In Vivo Accelerator-Based Boron Neutron Capture Therapy for Spontaneous Tumors in Large Animals: Case Series

(1) Background: accelerator-based neutron sources are a new frontier for BNCT but many technical issues remain. We aimed to study such issues and results in larger-animal BNCT (cats and dogs) with naturally occurring, malignant tumors in different locations as an intermediate step in translating current research into clinical practice. (2) Methods: 10 pet cats and dogs with incurable, malignant tumors that had no treatment alternatives were included in this study. A tandem accelerator with vacuum insulation was used as a neutron source. As a boron-containing agent, 10B-enriched sodium borocaptate (BSH) was used at a dose of 100 mg/kg. Animal condition as well as tumor progression/regression were monitored. (3) Results: regression of tumors in response to treatment, improvements in the overall clinical picture, and an increase in the estimated duration and quality of life were observed. Treatment-related toxicity was mild and reversible. (4) Conclusions: our study contributes to preparations for human BNCT clinical trials and suggests utility for veterinary oncology.

Design, Fabrication and Characterization of Nanoliposomes Containing Snake Venom of Pseudocereaster percius

Background: Development of antivenom or antidote requires the repetition of immunization of large animals, such as horses and goats, which ultimately releases the IgG immunoglobulin produced in the serum specimen. As snake venom involves a variety of proteins and enzymes getting administered into the animal, this process can inflict significant harm to the animal, therefore choosing carriers that can deliver the least amount of venom could be a safer option for animal immunization Objective: In this research, nanoliposomes were used to encapsulate venom as a protected cargo for immunization. We used two distinct liposomal formulations to entrap the venom: 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) associated with cholesterol in one formulation and dimethyldioctadecylamonium (Bromide salt) paired with cholesterol in the other. Method: Liposomal formulations prepared by solvent evaporation method and the venom was encapsulated in liposomes and evaluated for size and zeta potential. Meanwhile, encapsulation efficiency, venom release percentage, and phospholipase activity have all been analyzed. Results: The findings revealed that dimethyldioctadecylamonium (Bromide salt) combined with cholesterol had the highest encapsulation efficiency. In this formulation, the venom release rate had a steady-state profile. The lack of phospholipase activity in this formulation may be due to a bromide group in the liposomal structure that could be useful for immunization. Conclusion: Liposomal formulations, which do not have the active site of the snake venom enzymes, could be used for venom encapsulation.

Chimerism-Based Tolerance to Kidney Allografts in Humans: Novel Insights and Future Perspectives

Chronic rejection and immunosuppression-related toxicity severely affect long-term outcomes of kidney transplantation. The induction of transplantation tolerance – the lack of destructive immune responses to a transplanted organ in the absence of immunosuppression – could potentially overcome these limitations. Immune tolerance to kidney allografts from living donors has been successfully achieved in humans through clinical protocols based on chimerism induction with hematopoietic cell transplantation after non-myeloablative conditioning. Notably, two of these protocols have led to immune tolerance in a significant fraction of HLA-mismatched donor-recipient combinations, which represent the large majority of cases in clinical practice. Studies in mice and large animals have been critical in dissecting tolerance mechanisms and in selecting the most promising approaches for human translation. However, there are several key differences in tolerance induction between these models and humans, including the rate of success and stability of donor chimerism, as well as the relative contribution of different mechanisms in inducing donor-specific unresponsiveness. Kidney allograft tolerance achieved through durable full-donor chimerism may be due to central deletion of graft-reactive donor T cells, even though mechanistic data from patient series are lacking. On the other hand, immune tolerance attained with transient mixed chimerism-based protocols initially relies on Treg-mediated suppression, followed by peripheral deletion of donor-reactive recipient T-cell clones under antigenic pressure from the graft. These conclusions were supported by data deriving from novel high-throughput T-cell receptor sequencing approaches that allowed tracking of alloreactive repertoires over time. In this review, we summarize the most important mechanistic studies on tolerance induction with combined kidney-bone marrow transplantation in humans, discussing open issues that still need to be addressed and focusing on techniques developed in recent years to efficiently monitor the alloresponse in tolerance trials. These cutting-edge methods will be instrumental for the development of immune tolerance protocols with improved efficacy and to identify patients amenable to safe immunosuppression withdrawal.

Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models

The development in cryobiology in animal breeding had revolutionized the field of reproductive medicine. The main objective to preserve animal germplasm stems from variety of reasons such as conservation of endangered animal species, animal diversity, and an increased demand of animal models and/or genetically modified animals for research involving animal and human diseases. Cryopreservation has emerged as promising technique for fertility preservation and assisted reproduction techniques (ART) for production of animal breeds and genetically engineered animal species for research. Slow rate freezing and rapid freezing/vitrification are the two main methods of cryopreservation. Slow freezing is characterized by the phase transition (liquid turning into solid) when reducing the temperature below freezing point. Vitrification, on the other hand, is a phenomenon in which liquid solidifies without the formation of ice crystals, thus the process is referred to as a glass transition or ice-free cryopreservation. The vitrification protocol applies high concentrations of cryoprotective agents (CPA) used to avoid cryoinjury. This chapter provides a brief overview of fundamentals of cryopreservation and established methods adopted in cryopreservation. Strategies involved in cryopreserving germ cells (sperm and egg freezing) are included in this chapter. Last section describes the frontiers and advancement of cryopreservation in some of the important animal models like rodents (mouse and rats) and in few large animals (sheep, cow etc).

Myofibroblast Senescence Promotes Arrhythmogenic Remodeling in the Aged Infarcted Rabbit Heart

Progressive tissue remodeling after myocardial infarction (MI) promotes cardiac arrhythmias. This process is well studied in young animals, but little is known about pro-arrhythmic changes in aged animals. Senescent cells accumulate with age and accelerate age-associated diseases. Senescent cells interfere with cardiac function and outcome post-MI with age, but studies have not been performed in large animals, and the mechanisms are unknown. Here, we investigated the role of senescence in regulating inflammation, fibrosis, and arrhythmogenesis in young and aged infarcted rabbits. Aged rabbits exhibited increased peri-procedural mortality and arrhythmogenic electrophysiological remodeling at the infarct border zone (IBZ) compared to young rabbits. Studies of the aged infarct zone revealed persistent myofibroblast senescence and increased inflammatory signaling over a twelve-week timecourse. Senescent IBZ myofibroblasts in aged rabbits appear to be coupled to myocytes, and our computational modeling showed that senescent myofibroblast-cardiomyocyte coupling prolongs action potential duration (APD) and facilitates conduction block permissive of arrhythmias. Aged infarcted human ventricles show levels of senescence consistent with aged rabbits, and senescent myofibroblasts also couple to IBZ myocytes. Our findings suggest that senolytic drugs may mitigate arrhythmias post-MI.

Regeneration of Subcutaneous Cartilage in a Swine Model Using Autologous Auricular Chondrocytes and Electrospun Nanofiber Membranes Under Conditions of Varying Gelatin/PCL Ratios

The scarcity of ideal biocompatible scaffolds makes the regeneration of cartilage in the subcutaneous environment of large animals difficult. We have previously reported the successful regeneration of good-quality cartilage in a nude mouse model using the electrospun gelatin/polycaprolactone (GT/PCL) nanofiber membranes. The GT/PCL ratios were varied to generate different sets of membranes to conduct the experiments. However, it is unknown whether these GT/PCL membranes can support the process of cartilage regeneration in an immunocompetent large animal model. We seeded swine auricular chondrocytes onto different GT/PCL nanofiber membranes (GT:PCL = 30:70, 50:50, and 70:30) under the sandwich cell-seeding mode. Prior to subcutaneously implanting the samples into an autologous host, they were cultured in vitro over a period of 2 weeks. The results revealed that the nanofiber membranes with different GT/PCL ratios could support the process of subcutaneous cartilage regeneration in an autologous swine model. The maximum extent of homogeneity in the cartilage tissues was achieved when the G5P5 (GT: PC = 50: 50) group was used for the regeneration of cartilage. The formed homogeneous cartilage tissues were characterized by the maximum cartilage formation ratio. The extents of the ingrowth of the fibrous tissues realized and the extents of infiltration of inflammatory cells achieved were found to be the minimum in this case. Quantitative analyses were conducted to determine the wet weight, cartilage-specific extracellular matrix content, and Young’s modulus. The results indicated that the optimal extent of cartilage formation was observed in the G5P5 group. These results indicated that the GT/PCL nanofiber membranes could serve as a potential scaffold for supporting subcutaneous cartilage regeneration under clinical settings. An optimum GT/PCL ratio can promote cartilage formation.

