A masked, placebo-controlled, randomized clinical trial evaluating safety and the effect on cardiac function of low-dose rapamycin in 17 healthy client-owned dogs.

Introduction: Geroscience studies of low-dose rapamycin in laboratory species have identified numerous benefits, including reversing age-related cardiac dysfunction. Cardiovascular benefits have been observed in dogs with 10 weeks of treatment, raising questions about possible benefits and adverse effects of long-term use of low-dose rapamycin. The objectives of this study were to assess the impact of 6 months of low-dose rapamycin on echocardiographic indices of cardiac function in healthy dogs and to document the occurrence of adverse events. Methods: Seventeen client-owned dogs aged 6-10 years, weighing 18-36 kg, and without significant systemic disease were included in a prospective, randomized, placebo-controlled, masked clinical trial. Low-dose rapamycin (0.025 mg/kg) or placebo was administered three times per week for 6 months. Baseline, 6-month, and 12-month evaluation included physical examination, cardiology examination, and clinicopathology. Three-month evaluation included physical examination and clinicopathology. Owners completed online questionnaires every 2 weeks. Results: There were no statistically significant differences in echocardiographic parameters between rapamycin and placebo groups at 6 or 12 months. No clinically significant adverse events occurred. In 26.8% of the bi-weekly surveys owners whose dogs received rapamycin reported perceived positive changes in behavior or health, compared to 8.1% in the placebo group (p = 0.04). Discussion: While no clinically significant change in cardiac function was observed in dogs treated with low-dose rapamycin, the drug was well-tolerated with no significant adverse events.

Development and validation of a novel instrument to capture companion dog mortality data: the Dog Aging Project End of Life Survey.

OBJECTIVE: The researchers and clinicians within the Dog Aging Project (DAP), a longitudinal cohort study of aging in companion dogs, created and validated a novel survey instrument titled the End of Life Survey (EOLS) to gather owner-reported mortality data about companion dogs. SAMPLE: Bereaved dog owners who participated in the refinement, face validity assessment, or reliability assessment of the EOLS (n = 42) and/or completed the entire survey between January 20 and March 24, 2021 (646). PROCEDURES: The EOLS was created and modified by veterinary health professionals and human gerontology experts using published literature, clinical veterinary experience, previously created DAP surveys, and feedback from a pilot study conducted with bereaved dog owners. The EOLS was subjected to qualitative validation methods and post hoc free-text analysis to evaluate its ability to thoroughly capture scientifically relevant aspects of companion dogs' deaths. RESULTS: The EOLS was well received with excellent face validity as assessed by dog owners and experts. The EOLS had fair to substantial reliability for the 3 validation themes-cause of death (κ = 0.73; 95% CI, 0.5 to 0.95), perimortem quality of life (κ = 0.49; 95% CI, 0.26 to 0.73), and reason for euthanasia (κ = 0.3; 95% CI, 0.08 to 0.52)-and had no need for any substantial content alterations based on free-text analysis. CLINICAL RELEVANCE: The EOLS has proven to be a well-accepted, comprehensive, and valid instrument for capturing owner-reported companion dog mortality data and has the potential to enhance veterinarians' ability to care for the aging dog population by illuminating their understanding of companion dogs' end-of-life experiences.

Development and Validation of a Novel Instrument to Capture Companion Dog Mortality Data: The Dog Aging Project End of Life Survey.

Objective: The researchers and clinicians within the Dog Aging Project (DAP), a longitudinal cohort study of aging in companion dogs, created and validated a novel survey instrument titled the End of Life Survey (EOLS) to gather owner-reported mortality data about companion dogs. Sample: Bereaved dog owners who participated in the refinement, face validity assessment, or reliability assessment of the EOLS (n=42) and/or completed the entire survey between January 20 and March 24, 2021 (n=646). Procedures: The EOLS was created and modified by veterinary health professionals and human gerontology experts using published literature, clinical veterinary experience, previously created DAP surveys, and feedback from a pilot study conducted with bereaved dog owners. The EOLS was subjected to qualitative validation methods and post-hoc free-text analysis to evaluate its ability to thoroughly capture scientifically relevant aspects of companion dogs' death. Results: The EOLS was well-received with excellent face validity as assessed by dog owners and experts. The EOLS had fair to substantial reliability for the three validation themes: cause of death (kappa = 0.73; 95% CI [0.5-0.95]), perimortem quality of life (kappa = 0.49; 95% CI [0.26-0.73]), and reason for euthanasia (kappa = 0.3; 95% CI [0.08-0.52]) and had no need for any substantial content alterations based on free-text analysis. Clinical Relevance: The EOLS has proven to be a well-accepted, comprehensive, and valid instrument for capturing owner-reported companion dog mortality data and has the potential to enhance veterinarians' ability to care for the aging dog population by illuminating their understanding of companion dogs' end-of-life experiences.

Development and evaluation of a survey instrument to assess veterinary medical record suitability for multi-center research studies.

