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The Portable Extensible Toolkit for Scientific computation (PETSc) library delivers scalable solvers for nonlinear time-dependent differential and algebraic equations and for numerical optimization. The PETSc design for performance portability addresses fundamental GPU accelerator challenges and stresses flexibility and extensibility by separating the programming model used by the application from that used by the library, and it enables application developers to use their preferred programming model, such as Kokkos, RAJA, SYCL, HIP, CUDA, or OpenCL, on upcoming exascale systems. A blueprint for using GPUs from PETSc-based codes is provided, and case studies emphasize the flexibility and high performance achieved on current GPU-based systems.

Vesicular stomatitis virus (VSV) is a prototype RNA virus that has been instrumental in advancing our understanding of viral molecular biology and has applications in vaccine development, cancer therapy, antiviral screening, and more. Current VSV genome plasmids for purchase or contract virus services provide limited options for modification, restricted to predefined cloning sites and insert locations. Improved methods and tools to engineer VSV will unlock further insights into long-standing virology questions and new opportunities for innovative therapies. Here, we report the design and construction of a full-length VSV genome. The 11,161 base pair synthetic VSV (synVSV) was assembled from four modularized DNA fragments. Following rescue and titration, phenotypic analysis showed no significant differences between natural and synthetic viruses. To demonstrate the utility of a synthetic virology platform, we then engineered VSV with a foreign glycoprotein, a common use case for studying viral entry and developing anti-virals. To show the freedom of design afforded by this platform, we then modified the genome of VSV by rearranging the gene order, switching the positions of VSV-P and VSV-M genes. This work represents a significant technical advance, providing a flexible, cost-efficient platform for the rapid construction of VSV genomes, facilitating the development of innovative therapies.

Osteosarcoma (OS) is a primary bone malignancy characterized by an aggressive nature, limited treatment options, low survival rate, and poor patient prognosis. Conditionally replicative adenoviruses (CRAds) armed with immune checkpoint inhibitors hold great potential for enhanced therapeutic efficacy. The present study aims to investigate the anti-tumor efficacy of CAV2-AU-M2, a CAV2-based CRAd armed with an anti-PD-1 single-domain antibody (sdAb), against OS cell lines in vitro . The infection, conditional replication, cytopathic effects, and cytotoxicity of CAV2-AU-M2 were tested in four different OS cell lines in two-dimensional (2D) and three-dimensional (3D) cell cultures. CAV2-AU-M2 showed selective replication in the OS cells and induced efficient tumor cell lysis and death. Moreover, CAV2-AU-M2 produced an anti-PD-1 sdAb that demonstrated effective binding to the PD-1 receptors. This study demonstrated the first CRAd armed with an anti-PD-1 sdAb. This combined approach of two distinct immunotherapies is intended to enhance the anti-tumor immune response in the tumor microenvironment.

Introduction: Osteosarcoma (OSA) is an aggressive form of bone cancer in both dogs and humans. The treatment options for metastatic (stage III) OSA are currently limited and the prognosis is poor. Zoledronate, a second generation amino-bisphosphonate, is commonly used for palliation of cancer induced bone pain. Zoledronate has also demonstrated anti-cancer properties and possibly enhances the cytotoxicity of doxorubicin in a canine histiocytosis cell line and human prostatic cancer cell line. The goal of this study was to evaluate the combination effect of zoledronate and various chemotherapeutic drugs in canine OSA cells., Methods: Canine OSA cell line (D17), cells from two canine primary OSAs, and MDCK, a canine kidney cell line, were used to evaluate the therapeutic potential of these drugs. Carboplatin, doxorubicin, vinorelbine, toceranib, and isophosphoramide mustard (active metabolite of ifosfamide) were used as chemotherapeutic agents. First, cells were treated with either zoledronate or chemotherapy drug alone for 72 hours. Cell viability was assessed using CellTiter Glo and IC 5 , IC 10 , IC 20 , and IC 50 were calculated. Second, cells were treated with a combination of zoledronate and each chemotherapeutic agent at their IC 5 , IC 10 , IC 20 , and IC 50 concentrations. After 72 hours, cell viability was assessed by CellTiter Glo., Results and Discussion: Zoledronate, carboplatin, doxorubicin, vinorelbine, and isophosphoramide mustard showed concentration dependent decrease in cell viability. Toceranib showed decreased cell viability only at higher concentrations. When zoledronate was used in combination with chemotherapy drugs, while it showed potential synergistic effects with toceranib, potential antagonistic effects with vinorelbine and isophosphoramide mustard were observed. However, the results differed by cell line and thus, further evaluation is warranted to understand the exact mechanism of action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Iwaki, Lindley, Bergman, Smith and Pondugula.)

