult dogs received a 24-hour infusion of morphine, buprenorphine, or control solution (saline) in a randomized, controlled, crossover block design. Leukocyte apoptosis, phagocytosis, and oxidative burst were evaluated using flow cytometry. Lipopolysaccharide, lipoteichoic acid, and peptidoglycan-stimulated leukocyte production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were determined using canine specific multiplex assays. No significant treatment effects were detected among groups. These data suggest that healthy dogs could be less sensitive to the immunomodulatory effects of acute opioid administration compared with other species. Larger investigations in healthy and immunologically challenged dogs are recommended prior to application of these results in clinical patients.

Effects of tramadol and o-desmethyltramadol on canine innate immune system function

OBJECTIVE Tramadol is a commonly used opioid analgesic in dogs, particularly in dogs with a compromised immune system. An opioid may be selected for its immunomodulatory effects. Consequently, the objective of this study was to investigate the effects of tramadol on immune system function by evaluating the effect of tramadol and o-desmethyltramadol (M1) on the function of canine leukocytes in vitro. The hypothesis was that tramadol and M1 would not alter polymorphonuclear leukocyte (PMN) phagocytosis, PMN oxidative burst, or stimulated leukocyte cytokine production capacity of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10. STUDY DESIGN In vitro pharmacodynamic study. ANIMALS Six healthy dogs. METHODS Blood from six dogs was obtained and incubated with various concentrations of tramadol and M1. Phagocytosis and oxidative burst were assessed using flow cytometry, and lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay. RESULTS No differences were detected in phagocytosis or oxidative burst with any drug concentration. Tramadol did not alter leukocyte cytokine production, however, M1 significantly blunted IL-10 production. CONCLUSIONS Tramadol and its metabolite M1 were sparing to PMN phagocytosis and oxidative burst in dogs in vitro. Tramadol did not alter leukocyte cytokine production, however, M1 blunted IL-10 production at clinically achievable concentrations suggesting that M1 may promote a proinflammatory shift. CLINICAL RELEVANCE These data suggest that tramadol has minimal effect on phagocytosis and oxidative burst, and may promote a proinflammatory shift. Therefore, tramadol may be an ideal opioid analgesic in dogs at high risk of infection. Further investigation in vivo is warranted.

The health effects of fetal microchimerism can be modeled in companion dogs.

Fetal microchimerism (FMC) has been described to have a range of effects on health and disease. Y-chromosomal DNA has been detected in Golden Retrievers suggesting persistent FMC. In that report, nine dogs had evidence of microchimerism without prior pregnancy. To further understand this finding, a dam with prior male live births giving birth to her fourth litter of puppies, all females, was evaluated for FMC along with two of her daughters. All three female dogs had evidence of Y-chromosomal DNA in their blood. This suggests that male cells carried by the dam from previous pregnancy trafficked to her daughters to establish microchimerism in younger siblings. Companion dogs share many of the same cancers as humans, have out-bred genetics, and share the human environment, making them optimal models of human disease. Understanding the impact of FMC on health and disease of dogs could elucidate mechanisms useful for clinical interventions in humans.