Transcriptomic Insight into the Melon Morphology of Toothed Whales for Aquatic Molecular Developments

Aquatic habitats are home to large animals such as marine mammals. Toothed whales have special fat deposits in the forehead region (called the melon) of their heads that are associated with echolocation underwater. This fat is also important industrially for human use. Due to the lack of gene expression information on the melon fat of toothed whales, we investigated the melon morphology via the transcriptomic approach. Four parts of the melons of three individual Risso’s dolphins were used for total RNA extraction, cDNA library preparation, and sequencing via next-generation sequencing (NGS) technologies. After the downstream analysis of raw sequence data, we determined that the outer layer of the melon’s ML4 region played multifunctional roles. The 36 differentially expressed genes of outer melon included ASB5, MYH13, MYOM2, and MYOM3. These genes are associated with muscle function and energy metabolism. Gene clustering and functional enrichment analyses also represented enrichments, such as the pentose phosphate pathway and morphogenesis related to lipid metabolism and muscle functions. This study will be crucial for muscle and fat functional-related molecular studies on aquatic mammals. Additionally, the study presents potential pathways, such as melon fat biosynthesis, for sustainable future developments.

Genetically modified large animal models for investigating neurodegenerative diseases

Neurodegenerative diseases represent a large group of neurological disorders including Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington’s disease. Although this group of diseases show heterogeneous clinical and pathological phenotypes, they share important pathological features characterized by the age-dependent and progressive degeneration of nerve cells that is caused by the accumulation of misfolded proteins. The association of genetic mutations with neurodegeneration diseases has enabled the establishment of various types of animal models that mimic genetic defects and have provided important insights into the pathogenesis. However, most of genetically modified rodent models lack the overt and selective neurodegeneration seen in the patient brains, making it difficult to use the small animal models to validate the effective treatment on neurodegeneration. Recent studies of pig and monkey models suggest that large animals can more faithfully recapitulate pathological features of neurodegenerative diseases. In this review, we discuss the important differences in animal models for modeling pathological features of neurodegenerative diseases, aiming to assist the use of animal models to better understand the pathogenesis and to develop effective therapeutic strategies.

Contractile force of transplanted cardiomyocytes contributes to heart function after injury

Transplantation of pluripotent stem cell-derived cardiomyocytes represents an innovative therapeutic strategy for heart failure. Studies in small and large animals have demonstrated functional recovery of left ventricular function after cardiomyocyte transplantation, and first clinical studies are currently underway. Yet, the mechanism of action underlying graft-induced benefit is unknown. Here we demonstrate that transplanted cardiomyocytes actively contribute to heart function. We transplanted cardiomyocytes with an optogenetic off-on switch in a guinea pig cardiac injury model. Light-induced inhibition of engrafted cardiomyocyte contractility resulted in a rapid decrease of left ventricular function that was fully reversible with the offset of photostimulation. Hence, our optogenetic approach demonstrated that transplanted cardiomyocytes actively participate in heart function, supporting the hypothesis that the delivery of new force-generating myocardium can serve as a regenerative therapeutic strategy.

Study on the Application of Super-Resolution Ultrasound for Cerebral Vessel Imaging in Rhesus Monkeys

Background: Ultrasound is ideal for displaying intracranial great vessels but not intracranial microvessels and terminal vessels. Even with contrast agents, the imaging effect is still unsatisfactory. In recent years, significant theoretical advances have been achieved in super-resolution imaging. The latest commonly used ultrafast plane-wave ultrasound Doppler imaging of the brain and microbubble-based super-resolution ultrasound imaging have been applied to the imaging of cerebral microvessels and blood flow in small animals such as mice but have not been applied to in vivo imaging of the cerebral microvessels in monkeys and larger animals. In China, preliminary research results have been obtained using super-resolution imaging in certain fields but rarely in fundamental and clinical experiments on large animals. In recent years, we have conducted a joint study with the Xi'an Jiaotong University to explore the application and performance of this new technique in the diagnosis of cerebrovascular diseases in large animals.Objective: To explore the characteristics and advantages of microbubble-based super-resolution ultrasound imaging of intracranial vessels in rhesus monkeys compared with conventional transcranial ultrasound.Methods: First, the effectiveness and feasibility of the super-resolution imaging technique were verified by modular simulation experiments. Then, the imaging parameters were adjusted based on in vitro experiments. Finally, two rhesus monkeys were used for in vivo experiments of intracranial microvessel imaging.Results: Compared with conventional plane-wave imaging, super-resolution imaging could measure the inner diameters of cerebral microvessels at a resolution of 1 mm or even 0.7 mm and extract blood flow information. In addition, it has a better signal-to-noise ratio (5.625 dB higher) and higher resolution (~30-fold higher). The results of the experiments with rhesus monkeys showed that microbubble-based super-resolution ultrasound imaging can achieve an optimal resolution at the micron level and an imaging depth >35 mm.Conclusion: Super-resolution imaging can realize the monitoring imaging of high-resolution and fast calculation of microbubbles in the process of tissue damage, providing an important experimental basis for the clinical application of non-invasive transcranial ultrasound.