Background: Molecular biomarker analysis is standard of care in advanced nonsmall cell lung cancer (NSCLC). Pathologist-driven reflex testing protocols are one approach to initiating this analysis. Two years after insourcing genomic analysis at our institution, a reflex testing protocol for advanced NSCLC was initiated., Methods: A retrospective review of the records of 578 NSCLC biopsies was performed to assess the impact of 3 genomic testing workflows (send-out, in-house clinician-ordered, and in-house reflex) on time to initiation of molecular testing [initiation time (IT)], reporting time (RT), proportion of test failures, and test ordering practices. The proportion of test failures by test methodology was also assessed., Results: IT was lowest for reflex protocol orders (mean weekdays: 30.0 send-out, 27.4 in-house clinician-ordered, 0.95 reflex). Test failure was highest for send-out testing (31.7% vs. 10% each for in-house clinician-ordered and reflex). RT remained consistent across the 3 workflows (mean weekdays: 11.1 send-out, 11.9 in-house clinician-ordered, and 11.4 reflex). Guideline-congruent molecular testing increased upon insourcing genomic analysis and again upon implementing reflex testing with a reduction in nonbiomarker informed care (58.8% send-out, 19.5% in-house clinician-ordered, 11.5% reflex)., Conclusions: Implementation of reflex in-house genomic analysis for advanced NSCLC ensured consistency in RT and significantly decreased IT and proportion of test failures. Insourcing genomic analysis and thoughtful care pathway design improve equitable access to molecular biomarker analysis and mitigate nonbiomarker informed cancer care in NSCLC., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Osteosarcoma (OSA) is a highly aggressive bone tumor primarily affecting pediatric or adolescent humans and large-breed dogs. Canine OSA shares striking similarities with its human counterpart, making it an invaluable translational model for uncovering the disease's complexities and developing novel therapeutic strategies. Tumor heterogeneity, a hallmark of OSA, poses significant challenges to effective treatment due to the evolution of diverse cell populations that influence tumor growth, metastasis, and resistance to therapies. In this study, we apply single-nuclei multiome sequencing, encompassing ATAC (Assay for Transposase-Accessible Chromatin) and GEX (Gene Expression, or RNA) sequencing, to a treatment-naïve primary canine osteosarcoma. This comprehensive approach reveals the complexity of the tumor microenvironment by simultaneously capturing the transcriptomic and epigenomic profiles within the same nucleus. Furthermore, these results are analyzed in conjunction with bulk RNA sequencing and differential analysis of the same tumor and patient-matched normal bone. By delving into the intricacies of OSA at this unprecedented level of detail, we aim to unravel the underlying mechanisms driving intra-tumoral heterogeneity, opening new avenues for therapeutic interventions in both human and canine patients. This study pioneers an approach that is broadly applicable, while demonstrating significant heterogeneity in the context of a single individual's tumor.

Spindle cell/sclerosing rhabdomyosarcoma (SSRMS) is a clinicopathologically and molecularly heterogeneous disease. Gene fusions have been identified in intraosseous SSRMS, consisting predominantly of EWSR1 / FUS :: TFCP2 and MEIS1 :: NCOA2 . The former often follow an aggressive clinical course; there is limited clinical follow-up available for the latter. We report here a new case of the very rare intraosseous SSRMS with MEIS1 :: NCOA2 gene fusion and include the detailed treatment course and 52 months of clinical follow-up. SSRMS with MEIS1 :: NCOA2 gene fusion appears biologically distinct from other intraosseous SSRMS, following a course characterized by local recurrence with rare reports of metastasis to date.

MicroRNAs (miRNAs) are single-stranded, non-coding RNA molecules that regulate gene expression post-transcriptionally by binding to messenger RNAs. miRNAs are important regulators of gene expression, and their dysregulation is implicated in many human and canine diseases. Most cancers tested to date have been shown to express altered miRNA levels, which indicates their potential importance in the oncogenic process. Based on this evidence, numerous miRNAs have been suggested as potential cancer biomarkers for both diagnosis and prognosis. miRNA-based therapies have also been tested in different cancers and have provided measurable clinical benefits to patients. In addition, understanding miRNA biogenesis and regulatory mechanisms in cancer can provide important knowledge about resistance to chemotherapies, leading to more personalized cancer treatment. In this review, we comprehensively summarized the importance of miRNA in human and canine cancer research. We discussed the current state of development and potential for the miRNA as both a diagnostic marker and a therapeutic target.

Canine adenovirus type 2 (CAV2) is a nonhuman adenovirus with a known ability to infect human and canine cells. The cell surface receptors involved in CAV2 transduction are still unknown. Identification of these would provide valuable information to develop enhanced gene delivery tools and better understand CAV2 biology. CAV2 is erroneously grouped with Ad5 based on the knowledge that CAV2 may transduce using CAR. Therefore, we have evaluated CAV2 and Ad5 (CAV2GFP, Ad5G/L) infection patterns in various canine and human cell lines to determine their different tropisms. Our research demonstrates that CAV2 can successfully infect cells that Ad5 does not infect, and CAV2 infections do not correlate with CAR expression. CAV2 can infect cells that have a low or minimal expression of CAR. Our data suggest that CAV2 transduction is not dependent on the CAR receptor, and thus, it is crucial to find novel CAV2 receptors., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Madison D. Hogans et al.)

Recent breakthroughs in cancer immunotherapy have provided unprecedented clinical benefits to human cancer patients. Cancer is also one of the most common causes of death in pet dogs. Thus, canine-specific immune therapies targeting similar signaling pathways can provide better treatment options for canine cancer patients. Here, we describe the development and characterization of two canine-specific anti-OX40 agonists to activate OX40 signaling. We show that canine OX40, like human OX40, is not expressed on resting T cells, and its expression is markedly increased on canine CD4 T cells and Tregs after stimulation with concanavalin A (Con-A). cOX40 is also expressed on tumor-infiltrating lymphocytes (TILs) in canine osteosarcoma patients. The canine-specific OX40 agonists strongly activates cPBMCs by increasing IFN-γ expression and do not require Fc receptor-mediated cross-linking for OX40 agonism. Together, these results suggest that cFcOX40L proteins are potent OX40 agonists and have the potential to enhance antitumor immunity in canine cancer patients., Competing Interests: The authors declare that they have no competing interests., (© 2022 The Author(s).)

Despite significant advances in cancer diagnosis and treatment, osteosarcoma (OSA), an aggressive primary bone tumor, has eluded attempts at improving patient survival for many decades. The difficulty in managing OSA lies in its extreme genetic complexity, drug resistance, and heterogeneity, making it improbable that a single-target treatment would be beneficial for the majority of affected individuals. Precision medicine seeks to fill this gap by addressing the intra- and inter-tumoral heterogeneity to improve patient outcome and survival. The characterization of differentially expressed genes (DEGs) unique to the tumor provides insight into the phenotype and can be useful for informing appropriate therapies as well as the development of novel treatments. Traditional DEG analysis combines patient data to derive statistically inferred genes that are dysregulated in the group; however, the results from this approach are not necessarily consistent across individual patients, thus contradicting the basis of precision medicine. Spontaneously occurring OSA in the dog shares remarkably similar clinical, histological, and molecular characteristics to the human disease and therefore serves as an excellent model. In this study, we use transcriptomic sequencing of RNA isolated from primary OSA tumor and patient-matched normal bone from seven dogs prior to chemotherapy to identify DEGs in the group. We then evaluate the universality of these changes in transcript levels across patients to identify DEGs at the individual level. These results can be useful for reframing our perspective of transcriptomic analysis from a precision medicine perspective by identifying variations in DEGs among individuals.

