Minipig Research Articles

Experimental research on efficacy of intelligent human-machine collaborative robot-assisted cranio-tomy system

Objective To explore the accuracy, efficiency and safety of robot-assisted craniotomy system named Cranibot. Methods The system called Cranibot was developed by Department of Neurosurgery, The First Medical Center of Chinese PLA General Hospital and Institute of Intelligent Robotics, Beijing Institute of Technology and then employed to carry out randomized controlled experiments on 3 kinds of subjects including 8 3D-printing PVC (polyvinyl chloride) skull models, 5 cadaveric pig heads and 5 living Bama miniature pigs. Both left and right sides were symmetrically selected in each subject to perform craniotomy, and on each side 4 holes were drilled to form a square bone flap. One part of the skull on one side was randomly categorized into the experimental group in which craniotomy was performed by system, and its symmetrical part of skull on the other side belonged to the control group in which craniotomy was performed by the same neurosurgeon team. We observed and measured the drilling position error, time spent on craniotomy and incidence of tissue injury in the two groups. Results In skull model experiments, the average position error and time spent on craniotomy of robot group were 1.87±0.66 mm and 6.64±1.15 min, and those of surgeon group were 3.14±0.73 mm and 8.06±1.10 min respectively. The differences were statistically significant (both P 0.05). Conclusion The robot-assisted craniotomy system named Cranibot could improve the accuracy and efficiency of craniotomy, while its safety requires further research with large sample size. Key words: Robotics; Neurosurgical procedures; Man-machine systems; Models, animal

Titanium and zirconium release from titanium- and zirconia implants in mini pig maxillae and their toxicity in vitro

ObjectiveTitanium (Ti)- and Zirconia (ZrO2)-implants in mini pig maxillae were compared with respect to Ti/zirconium (Zr) release into the surrounding bone tissues, the resulting short term tissue responses and the potential toxicity. MethodsTi/Zr release from Ti- and ZrO2-implants in mini pig maxillae was determined with inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS). The spatial distribution of Ti and Zr in maxilla tissues near the implant surface was assessed with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). A histological analysis was performed to investigate the tissue responses after 12 weeks of implantation. The cytotoxicity and DNA damage of Ti particles and ZrO2 particles were studied with XTT and Comet assay. ResultsThe mean Ti content in the bone adjacent to Ti-implants was 1.67 mg/kg-bone weight. The highest Ti content detected was 2.17 mg/kg-bone weight. The mean Zr content in the bone adjected to ZrO2-implants was 0.59 mg/kg-bone weight. The highest Zr content was 0.75 mg/kg-bone weight. The spatial distribution of the Ti and Zr in bone showed mainly a higher intensity of Ti and Zr close to the screw thread outer tip rather. Histological analysis indicated that near both implant-types signs of bone marrow fibrosis were present. EC50 of commercially available ZrO2-nanoparticles (NPs, <100 nm) and ZrO2-microparticles (MPs, <5 μm) was 13.96 mg/ml and 80.99 mg/ml, respectively. ZrO2-NPs and ZrO2-MPs can induce DNA damage at 70 μg/ml and 810 μg/ml, respectively. SignificanceAfter 12-weeks of implantation, increased concentrations of Ti and Zr can be detected in bone/tissues near Ti- and ZrO2-implants in mini pig maxillae. Ti content released from Ti-implants is two times higher than the Zr content released from ZrO2-implants. ZrO2-NPs showed lower cytotoxicity and DNA damage compared to results reported for Ti-NPs in human cells.

Generation and delivery of “Yamanaka factor” recombinant proteins mediated with magnetic iron oxide nanoparticles (MIONPs)

