Wild-type Pigs Research Articles

A Comprehensive Map of CNS Transduction by Eight Recombinant Adeno-associated Virus Serotypes Upon Cerebrospinal Fluid Administration in Pigs

Cerebrospinal fluid administration of recombinant adeno-associated viral (rAAV) vectors has been demonstrated to be effective in delivering therapeutic genes to the central nervous system (CNS) in different disease animal models. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency, and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39, and rh.43 delivered into the cisterna magna of wild-type pigs. rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested. Moreover, rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful preclinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases.

20 SIALYLTRANSFERASE GENE EXPRESSION IN GGTA1 KNOCKOUT PIGS

It is considered that GGTA1 knockout (KO) pig production via somatic cell NT would overcome the problem of immune rejection after xenotransplantation. It is reported that although GGTA KO mice showed only a mild increase in sialyltransferase gene expression, GGTA1 deficiency in pig could increase the sialyltransferase activities, non-Gal epitope expression, consequently may raise non-Gal xenoantigenicity. Therefore, in the present study we investigated whether the expression level of Sia-containing glycoconjugate mRNA in transgenic pigs could be affected by knocking out the GGTA1 gene. Besides GGTA1 KO pigs, double genes overexpressing pigs (2TG) and GGTA1 KO with double genes overexpressing (KO+2TG) pigs were produced by somatic cell NT. For the present study, fibroblasts were isolated from wild-type pigs without gene modification, 2TG, GGTA1 KO, and KO+2TG pig. The GAPDH gene was used as an internal standard to normalise the real-time PCR (RT-qPCR) analysis reaction efficiency and to quantify mRNA in pigs-derived fibroblast. The expression levels were compared between them (RT-qPCR) in triplicate for each sample. Oligonucleotide primers for real-time PCR were designed for Hanganutziu-Deicher antigen (ST3Gal1–4, ST6Gal1) and Sialyl-Tn antigen (ST6GalNac1, ST6GalNac2, and ST6GalNac6) analysis. For statistical analysis, one-way ANOVA with Dunn’s multiple comparison test were used. The mRNA expression of GGTA1 KO and KO+2TG pig derived fibroblasts cells genes showed that ST3Gal1, ST3Gal2, ST3Gal3, and ST6Gal1 gene expression were significantly up-regulated compared to the wild and 2TG pigs (P < 0.05). However, ST3Gal4, Sialyl-Tn antigen including ST6GalNac1, ST6GalNac2, and ST6GalNac6 in KO+2TG pigs were not different compared with the wild pigs (P > 0.05), whereas only GGTA1 KO pigs showed significantly higher expressions than wild, 2TG, and KO+2TG pigs (P < 0.05). These results demonstrated that GGTA KO pig-derived cells exhibit a higher Hanganutziu-Deicher antigen on glycoprotein and glycolipid than controls, and KO+2TG pig exhibit no differences when compared with GGTA1 KO pig, indicating that they do not act as an immune antigen in xenograft. Overall, the increase in glycosyltransferase expression suggests a corresponding increase in the cell surface sialyation in GGTA KO pig cells. For xenotransplantation, KO+2TG pigs were more preferable because of absence of immune rejection for Sia-containing glycoconjugate on glycoprotein and glycolipid than GGTA KO pigs. This study was supported by the Ministry of Trade, Industry and Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

Clonidine inhibits anti-non-Gal IgM xenoantibody elicited in multiple pig-to-primate models.