Dogs are remarkable, adaptable, and dependable creatures that have evolved alongside humans while contributing tremendously to our survival. Our canine companions share many similarities to human disease, particularly cancer. With the advancement of next-generation sequencing technology, we are beginning to unravel the complexity of cancer and the vast intra- and intertumoral heterogeneity that makes treatment difficult. Consequently, precision medicine has emerged as a therapeutic approach to improve patient survival by evaluating and classifying an individual tumor's molecular profile. Many canine and human cancers share striking similarities in terms of genotypic, phenotypic, clinical, and histological presentations. Dogs are superior to rodent models of cancer because they are a naturally heterogeneous population in which tumors occur spontaneously, are exposed to similar environmental conditions, and show more similarities in key modulators of tumorigenesis and clinical response, including the immune system, drug metabolism, and gut microbiome. In this chapter, we will explore various canine models of human cancers and emphasize the dog's critical role in advancing precision medicine and improving the survival of both man and man's best friend., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Genetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time., (© 2021. The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.)

Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals., (© 2021. The Author(s).)

Cancer is the leading cause of death in the geriatric dog population. Currently, the use of immune checkpoint inhibitors (ICIs) such as anti-CTLA4 antibodies has markedly improved the prognosis of several cancers in their advanced stages. However, ICIs targeting CTLA4 blockade to treat canine cancer patients are yet to define. In this study, we sought to develop, characterize and assess whether chimeric heavy chain only antibodies (cHcAbs) against CTLA4 are viable therapeutic candidates for the treatment of canine cancers. Anti-CTLA4 nanobodies (Nbs) were identified from a yeast nanobody (Nb) library using magnetic-assisted cell sorting (MACS) and flow cytometry. cHcAbs were engineered by genetically fusing the DNA sequences coding for anti-CTLA4 Nbs with the Fc domain of the subclass B of canine IgG. Recombinant cHcAbs were purified from ExpiCHO-S cells. Stable cell lines expressing canine CTLA4 and FcγRI were used to elucidate the binding ability and specificity of cHcAbs. PBMCs isolated from healthy dogs were used to evaluate the ability of cHcAbs to activate canine PBMCs (cPBMCs). Novel Nbs were identified using the extracellular domain of canine CTLA4 protein to screen a fully synthetic yeast nanobody library. Purified Nbs bind specifically to natïve canine CTLA4. We report that chimeric HcAbs, which were engineered by fusing the anti-CTLA4 Nbs and Fc region of subclass B of canine IgG, were half the size of a conventional mAb and formed dimers. The chimeric HcAbs specifically binds both with canine CTLA4 and Fcγ receptors. As the binding of Nbs overlapped with the MYPPPY motif of canine CTLA4, these Nbs were expected to sterically disrupt the interaction of canine CTLA4 to B-7s. Like their human counterpart, canine CTLA4 was expressed on helper T cells and a small subset of cytotoxic T cells. Canine Tregs also constitutively expressed CTLA4, and stimulation with PMA/Ionomycin dramatically increased expression of CTLA4 on the cell surface. Stimulation of cPBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control. This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients., (© 2021. The Author(s).)

Delay in cancer diagnosis often results in metastasis and an inability to successfully treat the tumor. The use of broadly cancer-specific biomarkers at an early stage may improve cancer treatment and staging. This study has explored circulatory exosomal miRNAs as potential diagnostic biomarkers to identify cancer patients. Secretory exosomal miRNAs were isolated from 13 canine cancer cell lines (lymphoma, mast cell tumor, histiocytic cell line, osteosarcoma, melanoma, and breast tumor) and were sequenced by Next-Generation sequencing (NGS). We have identified 6 miRNAs (cfa-miR-9, -1841, -1306, -345, -132, and -26b) by NGS that were elevated in all cancer cell types. The miRNAs identified by NGS were then examined by Q-RT-PCR. The PCR data demonstrated similar expression patterns to those seen with NGS but provided fold differences that were much lower than those seen for NGS. Cfa-miR-9 was found to be the most consistently elevated miRNA in NGS and PCR, making it the most likely miRNA to prove diagnostic. In this study, we have demonstrated that it is possible to identify exosomal miRNAs with elevated secretion across multiple tumor types that could be used as circulatory diagnostic biomarkers for liquid biopsy in the future., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