Reprogramming mediated with proteins provides a novel, simpler, and safer strategy for generating induced pluripotent stem cells (iPSCs); in order to efficiently produce the recombination proteins and optimize the gene delivery system, different agents were screened to efficiently transfect plasmid DNA (pDNA) encoding “Yamanaka factor” (Oct 4, Sox 2, Klf 4, or c-Myc) into human embryonic kidney (HEK)-293T cells. The results indicated that 10 μg/mL Poly-MAG (polyethyleneimine (PEI)-MIONP) combined with exogenous magnetic field, 5 μg/mL Lipofectamine® 2000, and 40 μg/mL PEI were, respectively, more suitable to deliver the pDNA encoding Oct 4 into HEK-293T cells in the same transfection reagent groups, but the cell survival rate in 10 μg/mL Poly-MAG treatment group was significantly higher than that in the latter two groups (P < 0.01). “Yamanaka factor” recombination proteins could be extracted and purified from enhanced green fluorescent protein positive (EGFP+) HEK-293T cells. These recombination proteins could be delivered into fibroblasts of miniature pig via the actions of poly-arginine 9R, Poly-MAG, and exogenous magnetic field, producing iPSC-like cells. Furthermore, these iPSC-like cells with MIONPs would be enriched under the action of magnetic force. Therefore, the synergistic action of Poly-MAG and exogenous magnetic field could not only mediate the gene delivery of “Yamanaka factor”, but also enhance the transmembrane of functional recombination proteins and enrichment of generated iPSC-like cells, which laid a solid foundation for future safer iPSC-based targeted therapies.

Systematic Review of Human Dental Pulp Stem Cells for Cartilage Regeneration.

Background: Symptomatic cartilage lesions and early osteoarthritis produce significant clinical and economic burdens. Cartilage repair can improve the symptoms and delay arthroplasty. The complete healing of damaged cartilage with the consistent reproduction of normal hyaline cartilage has not yet been achieved. The choice of harvesting site might influence the cells' abilities to modulate immunologic and inflammatory responses. Recently, dental pulp has been shown to contain a stem cell niche consisting of dental pulp stem cells (DPSCs) that maintain their self-renewal capacity due to the active environment in the dental pulp of deciduous teeth. Objective: The aim of this study was to critically review the current literature on the potential and limitations of the use of dental pulp-derived mesenchymal stem cells in cell-based therapies for cartilage regeneration. Methods: An electronic, customized search of scientific articles was conducted using the PubMed/MEDLINE and EMBASE databases from their inception to December 2018. The inclusion criteria were applied, and the articles that described the use of DPSC in cartilage treatment were selected for complete evaluation. The articles were classified according to the scaffold used, experimental model, chondrogenic differentiation features, defect location, cartilage evaluation, and results. After the application of the eligibility criteria, a total of nine studies were selected and fully analyzed. Results: A variety of animal models were used, including mice, rats, rabbits, and miniature pigs, to evaluate the quality and safety of human DPSCs in the repair of cartilage defects. Among the articles, two studies focused on preclinical models of cartilage tissue engineering. Five studies implanted DPSCs in other animal sites. Conclusion: The use of DPSCs is a potential new stem cell therapy for articular cartilage repair. The preclinical evidence discussed in this article provides a solid foundation for future clinical trials. Impact statement Osteoarthritis presents an ever-increasing clinical and socioeconomic burden. While cartilage repair has the potential to improve symptoms and delay joint replacement, complete regeneration of hyaline cartilage has been an elusive goal. Dental pulp has been shown to contain a niche that protects dental pulp stem cells (DPSCs) from the cumulative effects of genetic and environmental factors and maintains their self-renewal capacity due to the active environment. Transplantation and preclinical trials have demonstrated the strong potential of regenerative tissue-engineering protocols using DPSCs.

SLA-1 Genetic Diversity in Pigs: Extensive Analysis of Copy Number Variation, Heterozygosity, Expression, and Breed Specificity

Swine leukocyte antigens play indispensable roles in immune responses by recognizing a large number of foreign antigens and thus, their genetic diversity plays a critical role in their functions. In this study, we developed a new high-resolution typing method for pig SLA-1 and successfully typed 307 individuals from diverse genetic backgrounds including 11 pure breeds, 1 cross bred, and 12 cell lines. We identified a total of 52 alleles including 18 novel alleles and 9 SLA-1 duplication haplotypes, including 4 new haplotypes. We observed significant differences in the distribution of SLA-1 alleles among the different pig breeds, including the breed specific alleles. SLA-1 duplication was observed in 33% of the chromosomes and was especially high in the biomedical model breeds such as SNU (100%) and NIH (76%) miniature pigs. Our analysis showed that SLA-1 duplication is associated with the increased level of SLA-1 mRNA expression in porcine cells compared to that of the single copy haplotype. Therefore, we provide here the results of the most extensive genetic analysis on pig SLA-1.