Here we describe the development and evaluation of a survey instrument to assess the research suitability of veterinary electronic medical records (EMRs) through the conduct of two studies as part of the Dog Aging Project (DAP). In study 1, four reviewers used the instrument to score a total of 218 records in an overlapping matrix of pairs to assess inter-rater agreement with respect to appropriate format (qualification), identification match (verification), and record quality. Based upon the moderate inter-rater agreement with respect to verification and the relatively large number of records that were incorrectly rejected the instrument was modified and more specific instructions were provided. In study 2, a modified instrument was again completed by four reviewers to score 100 different EMRs. The survey scores were compared to a gold standard of board-certified specialist review to determine receiver operating curve statistics. The refined survey had substantial inter-rater agreement across most qualification and verification questions. The cut-off value identified had a sensitivity of 95 and 96% (by reviewer 1 and reviewer 2, respectively) and a specificity of 82% and 91% (by reviewer 1 and reviewer 2, respectively) to predict gold standard acceptance or rejection of the record. Using just qualification and verification questions within the instrument (as opposed to full scoring) minimally impacted sensitivity and specificity and resulted in substantial time savings in the review process.

Human leukocyte antigen G expression in breast cancer: role in immunosuppression.

Human leukocyte antigen G (HLA-G) is an immunotolerant nonclassical major histocompatibility complex Class Ib molecule. It is expressed by trophoblastic placental cells during pregnancy to protect the fetus from maternal alloreactivity. HLA-G is overexpressed in tumors and involved in cancer immune evasion. Reverse transcription-polymerase chain reaction and immunohistochemistry (IHC) were used to examine HLA-G expression in normal mammary and breast cancer cell lines and normal and human breast cancer tissues. Reverse transcription-polymerase chain reaction confirmed that normal epithelial MCF-12A cells had no HLA-G mRNA expression, whereas cancer cell lines MCF-7, T47D, and MDA-MB-231 and NCI/Adr-Res had various levels of HLA-G mRNA expression. Twelve (12) normal and 38 breast cancer tissues were examined by IHC. Fifty-eight (58) percent (22/38) of cancers had medium to strong staining to HLA-G, whereas only 8% (1/12) of normal breast tissues had medium to strong staining, and the difference was significant (p < 0.05). HLA-G staining was found in the membranes and cytoplasm of cancer cells. In conclusion, breast cancer cells overexpress HLA-G mRNA and protein, and this probably contributes to immune evasion.

Altering regulatory T cell function in cancer immunotherapy: a novel means to boost the efficacy of cancer vaccines.

Cancers express tumor associated antigens that should elicit immune attack, but spontaneous immune rejection of established cancer is rare. Recent data demonstrate that specific and active tumor-mediated mechanisms hinder host anti-tumor immunity. CD4+CD25+ T regulatory cells (Tregs) are important mediators of active immune evasion in cancer. Disrupting tumor-mediated mechanisms hindering host immunity is a novel approach to tumor immunotherapy. Treg depletion improves endogenous anti-tumor immunity and the efficacy of active immunotherapy in animal models for cancer, suggesting that inhibiting Treg function could also improve the limited successes of human cancer immunotherapy. We have identified five strategies to block Treg activity: depletion, interference with trafficking, inhibition of differentiation, blockade of function or raising the effector T cell threshold for suppression. Discovery of additional regulatory cell populations expands the potential targets for these approaches. The fusion toxin denileukin diftitox (Ontak) reduces Treg numbers and function in the blood of some patients with cancer. We discuss specific strategies to block Treg activity and present some of our preliminary data in this area. Combining Treg depletion with active vaccination and other approaches poses additional challenges that are discussed.

Dual targeting of adenoviral vectors at the levels of transduction and transcription enhances the specificity of gene expression in cancer cells.

Adenoviral (Ad) vector-mediated strategies for cancer gene therapy mandate a vector that is capable of efficient expression of the therapeutic gene specifically within the target tumor cells. In one approach to the development of cancer cell-specific vectors, Ad vectors have been targeted at the level of transduction to achieve the selective delivery of the therapeutic gene. In an alternative approach to the derivation of cancer cell-specific vectors, Ad vectors have been targeted at the level of transcription by placing the therapeutic gene under the control of transcriptional regulatory sequences that are activated in tumor cells, but not in normal cells, and therefore target expression selectively to the tumor cell. In this report, we demonstrate that a higher degree of specificity for cancer cells can be achieved by combining the complementary approaches of transductional and transcriptional targeting, each of which is imperfect or "leaky" by itself.

Targeted adenoviral vectors.

Replication-defective vectors based on human adenovirus serotypes 2 and 5 (Ad2 and Ad5) possess a number of attributes which favor their use as gene delivery vehicles in gene therapy applications. However, the widespread distribution of the primary cellular receptor for Ad, the coxsackievirus and adenovirus receptor (CAR), allows Ad vectors to infect a broad range of cells in the host. Conversely, a number of tissues which represent important targets for gene therapy, such as the airway epithelium and cancer cells, are refractory to Ad infection due a paucity of CAR. Thus, there is a strong rationale for the development of CAR-independent Ad vectors capable of enhanced specificity and efficiency of gene transfer to target cells. In this article we review the approaches which have been employed to generate tropism-modified Ad vectors. These targeting strategies have led to improvements in the safety and efficacy of Ad vectors and have the potential to yield an increased therapeutic benefit in the human clinical context.