The University of Missouri (MU) has established a colony of dystrophin-deficient dogs with a mixed breed background to mirror the variable pathologic effects of dystrophinopathies between persons of a given kindred to further the understanding of the genetic and molecular basis of the variable phenotype; thus to facilitate discovery of an effective therapeutic strategy. Herein we report the phenotype and genotype of a normal-appearing 10-month-old colony female that died suddenly. At necropsy examination, there were reduced skeletal and laryngeal muscle volume and mild dilatation of the oesophagus. Microscopic findings consisted of extensive degeneration and regeneration of the axial skeletal, tongue, oesophageal, and laryngeal muscles that were characterized by considerable central nucleation, individual fibre mineralization and interstitial fibrosis. The myocardial findings were limited to infiltration of adipose cells in the interstitium. The female dog was a compound heterozygote with one X chromosome carrying a point mutation in intron 6 of the dystrophin gene and the other X chromosome carrying a repetitive element insertion in intron 13 of the dystrophin gene. Although the direct cause of death was uncertain, it might likely be due to sudden cardiac death as has been seen in Duchenne muscular dystrophy patients. This case demonstrated dystrophinopathy in female dogs that have no ameliorating normal X chromosome., (© 2021 The Authors Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Osteosarcoma is one among the most common neoplasms in dogs. Current treatments show limited efficacy and fail to prevent metastasis. Conditionally replicative adenoviruses (CRAd) replicate exclusively in targeted tumor cells and release new virus particles to infect additional cells. We proposed that OC-CAVE1 (CAV2 with the E1A promoter replaced with the osteocalcin promotor) may also enhance existing immunity against tumors by overcoming immune tolerance via exposure of new epitopes and cytokine signaling. Eleven client-owned dogs with spontaneously occurring osteosarcomas were enrolled in a pilot study. All dogs were injected with OC-CAVE1 following amputation of the affected limb or limb-sparing surgery. Dogs were monitored for viremia and viral shedding. There was minimal virus shedding in urine and feces by the 6th day and no virus was present in blood after 4 weeks. CAV-2 antibody-titers increased in all of the patients, post-CRAd injection. Immunological assays were performed to monitor 1) humoral response against tumors, 2) levels of circulatory CD11c + cells, 3) levels of regulatory T cells, and 4) cytotoxic activity of tumor specific T cells against autologous tumor cells between pre-CRAd administration and 4 weeks post-CRAd administration samples. Administration of the CRAd OC-CAVE1 resulted in alteration of some immune response parameters but did not appear to result in increased survival duration. However, 2 dogs in the study achieved survival times in excess of 1 year. Weak replication of OC-CAVE1 in metastatic cells and delay of chemotherapy following CRAd treatment may contribute to the lack of immune response and improvement in survival time of the clinical patients., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

Gene therapy is a promising treatment option for cancer. However, its utility may be limited due to expression in off-target cells. Cancer-specific promoters such as telomerase reverse transcriptase (TERT), survivin, and chemokine receptor 4 (CXCR4) have enhanced activity in a variety of human and murine cancers, however, little has been published regarding these promoters in dogs. Given the utility of canine cancer models, the activity of these promoters along with adenoviral E2F enhanced E1a promoter (EEE) was evaluated in a variety of canine tumors, both from the endogenous gene and from exogenously administered constructs. Endogenous expression levels were measured for cTERT, cSurvivin, and cCXCR4 and were low for all three, with some non-malignant and some tumor cell lines and tissues expressing the gene. Expression levels from exogenously supplied promoters were measured by both the number of cells expressing the construct and the intensity of expression in individual cells. Exogenously supplied promoters were active in more cells in all tumor lines than in normal cells, with the EEE promoter being most active, followed by cTERT. The intensity of expression varied more with cell type than with specific promoters. Ultimately, no single promoter was identified that would result in reliable expression, regardless of the tumor type. Thus, these findings imply that identification of a pan-cancer promoter may be difficult. In addition, this data raises the concern that endogenous expression analysis may not accurately predict exogenous promoter activity., Competing Interests: The authors have declared that no competing interests exist

Extracting sufficient quantity and quality RNA from bone is essential for downstream application, such as transcriptomic sequencing, to evaluate gene expression. Isolation of RNA from bone presents a unique challenge owing to the hypocellular, brittle and mineralized matrix, which makes homogenizing the tissue difficult and provides little RNA to work with. Removal of contaminating tissue, such as bone marrow and connective tissue, is essential for isolating RNA that is unique to osteoblasts, osteoclasts and osteocytes. This study established a method to effectively isolate RNA from normal canine bone cells using the phalanges, without contamination from other tissue types, for downstream transcriptomic analysis.

Introduction: Mechanical thrombectomy (MT) has dramatically improved the prognosis for acute ischemic stroke (AIS) patients. Despite high recanalization rates, up to half of the patients will not present a good neurological outcome after MT. Therapeutic hypothermia is perhaps the most robust neuroprotectant studied preclinically., Materials and Methods: We explored various warming effects that can reduce the effectiveness or potency of selective hypothermia during AIS under conditions similar to actual clinical care. Four different selective hypothermia layouts were chosen. Layouts 1 and 2 used a single catheter without and with an insulated IV bag. Layouts 3 and 4 used two catheters arrange coaxially, without and with an insulated IV bag. Independent variables measured were IV bag exit temperature, catheter inlet temperature, and catheter outlet temperature at four different flow rates ranging from 8 to 25 ml/min over an infusion duration of 20 min., Results: Dominant warming occurs along the catheter pathway compared to warming along the infusion line pathway, ranging from 66% to 72%. Coaxial configurations provided an approximate 4°C cooler temperature benefit on delivered infusate over a single catheter. Brain tissue temperature predictions show that the maximum cooling layout, Layout 4 at maximum flow provides a 1°C within 5 min., Conclusion: Significant rewarming effects occur along the infusate flow path from IV bag to site of injury in the brain. Previous selective hypothermia clinical work, using flow rates and equipment at conditions similar to our study, likely produced rapid but not deep tissue cooling in the brain (~ 1°C)., Competing Interests: TL Merrill, JE Mitchell, and DR Merrill are all employees of FocalCool, LLC, a company developing brain cooling technology., (Copyright: © 2019 Brain Circulation.)