Long-term case study of a Wuzhishan miniature pig with diabetes.

BackgroundMiniature pigs are attractive animal models for exploring diabetes because they are similar to humans in terms of physiological structure and metabolism. However, little is known about the complications of diabetes in pigs.MethodsIn this study, a 28‐month observation of a Wuzhishan miniature pig with streptozotocin (STZ)‐induced (120 mg/kg) diabetes was conducted, to investigate diabetes‐related complications and the possibility of self‐recovery in miniature pigs. Blood glucose, serum and urinary biochemistry was measured, and histopathologic examinations of eyes, kidney and pancreas were made.ResultsDuring the observation, diabetic complications of eyes and kidney were observed. The eye complications included bilateral cataracts in the 15th month and degeneration of inner retina and microaneurysm in the 28th month. Kidney complications included glomerular mesangial expansion, focal segmental glomerular sclerosis, and renal tubular epithelial degeneration, but no proteinuria was observed. By 28 months after the application of STZ, with no treatment given, blood glucose had recovered and the number of pancreatic islet beta‐cells had increased significantly.ConclusionsWe showed that the STZ‐induced diabetes model in miniature pigs could accurately mimic the pathological changes of human diabetes, and that pancreatic islet beta‐cell regeneration did occur in an adult miniature pig, providing a new means for exploring diabetic complications and pancreatic islet beta‐cell regeneration.

Photobiomodulation by diffusing optical fiber on spinal cord: A feasibility study in piglet model.

Previous studies on spinal cord injury (SCI) have confirmed that percutaneous photobiomodulation (PBM) therapy can ameliorate immunoinflammatory responses at sites of injury, accelerate nerve regeneration, suppress glial scar formation and promote the subsequent recovery of locomotor function. The current study was performed to evaluate a large-animal model employing implanted optical fibers to accurately irradiate targeted spinal segments. The method's feasibility and irradiation parameters that do not cause phototoxic reaction were determined, and the methodology of irradiating the spinal cord with near-infrared light was investigated in detail. A diffusing optical fiber was implanted above the T9 spinal cord of Bama miniature pigs and used to transfer near-infrared light (810 nm) onto the spinal cord surface. After daily irradiation with 200, 300, 500 or 1000 mW for 14 days, both sides of the irradiated area of the spinal cord were assessed for temperature changes. The condition of the spinal cord and the position of optical fiber were investigated by magnetic resonance imaging (MRI), and different parameters indicating temperature increases or phototoxicity were measured on the normal spinal cord surface due to light irradiation (ie, heat shock responses, inflammatory reactions and neuronal apoptosis), and the animals' lower-limb neurological function and gait were assessed during the irradiation process. The implanted device was stable inside the freely moving animals, and light energy could be directly projected onto the spinal cord surface. The screening of different irradiation parameters preliminary showed that direct irradiation onto the spinal cord surface at 200 and 300 mW did not significantly increase the temperature, stress responses, inflammatory reactions and neural apoptosis, whereas irradiation at 500 mW slightly increased these parameters, and irradiation at 1000 mW induced a significant temperature increase, heat shock, inflammation and apoptosis responses. HE staining of spinal cord tissue sections did not reveal any significant structural changes of the tissues compared to the control group, and the neurological function and gait of all irradiated animals were normal. In this study, we established an in-vivo optical fiber implantation method, which might be safe and stable and could be used to directly project light energy onto the spinal cord surface. This study might provide a new perspective for clinical applications of PBM in acute SCI.

Targeted Optical Imaging of the Glucagonlike Peptide 1 Receptor Using Exendin-4-IRDye 800CW.