Survival of vascularized xenografts is dependent on pre-emptive inhibition of the xenoantibody response against galactosyltransferase knockout (GTKO) porcine organs. Our analysis in multiple GTKO pig-to-primate models of xenotransplantation has demonstrated that the anti-non-gal-α-1,3-gal (anti-non-Gal) xenoantibody response displays limited structural diversity. This allowed our group to identify an experimental compound which selectively inhibited induced anti-non-Gal IgM xenoantibodies. However, because this compound had an unknown safety profile, we extended this line of research to include screening small molecules with known safety profiles allowing rapid advancement to large animal models. The NIH clinical collections of small molecules were screened by ELISA for their ability to inhibit xenoantibody binding to GTKO pig endothelial cells. Serum collected from non-immunosuppressed rhesus monkeys at day 14 post-injection with GTKO pig endothelial cells was utilized as a source of elicited xenoantibody for initial screening. Virtual small molecule screening based on xenoantibody structure was used to assess the likelihood that the identified small molecules bound xenoantibody directly. As a proxy for selectivity, ELISAs against tetanus toxoid and the natural antigens laminin, thyroglobulin, and single-stranded DNA (ssDNA) were utilized to assess the ability of the identified reagents to inhibit additional antibody responses. The identified inhibitory small molecules were further tested for their ability to inhibit xenoantibody elicited in multiple settings, including rhesus monkeys pre-treated with an anti-non-Gal selective anti-idiotypic antibody, non-immunosuppressed rhesus monkeys immunized with wild-type fetal pig isletlike cell clusters, and non-immunosuppressed baboons transplanted with GTKO multiple transgenic pig kidneys. Four clinically relevant small molecules inhibited anti-non-Gal IgM binding to GTKO pig endothelial cells in vitro. Three of these drugs displayed a limited region of structural similarity suggesting they may inhibit xenoantibody by a similar mechanism. One of these, the anti-hypertensive agent clonidine, displayed only minimal inhibition of antibodies elicited by vaccination against tetanus toxoid or pre-existing natural antibodies against laminin, thyroglobulin, or ssDNA. Furthermore, clonidine inhibited elicited anti-non-Gal IgM from all animals that demonstrated a xenoantibody response in each experimental setting. Clinically relevant small molecule drugs with known safety profiles can inhibit xenoantibody elicited against non-Gal antigens in diverse experimental xenotransplantation settings. These molecules are ready to be tested in large animal models. However, it will first be necessary to optimize the timing and dosing required to inhibit xenoantibodies in vivo.

Overexpressed human heme Oxygenase-1 decreases adipogenesis in pigs and porcine adipose-derived stem cells

Adipose-derived mesenchymal stem cells (ADSC) are multipotent, which means they are able to differentiate into several lineages in vivo and in vitro under proper conditions. This indicates it is possible to determine the direction of differentiation of ADSC by controlling the microenvironment. Heme oxygenase 1 (HO-1), a type of antioxidant enzyme, attenuates adipogenicity and obesity. We produced transgenic pigs overexpressing human HO-1 (hHO-1-Tg), and found that these animals have little fatty tissue when autopsied. To determine whether overexpressed human HO-1 suppresses adipogenesis in pigs, we analyzed body weight increases of hHO-1-Tg pigs and wild type (WT) pigs of the same strain, and induced adipogenic differentiation of ADSC derived from WT and hHO-1-Tg pigs. The hHO-1-Tg pigs had lower body weights than WT pigs from 16 weeks of age until they died. In addition, hHO-1-Tg ADSC showed reduced adipogenic differentiation and expression of adipogenic molecular markers such as PPARγ and C/EBPα compared to WT ADSC. These results suggest that HO-1 overexpression reduces adipogenesis both in vivo and in vitro, which could support identification of therapeutic targets of obesity and related metabolic diseases.

In vitro testing of an anti-CD40 monoclonal antibody, clone 2C10, in primates and pigs

BackgroundThe CD40/CD154 and CD28/B7 pathways are important in allo- and xeno-transplantation. Owing to the thrombotic complications of anti-CD154mAb, anti-CD40mAb has emerged as a promising inhibitor of costimulation. Various clones of anti-CD40mAb have been developed against primate species, e.g., clone 2C10 against rhesus monkeys. We have compared the in vitro efficacy of 2C10 to prevent a T cell response in primates and pigs. MethodsThe binding of 2C10 to antigen-presenting cells (PBMCs [B cells]) of humans, rhesus and cynomolgus monkeys, baboons, and pigs was measured by flow cytometry, and was also tested indirectly by a blocking assay. The functional capacity of 2C10 was tested by mixed lymphocyte reaction (MLR) with polyclonal stimulation by phytohemagglutinin (PHA) and also with wild-type pig aortic endothelial cells (pAECs) as stimulators. ResultsThere was a significant reduction in binding of 2C10 to baboon PBMCs compared to rhesus, cynomolgus, and human PBMCs, and minimal binding to pig PBMCs. The blocking assay confirmed that the binding of 2C10 was significantly lower to baboon PBMCs when compared to the other primate species tested. The functional assay with PHA showed significantly reduced inhibition of PBMC proliferation in humans, cynomolgus monkeys, and baboons compared to rhesus monkeys, which was confirmed on MLR with pAECs. ConclusionsSince both the binding and functional activity of 2C10 in the baboon is lower than in rhesus monkeys, in vivo treatment using 2C10 in the baboon might require a higher dose or more frequent administration in comparison to rhesus monkeys. It may also be beneficial to develop species-specific clones of anti-CD40mAb.