Breast cancer is among the most common malignancies affecting women and reproductively intact female dogs, resulting in death from metastatic disease if not treated effectively. To better manage the disease progression, canine mammary tumor (CMT) cells derived from malignant canine mammary cancers were fused to autologous dendritic cells (DCs) to produce living hybrid-cell fusion vaccines for canine patients diagnosed with spontaneous mammary carcinoma. The high-speed sorting of rare autologous canine patient DCs from the peripheral blood provides the autologous component of fusion vaccines, and fusion to major histocompatibility complex-unmatched CMT cells were produced at high rates. The vaccinations were delivered to each patient following a surgical resection 3 times at 3-week intervals in combination with immuno-stimulatory oligonucleotides and Gemcitabine adjunct therapy. The immunized patient animals survived 3.3-times longer (median survival 611 days) than the control patients (median survival 184 days) and also appeared to exhibit an enhanced quality of life. A comparison of vaccinated patients diagnosed with inflammatory mammary carcinoma resulted in a very short median survival (42 days), suggesting no effect of vaccination. The data showed that the development of autologous living DC-based vaccine strategies in patient animals designed to improve the management of canine mammary carcinoma can be successful and may allow an identification of the antigens that can be translatable to promote effective immunity in canine and human patients., Competing Interests: The authors declare no conflicts of interest., (© 2019 The Korean Society of Veterinary Science.)

In humans, dystrophin mutations cause the X-linked recessive disorder known as Duchenne muscular dystrophy (DMD). These mutations result in skeletal and cardiac muscle damage with mortality increasingly associated with cardiomyopathy. We have identified a novel dystrophin mutation in exon 21 in a line of Australian Labradoodles; affected dogs develop progressive clinical signs including poor weight gain and weight loss, gait abnormalities, exercise intolerance, skeletal muscle atrophy, macroglossa, ptyalism, dysphagia, kyphosis, and a plantigrade stance. Echocardiographic abnormalities include hyperechoic foci in the left ventricular papillary muscles, septal hypokinesis, and decreased left ventricular systolic and diastolic volume and internal diameter. Holter recordings found a Mobitz type II second-degree atrioventricular (AV) block in one affected dog. Analysis of phosphocreatine-to-ATP ratios (PCr/ATP) (obtained via cardiac magnetic resonance imaging and spectroscopy evaluation), found no statistically significant difference in the mean PCr/ATP between groups. Histopathologic skeletal muscle changes included fibrofatty infiltration, myocyte degeneration, necrosis, and regeneration, lymphohistiocytic inflammation, and mineralization; cardiac changes were limited to a focal area of mineralization adjacent to the sinoatrial node in the dog with a second-degree AV block. Due to rapidly progressive clinical signs, a severe phenotype, and potential for cardiac involvement, Australian Labradoodle dystrophinopathy may be a useful model to further study DMD pathogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Adenoviruses are the most widely used vectors in cancer gene therapy. Adenoviruses vectors are well characterized and are easily manipulated. Adenovirus serotype 5 (Ad5) is the most commonly used human serotype. Ad5 internalization into host cells is a combined effect of binding of Ad5 fiber knob with the coxsackie virus and adenovirus receptor (CAR) and binding of RGD motifs in viral penton to cell surface integrins (αvβ3, αvβ5). Ad5's wide range of host-cell transduction and lack of integration into the host genome have made it an excellent choice for cancer therapeutics. However, Ad5 has limited ability to transduce cells of hematopoietic origin. It has been previously reported that low or no expression of CAR is a potential obstacle to Ad5 infection in hematopoietic origin cells. In addition, we have previously reported that low levels of cell surface integrins (αvβ3, αvβ5) may inhibit Ad5 infection in canine lymphoma cell lines. In the current report we have examined the ability of an Ad5 vector to infect human (HEK293) and canine non-cancerous (NCF and PBMC), canine non-hematopoietic origin cancer (CMT28, CML7, and CML10), and canine hematopoietic origin cancer (DH82, 17-71, OSW, MPT-1, and BR) cells. In addition, we have quantified CAR, αvβ3 and αvβ5 integrin transcript expression in these cells by using quantitative reverse transcriptase PCR (q-RT-PCR). Low levels of integrins were present in MPT1, 17-71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. CAR mRNA levels were comparatively higher in MPT1, 17-71, OSW, and PBMC cells. This report confirms and expands the finding that low or absent expression of cell surface integrins may be the primary reason for the inability of Ad5-based vectors to transduce cells of lymphocytic origin and some myeloid cells but this is not true for all hematopoietic origin cells. For efficient use of Ad5-based therapeutic vectors in cancers of lymphocytic origin, it is important to address the defects in cell surface integrins., Competing Interests: The authors have declared that no competing interests exist.

Background: Splenic masses are common in older dogs; yet diagnosis preceding splenectomy and histopathology remains elusive. MicroRNAs (miRNAs) are short, non-coding RNAs that play a role in post-transcriptional regulation, and differential expression of miRNAs between normal and tumor tissue has been used to diagnose neoplastic diseases. The objective of this study was to determine differential expression of miRNAs by use of RNA-sequencing in canine spleens that were histologically confirmed as hemangiosarcoma, nodular hyperplasia, or normal., Results: Twenty-two miRNAs were found to be differentially expressed in hemangiosarcoma samples (4 between hemangiosarcoma and both nodular hyperplasia and normal spleen and 18 between hemangiosarcoma and normal spleen only). In particular, mir-26a, mir-126, mir-139, mir-140, mir-150, mir-203, mir-424, mir-503, mir-505, mir-542, mir-30e, mir-33b, mir-365, mir-758, mir-22, and mir-452 are of interest in the pathogenesis of hemangiosarcoma., Conclusions: Findings of this study confirm the hypothesis that miRNA expression profiles are different between canine splenic hemangiosarcoma, nodular hyperplasia, and normal spleens. A large portion of the differentially expressed miRNAs have roles in angiogenesis, with an additional group of miRNAs being dysregulated in vascular disease processes. Two other miRNAs have been implicated in cancer pathways such as PTEN and cell cycle checkpoints. The finding of multiple miRNAs with roles in angiogenesis and vascular disease is important, as hemangiosarcoma is a tumor of endothelial cells, which are driven by angiogenic stimuli. This study shows that miRNA dysregulation is a potential player in the pathogenesis of canine splenic hemangiosarcoma.