The treatment of choice for insulinomas and focal lesions in congenital hyperinsulinism (CHI) is surgery. However, intraoperative detection can be challenging. This challenge could be overcome with intraoperative fluorescence imaging, which provides real-time lesion detection with a high spatial resolution. Here, a novel method for targeted near-infrared (NIR) fluorescence imaging of glucagonlike peptide 1 receptor (GLP-1R)–positive lesions, using the GLP-1 agonist exendin-4 labeled with IRDye 800CW, was examined in vitro and in vivo. Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with GLP-1R. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. In vivo NIR fluorescence imaging of CHL-GLP-1R xenografts was performed. Localization of the tracer in the pancreatic islets of BALB/c nude mice was examined using fluorescence microscopy. Laparoscopic imaging was performed to detect the fluorescent signal of the tracer in the pancreas of mini pigs. Results: Exendin-4-IRDye 800CW binds GLP-1R with a half-maximal inhibitory concentration of 3.96 nM. The tracer accumulates in CHL-GLP-1R xenografts. Subcutaneous CHL-GLP-1R xenografts were visualized using in vivo NIR fluorescence imaging. The tracer accumulates specifically in the pancreatic islets of mice, and a clear fluorescent signal was detected in the pancreas of mini pigs. Conclusion: These data provide the first in vivo evidence of the feasibility of targeted fluorescence imaging of GLP-1R–positive lesions. Intraoperative lesion delineation using exendin-4-IRDye 800CW could benefit open as well as laparoscopic surgical procedures for removal of insulinomas and focal lesions in CHI.

A Regenerative Medicine Approach to the Treatment of Hearing, Balance, and Olfactory Disorders: What Is in the Future for Otolaryngology?

Regenerative medicine is being applied to many fields of medicine and is now starting to be considered and developed for application to treat hearing, balance, olfaction, and voice disorders. This special issue of the Anatomical Record with a series of over 20 papers covers many aspects of gene and stem cell therapies as they are developed for clinical applications in both in vitro and in vivo laboratory studies. These studies cover a wide range of approaches from gene editing in zebrafish with the latest technology (i.e., CRISPR/Cas9) to the isolation of human inner ear progenitor cells, to tracking transplanted human umbilical cord stem cells in mini pigs, to the in vitro building of graft tissues to repair tracheal defects with adipose tissue-derived stem cells. Anat Rec, 303:385-389, 2020. © 2019 American Association for Anatomy.

Effects of supplemental creatine and guanidinoacetic acid on spatial memory and the brain of weaned Yucatan miniature pigs.

The emergence of creatine as a potential cognitive enhancement supplement for humans prompted an investigation as to whether supplemental creatine could enhance spatial memory in young swine. We assessed memory performance and brain concentrations of creatine and its precursor guanidinoacetic acid (GAA) in 14-16-week-old male Yucatan miniature pigs supplemented for 2 weeks with either 200 mg/kg∙d creatine (+Cr; n = 7) or equimolar GAA (157 mg/kg∙d) (+GAA; n = 8) compared to controls (n = 14). Spatial memory tests had pigs explore distinct sets of objects for 5 min. Objects were spatially controlled, and we assessed exploration times of previously viewed objects relative to novel objects in familiar or novel locations. There was no effect of either supplementation on memory performance, but pigs successfully identified novel objects after 10 (p < 0.01) and 20 min (p < 0.01) retention intervals. Moreover, pigs recognized spatial transfers after 65 min (p < 0.05). Regression analyses identified associations between the ability to identify novel objects in memory tests and concentrations of creatine and GAA in cerebellum, and GAA in prefrontal cortex (p < 0.05). The concentration of creatine in brain regions was not influenced by creatine supplementation, but GAA supplementation increased GAA concentration in cerebellum (p < 0.05), and the prefrontal cortex of +GAA pigs had more creatine/g and less GAA/g compared to +Cr pigs (p < 0.05). Creatine kinase activity and maximal reaction velocity were also higher with GAA supplementation in prefrontal cortex (p < 0.05). In conclusion, there appears to be a relationship between memory performance and guanidino compounds in the cerebellum and prefrontal cortex, but the effects were unrelated to dietary supplementation. The cerebellum is identified as a target site for GAA accretion.

Low-Flow Nasal Cannula Hydrogen Therapy.