Generation of functional organs from pluripotent stem cells

Hematopoietic stem cells (HSCs) have played a major role in stem cell biology, providing many conceptual ideas and models. Among them is the concept of the "niche", a special bone-marrow microenvironment that by exchanging cues regulates stem-cell fate. The HSC niche also plays an important role in HSC transplantation. Successful engraftment of donor HSCs depends on myeloablative pretreatment to empty the niche. The concept of the stem-cell niche has now been extended to the generation of organs. We postulated that an empty "organ niche" exists in a developing animal when development of an organ is genetically disabled. This organ niche should be developmentally compensated by blastocyst complementation using wild-type primary stem cells (PSCs). We proved the principle of organogenesis from xenogeneic PSCs in an embryo unable to form a specific organ, demonstrating the generation of functionally normal rat pancreas by injecting rat PSCs into pancreatogenesis-disabled mouse embryos. This principle has held in pigs. When pancreatogenesis-disabled pig embryos underwent complementation with blastomeres from wild-type pig embryos to produce chimeric pigs, the chimeras had normal pancreata and survived to adulthood. Demonstration of the generation of a functional organ from PSCs in pigs is a very important step toward generation of human cells, tissues, and organs from individual patients' own PSCs in large animals.

Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions.

Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.

Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs.

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN−/−) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN+/+) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.

Integration of Perforated Subretinal Prostheses With Retinal Tissue.

To investigate the integration of subretinal implants containing full-depth perforations of various widths with rat and pig retina across weeks of implantation. In transgenic P23H rhodopsin line 1 (TgP23H-1) rats and wild-type (WT) pigs, we examined four subretinal implant designs: solid inactive polymer arrays (IPA), IPAs with 5- or 10-μm wide perforations, and active bipolar photovoltaic arrays (bPVA) with 5-μm perforations. We surgically placed the implants into the subretinal space using an external approach in rats or a vitreoretinal approach in pigs. Implant placement in the subretinal space was verified with optical coherence tomography and retinal perfusion was characterized with fluorescein angiography. Rats were sacrificed 8 or 16 weeks post-implantation (wpi) and pigs 2, 4, or 8 wpi, and retinas evaluated at the light microscopic level. Regardless of implant design, retinas of both species showed normal vasculature. In TgP23H-1 retinas implanted with 10-μm perforated IPAs, inner nuclear layer (INL) cells migrated through the perforations by 8 wpi, resulting in significant INL thinning by 16 wpi. Additionally, these retinas showed greater pseudo-rosette formation and fibrosis compared with retinas with solid or 5-μm perforated IPAs. TgP23H-1 retinas with bPVAs showed similar INL migration to retinas with 5-μm perforated IPAs, with less fibrosis and rosette formation. WT pig retina with perforated IPAs maintained photoreceptors, showed no migration, and less pseudo-rosette formation, but more fibrosis compared with implanted TgP23H-1 rat retinas. In retinas with photoreceptor degeneration, solid implants, or those with 5-μm perforations lead to the best biocompatibility.

Net lactate accumulation and low buffering capacity explain low ultimate pH in the longissimus lumborum of AMPKγ3R200Q mutant pigs

Postmortem lactate accumulation in skeletal muscle is linearly associated with the extent of pH decline. Yet, pigs harboring the AMPKγ3R200Q mutation produce meat with similar lactate levels to that of wild-type pigs but have a lower ultimate pH. We hypothesized that lower initial lactate levels and (or) lower buffering capacity in muscle of these pigs may help explain this discrepancy. Longissimus lumborum muscle samples were harvested at 0 and 1440min postmortem from AMPKγ3R200Q and wild-type pigs. As expected, AMPKγ3R200Q muscle exhibited a lower ultimate pH but similar lactate levels to that of wild-type pigs at 1440min postmortem. However, the total net lactate produced postmortem was greater in the AMPKγ3R200Q muscle due to lower initial lactate levels at 0min postmortem. Buffering capacity measured over the pH range of 5.5–7.0 was also lower in AMPKγ3R200Q muscle. Greater net lactate accumulation postmortem (i.e., glycolytic flux) coupled with a lower buffering capacity explains the lower ultimate pH of meat from AMPKγ3R200Q pigs.

Reduced noise susceptibility in littermate offspring from heterozygous animals of the German waltzing guinea pig.