Lymphoma is the most common hematopoietic tumor in dogs and humans, with similar pathogenesis and therapeutic responses. Anticancer drugs like vincristine (VCR) and doxorubicin (DOX) are often used in treating lymphoma. However, the cure rate is generally poor due to chemoresistance. Here, we sought to determine whether stearidonic acid (SDA), a plant-based dietary fatty acid, sensitizes chemoresistant canine lymphoid-tumor cells. GL-1 B-cell lymphoid-tumor cells were found to be highly sensitive to the antitumor-activity of VCR and DOX, while OSW T-cell and 17-71 B-cell lymphoid-tumor cells were moderately and fully resistant, respectively. SDA, at its non-toxic concentrations, significantly promoted the antitumor action of VCR and DOX in both OSW and 17-71 cells. SDA-mediated chemosensitization was associated with SDA inhibition of P-glycoprotein (P-gp) function. This was confirmed in HEK293 cells stably expressing P-gp as well as by increased binding-affinity of SDA to P-gp in P-gp docking analysis. SDA at its chemosensitizing concentrations did not affect the viability of healthy dog peripheral blood mononuclear cells, suggesting that SDA is non-toxic to normal dog peripheral blood leucocytes at its chemosensitizing concentrations. Our study identifies a novel dietary fatty acid that may be used as a dietary supplement in combination with chemotherapy to promote the antitumor efficacy of the chemotherapy drugs in dogs and possibly in humans with chemoresistant lymphoma., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Human melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) displays potent growth suppressing and cell killing activity against a wide variety of human and rodent cancer cells. In this study, we identified a canine ortholog of the human mda-7/IL-24 gene located within a cluster of IL-10 family members on chromosome 7. The full-length mRNA sequence of canine mda-7 was determined, which encodes a 186-amino acid protein that has 66% similarity to human MDA-7/IL-24. Canine MDA-7 is constitutively expressed in cultured normal canine epidermal keratinocytes (NCEKs), and its expression levels are increased after lipopolysaccharide stimulation. In cultured NCEKs, the canine mda-7 pre-mRNA is differentially spliced, via exon skipping and alternate 5'-splice donor sites, to yield five splice variants (canine mda-7sv1, canine mda-7sv2, canine mda-7sv3, canine mda-7sv4 and canine mda-7sv5) that encode four protein isoforms of the canine MDA-7 protein. These protein isoforms have a conserved N-terminus (signal peptide sequence) and are dissimilar in amino acid sequences at their C-terminus. Canine MDA-7 is not expressed in primary canine tumor samples, and most tumor derived cancer cell lines tested, like its human counterpart. Unlike human MDA-7/IL-24, canine mda-7 mRNA is not expressed in unstimulated or lipopolysaccharide (LPS), concanavalin A (ConA) or phytohemagglutinin (PHA) stimulated canine peripheral blood mononuclear cells (PBMCs). Furthermore, in-silico analysis revealed that canonical canine MDA-7 has a potential 28 amino acid signal peptide sequence that can target it for active secretion. This data suggests that canine mda-7 is indeed an ortholog of human mda-7/IL-24, its protein product has high amino acid similarity to human MDA-7/IL-24 protein and it may possess similar biological properties to human MDA-7/IL-24, but its expression pattern is more restricted than its human ortholog., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background and Study Aims: Capsule endoscopy is an attractive alternative to colorectal cancer screening by conventional colonoscopy, but is currently limited by compromised mucosal visibility because of the lack of safe, controlled colonic insufflation. We have therefore developed a novel system of untethered, wireless-controlled carbon dioxide (CO2) insufflation for use in colonic capsule endoscopy, which this study aims to assess in vivo., Material and Methods: This observational, nonsurvival, in vivo study used five Yorkshire-Landrace cross swine. A novel insufflation capsule was placed in the porcine colons, and we recorded volume of insufflation, time, force, visualization, and a pathologic assessment of the colon., Results: The mean (standard deviation [SD]) diameter of insufflation was 32.1 (3.9) mm. The volume of CO2 produced successfully allowed complete endoscopic visualization of the mucosa and safe proximal passage of the endoscope. Pathologic examination demonstrated no evidence of trauma caused by the capsule., Conclusions: These results demonstrate the feasibility of a novel method of controlled colonic insufflation via an untethered capsule in vivo. This technological innovation addresses a critical need in colon capsule endoscopy., (© Georg Thieme Verlag KG Stuttgart · New York.)

Type 2 diabetes (T2DM) diagnoses are skyrocketing, making treatment of this disease an increasing focus of primary care visits. Guidelines recommend insulin intensification over time to achieve HbA1c targets. We conducted a systematic review regarding patterns and trends of insulin intensification and barriers to intensification. Providers across primary and specialty care settings often did not intensify insulin regimens despite patients' clinical status. Even among progressed patients, HbA1c values remained high. The paucity of available studies prevented a comprehensive understanding of patterns and trends in insulin intensification. Such information is needed to assess the quality of pharmacologic care for patients with T2DM., (Copyright © 2013 Primary Care Diabetes Europe. Published by Elsevier Ltd. All rights reserved.)

The muscular dystrophies are a group of devastating genetic disorders that affect both skeletal and cardiac muscle. An effective gene therapy for these diseases requires bodywide muscle delivery. Tyrosine mutant adeno-associated virus (AAV) has been considered as a class of highly potent gene transfer vectors. Here, we tested the hypothesis that systemic delivery of tyrosine mutant AAV can result in bodywide muscle transduction in newborn dogs. Three tyrosine mutant AAV vectors (Y445F/Y731F AAV-1, Y445F AAV-6, and Y731F AAV-9) were evaluated. These vectors expressed the alkaline phosphatase reporter gene under transcriptional regulation of either the muscle-specific Spc5-12 promoter or the ubiquitous Rous sarcoma virus promoter. Robust skeletal and cardiac muscle transduction was achieved with Y445F/Y731F AAV-1. However, Y731F AAV-9 only transduced skeletal muscle. Surprisingly, Y445F AAV-6 resulted in minimal muscle transduction. Serological study suggests that the preexisting neutralization antibody may underlie the limited transduction of Y445F AAV-6. In summary, we have identified Y445F/Y731F AAV-1 as a potentially excellent systemic gene transfer vehicle to target both skeletal muscle and the heart in neonatal puppies. Our findings have important implications in exploring systemic neonatal gene therapy in canine models of muscular dystrophy.