BackgroundMolecular hydrogen (H2) is a biologically active gas that is widely used in the healthcare sector. In recent years, on-site H2 gas generators, which produce high-purity H2 by water electrolysis, have begun to be introduced in hospitals, clinics, beauty salons, and fitness clubs because of their ease of use. In general, these generators produce H2 at a low-flow rate, so physicians are concerned that an effective blood concentration of H2 may not be ensured when the gas is delivered through a nasal cannula. Therefore, this study aimed to evaluate blood concentrations of H2 delivered from an H2 gas generator via a nasal cannula.MethodsWe administered 100% H2, produced by an H2 gas generator, at a low-flow rate of 250 mL/min via a nasal cannula to three spontaneously breathing micro miniature pigs. An oxygen mask was placed over the nasal cannula to administer oxygen while minimizing H2 leakage, and a catheter was inserted into the carotid artery to monitor the arterial blood H2 concentration.ResultsDuring the first hour of H2 inhalation, the mean (standard error (SE)) H2 concentrations and saturations in the arterial blood of the three pigs were 1,560 (413) nL/mL and 8.85% (2.34%); 1,190 (102) nL/mL and 6.74% (0.58%); and 1,740 (181) nL/mL and 9.88% (1.03%), respectively. These values are comparable to the concentration one would expect if 100% of the H2 released from the H2 gas generator is taken up by the body.ConclusionsInhalation of 100% H2 produced by an H2 gas generator, even at low-flow rates, can increase blood H2 concentrations to levels that previous non-clinical and clinical studies demonstrated to be therapeutically effective. The combination of a nasal cannula and an oxygen mask is a convenient way to reduce H2 leakage while maintaining oxygenation.

Effect of Shenfu Injection on Isolated Empty Beating Hearts from Miniature Pigs.

ObjectiveTo investigate the effect of Shenfu (SF) injection on donor heart preservation.MethodsTwelve pigs were randomly divided into SF group (n=6) and control group (n=6). After eight hours of perfusion, the differences in hemoglobin, the expression of Bcl-2 and BAX, and changes in the myocardial ultrastructure were compared to illustrate the effects of SF injection in heart preservation.ResultsThe differences in free hemoglobin between the SF group and the control group were statistically significant (P=0.001), and there was significant interaction of groups with times (P=0.019), but the perfusion time may not be associated with the hemoglobin concentration (P=0.616). According to Western blotting analysis, the expression of Bcl-2 was higher in the SF group than in the control group, while the expression of BAX was not different between the two groups. As to ultrastructural changes, both groups exhibited mitochondrial swelling and myofilament lysis, but the degree of damage in the SF group was smaller.ConclusionOur study suggests that the application of SF injection for heart preservation may protect against cardiomyocytes and erythrocytes apoptosis, and Bcl-2 protein may play a role in these physiological processes.

A case of a linear foreign body removal in a miniature companion pig

Despite the recent rise in popularity of miniature pigs as companion animals, there are few published reports available regarding gastrointestinal surgery in these animals. This case report describes a 4-year-old castrated male companion potbellied pig who was diagnosed with a small bowel linear foreign body and associated pyloric outflow obstruction. A gastrotomy and three enterotomies involving the proximal and mid-jejunum and proximal centripetal loop of the spiral colon were performed to remove the linear foreign body. The surgical techniques and post-operative care described in this case report provided effective treatment of the linear foreign body and adequately prevented clinical evidence of post-operative complications in the companion potbellied pig. Follow-up information was obtained via telephone interviews and a recheck appointment one year later revealed a healthy animal with no abdominal radiographic or ultrasonographic abnormalities.

Y chromosome effect on prenatal live weight growth of laboratory minipigs

This paper describes the results of the influence of Y-chromosome gene complex on pre- and postnatal growth of piglets of laboratory mini-pigs ICG SB RAS. Breeding group includes four genealogical lines of boars united by a successive father-son relationship. Three lines: MS2853, MS2987, and VTN300 inherited their Y chromosomes from boars of Vietnamese-South Asian breed. The fourth line (LNDR07) received Y chromosome from the Landrace boar. This study revealed that all three lines of boars carrying Asian Y chromosome did not differ in weight of newborn offspring, while the weight and, correspondingly, prenatal growth of newborns of boars carrying European Y chromosome were statistically significantly less. Thus, at this stage of research, there is reason to believe that the selection group of mini-pigs ICG SB RAS contains polymorphism in the complex of Y-chromosome genes involved in the control of prenatal growth process. An assumption was considered that growth retardation during prenatal and early postnatal periods, as well as an increased proportion of culled offspring of boars carrying European Y chromosome can be caused by poor compatibility of its gene complex with the allele pool inherited from the mini pigs ICG SB RAS from Vietnamese breed.