Value-based insurance design (VBID) plans selectively lower cost sharing to increase medication adherence. Existing plans have been structured in a variety of ways, and these variations could influence the effectiveness of VBID plans. We evaluated seventy-six plans introduced by a large pharmacy benefit manager during 2007-10. We found that after we adjusted for the other features and baseline trends, VBID plans that were more generous, targeted high-risk patients, offered wellness programs, did not offer disease management programs, and made the benefit available only for medication ordered by mail had a significantly greater impact on adherence than plans without these features. The effects were as large as 4-5 percentage points. These findings can provide guidance for the structure of future VBID plans.

To study the canine immune system we generated a mouse model engrafted with canine lymphocytes using NOD SCID IL2R common gamma chain -/- (NSG) mice as recipients (Ca-PBL-SCID). Engraftment of canine peripheral blood lymphocytes (PBLs) was determined post-injection with 10(7) peripheral blood mononuclear cells (PBMCs) into irradiated NSG mice using flow cytometry and fluorescently labeled antibodies specific to canine helper T cells (CD45(+) CD4(+)), cytotoxic lymphocytes (CD45(+) CD8(+)), regulatory T cells (CD45(+) CD4(+) Foxp3(+)), and B cells (CD45(+) Ig(+) CD21lo). Canine CD45(+) lymphocytes were detectable as early as day 1 in the peritoneal cavity, and beginning at 9 days in the blood, bone marrow, and spleen. CD4(+) T cells, of which Foxp-3(+) CD25hi cells constituted a minor percentage, were the predominant lymphocyte population at 9 days post engraftment contrasting with increasing proportions of CD8(+) CTL's and Ig(+) B cells beginning at 16 days. Canine immunoglobulin was initially detected in the serum of Ca-PBL-SCID mice at 9 days post-engraftment and peaked in concentration at day 36. From day 28 to 52 post-engraftment 30% of the Ca-PBL-SCID mice became markedly anemic and thrombocytopenic, yet gross and histopathologic examination of bone marrow, kidneys, spleen, liver, and intestine revealed no obvious lesions. Blood smear evaluation revealed agglutination of mature red blood cells, reticulocytes and a regenerative anemia. These findings demonstrate that NSG mice are capable of engraftment of canine PBLs yet develop graft versus host disease similar to Hu-PBL-SCID mice., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Despite clear patient experience advantages, low specificity rates have thus far prevented swallowable capsule endoscopes from replacing traditional endoscopy for diagnosis of colon disease. One explanation for this is that capsule endoscopes lack the ability to provide insufflation, which traditional endoscopes use to distend the intestine for a clear view of the internal wall. To provide a means of insufflation from a wireless capsule platform, in this paper we use biocompatible effervescent chemical reactions to convert liquids and powders carried onboard a capsule into gas. We experimentally evaluate the quantity of gas needed to enhance capsule visualization and locomotion, and determine how much gas can be generated from a given volume of reactants. These experiments motivate the design of a wireless insufflation capsule, which is evaluated in ex vivo experiments. These experiments illustrate the feasibility of enhancing visualization and locomotion of endoscopic capsules through wireless insufflation.

Purpose: Treatment guidelines recommend insulin progression (switching from basal to a premixed insulin regimen, adding bolus doses, and/or increasing dosing frequency) to achieve A1C targets as type 2 diabetes progresses, but fewer patients are being progressed than would be indicated based on their disease status. This systematic review proposes 2 questions regarding insulin progression among patients with type 2 diabetes: (1) What are the patient, provider, and health system barriers to insulin progression? (2) Do insulin progression barriers differ between insulin-naive and insulin-experienced patients?, Methods: We conducted a systematic review in the MEDLINE, EMBASE, Science Citation Index, PsycINFO, CINAHL, and Cochrane Library databases through July 2011., Results: Of 745 potentially relevant articles, 10 met inclusion criteria: 7 evaluated patient and 2 evaluated provider barriers, and 1 was an intervention to reduce barriers among physicians. Patients with prior insulin experience had fewer barriers arising from injection-related concerns and worries about the burden of insulin progression than did insulin-naive patients. Physician barriers included concerns about patients' ability to follow more complicated regimens as well as physicians' own inexperience with insulin and progression algorithms. The cross-sectional nature, narrow scope, and failure of all studies to examine patient, provider, and health systems barriers concurrently limited both barrier identification and an assessment of their impact on progression., Conclusions: Patient and physician experience with insulin and diabetes/insulin education were associated with fewer perceived barriers to insulin progression. Future studies should use multilevel longitudinal designs to quantify the relative impact of potential patient, provider, and health system factors on progression and health outcomes.