Femoral bone defect healing using two novel biocompatible degradable materials

This study was conducted as anin vivoexperiment in adult miniature pigs with the aim to test two new biomaterials. An iatrogenic defect was made into the central femoral diaphysis in the experimental animals and subsequently fixated by bridging plate osteosynthesis. Into the defect we implanted a cancellous autograft (control group), a pasty injectable scaffold (EXP A), and a porous 3D cylinder (EXP B). Radiological examination was performed in all animals at 0, 10, 20, 30 weeks after surgical procedure and histological assessment was performed. In the newly formed bone the osteoblastic activity was monitored. In terms of radiology, the most effective method was observed in the control group (completely healed 100%) compared to experimental groups EXP A (70.0%) and EXP B (62.5%). Histological assessment showed a higher cell count in the place of bone defect in the control group compared to experimental groups. Between the experimental groups, a higher count of bone marrow cells was found in group EXP B. Both newly developed biomaterials seem to be suitable as replacements for large bone defects, having good workability and applicability. However, compared to the control group treated with a cancellous autograft, the newly formed bone did not reach the same number of cells settling in and in some cases, full radiological healing was not reached. Nevertheless, the material was found to be grown into the original bone in all cases within the experimental groups. The new biomaterials have a great potential as a substitute in the treatment of large bone defects.

Feasibility of computed tomography-guided irreversible electroporation for porcine kidney ablation.

The objective was to evaluate the feasibility and safety of computed tomography (CT)-guided percutaneous irreversible electroporation (IRE) in porcine kidneys. Under CT guidance, two monopole probes were used to precisely puncture through the renal parenchyma into the renal hilum in nine anesthetized adult Bama miniature pigs. After which, IRE ablation was performed. Biochemical and pathological examinations were carried out 2 h, 2, 7, and 14 days after the procedure. All procedures were performed successfully without any serious complications such as bleeding, infection, or death. All pigs survived until the end of the study. Pathological examinations showed that cells in the ablation area were dead within 2 days after the procedure, whereas the vascular endothelium showed only slight damage. After 2 days, endothelialization ensued and regrowth of smooth muscle cells was observed after 14 days. Hemogram tests indicated a transient increase but gradually returned to baseline levels 14 days after the procedure. IRE was essentially safe, however further studies on tumor ablation using several different animal models are needed.

A SNP in the 3′UTR of the porcine IGF-1 gene interacts with miR-new14 to affect IGF-1 expression, proliferation and apoptosis of PK-15 cells

The kidney of miniature pigs has been considered the most likely potential kidney source for patients needing kidney transplantation. Insulin-like growth factor 1 (IGF-1) is involved in regulating the growth of miniature pigs and inducing growth of kidneys. There are evidences showing that the SNPs in the 3′UTR of a gene may affect the gene expression by affecting the binding to a miRNA target site. In this study, one SNP (rs34142920) was screened in the IGF-1 3′UTR between 2 different body types of porcine breeds, Bama Xiang (BX) pigs, a miniature pig breed, and Large White (LW) pigs by sequencing. The secondary structure of the IGF-1 3′UTR mRNA containing the SNP in BX pigs is different from that of LW pigs. We then verified that there was a porcine miRNA (miR-new14) binding to this SNP in the 3′UTR of IGF-1 via cotransfecting the 3′UTR from the 2 breeds and miR-new14. We further found that the SNP downregulated mRNA and protein levels of IGF-1 by affecting the binding of miR-new14. To understand the function of miR-new14 in porcine kidney (PK-15) cells and its mechanism, cell proliferation and cell apoptosis assays were employed and results showed that proliferation viability of PK-15 cells was weakened and the apoptotic percentage of PK-15 cells was higher in the miR-new14 group. Porcine miRNA reduced the mRNA expression of AKT/ERK and protein levels of p-AKT/p-ERK. These results suggested that the expression of IGF-1 is influenced by this SNP and miR-new14 and that miR-new14 may suppress cell proliferation and promote cell apoptosis in PK-15 cells through regulating AKT and ERK signaling pathways, in which IGF-1 is involved.

Clinical Development of Sphingosine as Anti-Bacterial Drug: Inhalation of Sphingosine in Mini Pigs has no Adverse Side Effects.

Pulmonary infections with Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus) are of utmost clinical relevance in patients with cystic fibrosis, chronic obstructive pulmonary disease, after trauma and burn, upon ventilation or in immuno-compromised patients. Many P. aeruginosa and S. aureus strains are resistant to many known antibiotics and it is very difficult or often impossible to eradicate the pathogens in patient´s lungs. We have recently shown that the sphingoid base sphingosine very efficiently kills many pathogens, including for instance P. aeruginosa, S. aureus or Acinetobacter baumannii, in vitro. In vivo experiments of our group on cystic fibrosis mice indicated that inhalation of sphingosine prevents or eliminates existing acute or chronic pneumonia with P. aeruginosa or S. aureus in these mice. We also demonstrated that sphingosine is safe to use for inhalation up to high doses, at least in mice. To facilitate development of sphingosine to an anti-bactericidal drug that can be used in humans for inhalation, safety data on non-rodents, larger animals are absolutely required. Here, we inhaled mini pigs with increasing doses of sphingosine for 10 days and analyzed the uptake of sphingosine into epithelial cells of bronchi as well as into the trachea and lung and the systemic circulation. Moreover, we measured the generation of ceramide and sphingosine 1-phosphate that potentially mediate inflammation, the influx of leukocytes, epithelial cell death and disruption of the epithelial cell barrier. We demonstrate that inhalation of sphingosine results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea, but not in systemic accumulation. Inhaled sphingosine had no side effects up to very high doses. In summary, we demonstrate that inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no systemic or local side effects.

Two types of autologous cells in stricture development prevention after complete circular endoscopic dissection in minipig.

Complete circular endoscopic dissection (CED) is frequently accompanied with post-operative strictures formation in the esophagus. Various types of therapeutic approaches have recently been tested to prevent these strictures, e.g. cell therapy or stenting. Miniature pigs of Gottingen/Minnesota origin (n=10) were used in the study. First, we made the complete CED in the mid esophagus; next, the defect was left untreated or covered with mesenchymal stem cells (MSCs) or amixture of MSCs and primary oral keratinocytes (pOKs) suspension without/with fully covered self-expandable metallic stent (SEMS). Consequently, we performed acontrol endoscopy with astent removal, and necropsy was performed 17-36 days after cells application. All CED procedures were completed successfully without serious complications. Although we were able to detect MSCs or pOKs in the post-CED defects up to the 36th day after transplantation, the combination of MSCs or MSCs/pOKs with or without SEMS application did not prevent post-CED strictures development. The mixture of MSCs and pOKs resulted in the formation of cellular aggregates, which were mainly observed in submucosa, and the post-CED defect was covered with collagen fibers containing athin scarred epithelium, accompanied by various degrees of reconstruction and integrity. Suspension application of autologous MSCs alone or in combination with pOKs with or without SEMS was ineffective in the prevention of strictures formation after complete CED. Nevertheless, the presence of MSCs or pOKs in the post-CED defect was confirmed even 5 weeks after transplantation.

Whole-genome resequencing analysis of 20 Micro-pigs.

Miniature pigs have been increasingly used as mammalian model animals for biomedical research because of their similarity to human beings in terms of their metabolic features and proportional organ sizes. However, despite their importance, there is a severe lack of genome-wide studies on miniature pigs. In this study, we performed whole-genome sequencing analysis of 20 Micro-pigs obtained from Medi Kinetics to elucidate their genomic characteristics. Approximately 595 gigabase pairs (Gb) of sequence reads were generated to be mapped to the swine reference genome assembly (Sus scrofa 10.2); on average, the sequence reads covered 99.15% of the reference genome at an average of 9.6-fold coverage. We detected a total of 19,518,548 SNPs, of which 8.7% were found to be novel. With further annotation of all of the SNPs, we retrieved 144,507 nonsynonymous SNPs (nsSNPs); of these, 5968 were found in all 20 individuals used in this study. SIFT prediction for these SNPs identified that 812 nsSNPs in 402 genes were deleterious. Among these 402 genes, we identified some genes that could potentially affect traits of interest in Micro-pigs, such as RHEB and FRAS1. Furthermore, we performed runs of homozygosity analysis to locate potential selection signatures in the genome, detecting several loci that might be involved in phenotypic characteristics in Micro-pigs, such as MSTN, GDF5, and GDF11. In this study, we identified numerous nsSNPs that could be used as candidate genetic markers with involvement in traits of interest. Furthermore, we detected putative selection footprints that might be associated with recent selection applied to miniature pigs.