The absence of dystrophin in the heart leads to Duchenne cardiomyopathy. Dystrophin-deficient dogs represent a critical model to translate novel therapies developed in mice to humans. Unfortunately, little is known about cardiophysiology changes in these dogs. We performed prospective electrocardiographic and echocardiographic examinations at 3, 6 and 12 months of age in four normal and three affected dogs obtained from the same litter. Affected dogs showed growth retardation and serum creatine kinase elevation. Necropsy confirmed cardiac dystrophin deficiency and histopathology. Q/R ratio elevation and diastolic left ventricular (LV) internal diameter reduction were the most consistent findings in affected dogs at all ages. At 6 and 12 months, dystrophic dogs also showed significant reduction of PR intervals, LV end diastolic/systolic volumes and systolic LV internal diameters. Epicardial and endocardial slope times were significantly reduced in affected dogs at 12 months. These results establish the baseline for evaluating experimental therapies in the future., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Recombinant adenovirus vectors (Ad) have been recognized as effective in vivo gene delivery vehicles and utilized as gene therapy agents for a number of cancers. The elucidation of viral entry mechanisms has allowed the development of recombinant vectors that exploit existing cell surface receptors to achieve entry into the cell. B lymphocytes are normally resistant to infection by adenovirus 5, likely due to the lack of the Coxsackie and Adenovirus receptor (CAR). Using reverse-transcriptase PCR and flow cytometry, the CD40 receptor has been shown to be expressed on many lymphoma cells. We exploited this finding to develop a gene therapy strategy for treatment of canine B cell lymphoma. Ad5 was targeted to cells expressing CD40 via CD40 ligand (CD40L) and was effective in infecting CD40-expressing control cells; however, both primary canine lymphoma cells and cell lines demonstrated limited evidence of transduction. Following receptor binding, adenovirus entry into cells may require interaction with α(v)β(3/5) integrins; we demonstrate that canine lymphoma cells are deficient in these integrins. Reduced α(v)β(3) integrin expression may render these cells incapable of internalizing Ad vectors. Thus, any viral targeting approaches for treatment of canine lymphoma must also take into account the potential lack of internalization signals.

Duchenne muscular dystrophy (DMD) is a dystrophin-deficient lethal muscle disease. To date, the catastrophic muscle wasting phenotype has only been seen in dystrophin-deficient humans and dogs. Although Duchenne-like symptoms have been observed in more than a dozen dog breeds, the mutation is often not known and research colonies are rarely established. Here, we report an independent canine DMD model originally derived from the Pembroke Welsh corgi breed. The affected dogs presented clinical signs of muscular dystrophy. Immunostaining revealed the absence of dystrophin and upregulation of utrophin. Histopathologic examination showed variable fiber size, central nucleation, calcification, fibrosis, neutrophil and macrophage infiltration and cardiac focal vacuolar degeneration. Carrier dogs also displayed mild myopathy. The mutation was identified as a long interspersed repetitive element-1 (LINE-1) insertion in intron 13, which introduced a new exon containing an in-frame stop codon. Similar mutations have been seen in human patients. A colony was generated by crossing carrier females with normal males. Affected puppies had a normal birth weight but they experienced a striking growth delay in the first 5 days. In summary, the new corgi DMD model offers an excellent opportunity to study DMD pathogenesis and to develop novel therapies.

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell-tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c(+) expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.

Objective: To determine the earliest day of gestation at which relaxin could be detected in pregnant queens by use of a commercially available point-of-care test designed for use in dogs, and to calculate sensitivity and specificity of the test for pregnancy detection on any specified day of gestation., Design: Evaluation study., Animals: 162 female cats (24 queens from a breeding colony, 128 stray and feral queens undergoing ovariohysterectomy, and 10 ovariohysterectomized cats)., Procedures: 24 queens were monitored for pregnancy. Blood samples were collected daily and tested for relaxin until 2 consecutive positive test results were obtained. The earliest day of pregnancy detection was estimated by counting backward from the day of parturition to the day of the first positive test. The uteri, ovaries, and any fetuses of 128 stray and feral queens undergoing ovariohysterectomy were examined grossly, and gestational day in pregnant queens was determined on the basis of fetal crown-rump length. Blood samples from these queens and from 10 cats ovariohysterectomized prior to the study were collected for relaxin testing., Results: Pregnancy was detected by use of the relaxin test kit as early as gestational day 20; sensitivity of the test was 100% on and after gestational day 29. False-positive results were detected in 3 queens, 2 of which had large (approx 2×3-cm) ovarian cysts, resulting in a specificity of 95.9%., Conclusions and Clinical Relevance: A commercially available relaxin test kit designed for use in dogs can be used to reliably detect pregnancy in cats.

Toward increasing the diagnostic ability of wireless capsule endoscopy, we propose a method to wirelessly insufflate the Gastrointestinal Tract. By increasing the viewable surface area, it appears likely that capsule-based insufflation may reduce the number of false negative diagnoses made by endoscopic capsules. Our approach to wireless insufflation is to utilize controlled phase transition of a small volume of fluid stored onboard the capsule to a large volume of gas that is then emitted into the intestine. We begin by describing experiments designed to evaluate the amount of gas a capsule must produce to have a beneficial impact on visualization in the colon. We then describe experiments evaluating how much gas can be generated from a given volume of fluid, using Hydrogen Peroxide as our working fluid. We also evaluate thermal effects of the Hydrogen Peroxide reaction. The cumulative result of these experiments is an illustration of the feasibility of carrying a sufficient volume of fluid onboard a wireless capsule to generate a beneficial enhancement in visualization of the interior of the Gastrointestinal Tract, and specifically the colon.

Targeting viral vectors encoding tumor-associated antigens to dendritic cells (DCs) in vivo is likely to enhance the effectiveness of immunotherapeutic cancer vaccines. We have previously shown that genetic modification of adenovirus (Ad) 5 to incorporate CD40 ligand (CD40L) rather than native fiber allows selective transduction and activation of DCs in vitro. Here, we examine the capacity of this targeted vector to induce immune responses to the tumor antigen CEA in a stringent in vivo canine model. CD40-targeted Ad5 transduced canine DCs via the CD40-CD40L pathway in vitro, and following vaccination of healthy dogs, CD40-targeted Ad5 induced strong anti-CEA cellular and humoral responses. These data validate the canine model for future translational studies and suggest targeting of Ad5 vectors to CD40 for in vivo delivery of tumor antigens to DCs is a feasible approach for successful cancer therapy.