The German waltzing guinea pig is a spontaneously mutated strain with severe auditory and vestibular impairment caused by a so far unknown genetic mutation. The animals are born deaf and show a circling behavior. The heterozygote animals of this guinea pig strain have functionally normal hearing and balance. However, these animals have, in earlier studies, shown an increased resistance to noise compared with normal wild-type guinea pigs. In the present study, we explored the functional hearing with auditory brainstem response thresholds before and at different time points after noise exposure. Symptom-free littermates from heterozygote couples of the German waltzing guinea pigs were exclusively used for the study, which, after the hearing test, were sent back for breeding to confirm their genotype (i.e. heterozygote or normal). The aim of this paper was to ascertain that the previously shown reduced susceptibility to noise trauma in the heterozygote animals of the German waltzing guinea pig was also evident when littermates were used as control animals. The findings are important for further analysis of the heterozygote animals of this strain and for future investigations of the underlying mechanisms behind the diverse susceptibility to exposures of loud sound.

Xenotransplantation literature update, May-June 2015.

Xenotransplantation literature update, May-June 2015.

WS06.6 Mucociliary transport is impaired in cystic fibrosis pig airways

Objectives The basic defect in cystic fibrosis (CF) is well characterized, but the link between defects in the CF transmembrane conductance regulator (CFTR) and the phenomenon of stagnant mucus is not well understood. We have shown that the ileal mucus in CFTR mutant mice adheres to the epithelium, is denser, and is less penetrable than that of wildtype mice and that addition of 115 mM NaHCO 3 to mucus partially normalizes this mucus phenotype. Using this knowledge of effects on ileal mucus, we are developing an airway experimental set-up. Methods Excised airways from CFTR knockout (CF) and wildtype (WT) pigs are pinned to a chamber, alcian blue mucosal stain is added to visualize the mucus and the transport is recorded. During the experiment, mucus is collected for proteomics using masspectrometry. Scanning electron microscopy is used to visualize the properties of the mucus, and transmission electron microscopy is used to study mucin secretion in detail. Results Average clearance rates of strands in CF pig airways are lower than in WT pig airways. Preliminary results suggest that the initial defect in newborn pigs involves clearance of Muc5b from the glands. Furthermore, we show that removal of bicarbonate from WT airways reduces the average clearance rate to the level of CF airways. Conclusion Evidence suggests the importance of bicarbonate in the proper unpacking and secretion of mucins. Thus, restoration of bicarbonate to the apical surface of the epithelium in combination with osmolytes may induce proper mucin unpacking in CF epithelia, and therefore could relieve the mucus obstruction that causes clinical problems for cystic fibrosis patients.

Apolipoprotein CIII regulates lipoprotein-associated phospholipase A2 expression via the MAPK and NFκB pathways.

Apolipoprotein CIII (apo CIII), a small glycoprotein that binds to the surfaces of certain lipoproteins, is associated with inflammatory and atherogenic responses in vascular cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been proposed as an inflammatory biomarker and potential therapeutic target for cardiovascular disease (CVD). Here, we report that apo CIII increases Lp-PLA2 mRNA and protein levels in dose- and time- dependent manner in human monocytic THP-1 cells, and the increase can be abolished by MAPK and NFκB pathway inhibitors. Lp-PLA2 inhibitor, 1-linoleoyl glycerol attenuates the inflammation induced by apo CIII. In turn, exogenous Lp-PLA2 expression upregulates apo CIII and the upregulation can be inhibited by 1-linoleoyl glycerol in HepG2 cells. Moreover, plasma Lp-PLA2 level is correlated with apo CIII expression in pig liver. In vivo, Lp-PLA2 expression in monocytes and its activity in serum were significantly increased in human apo CIII transgenic porcine models compared with wild-type pigs. Our results suggest that Lp-PLA2 and apo CIII expression level is correlated with each other in vitro and in vivo.

7. Lentiviral Vector-Mediated CFTR Gene Transfer to CF Pig Airways Corrects the Anion Transport Defect In Vivo

Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasian populations and is caused by mutations in CFTR. Complementation of CFTR rescues the anion transport defect in vitro, making this monogenetic disease a candidate for CF gene therapy. Lentiviral vector-mediated CFTR gene transfer has long been discussed as a promising strategy for gene therapy. Lentiviral vectors can efficiently transduce non-dividing cells and persistently express a therapeutic transgene. We previously demonstrated that the non-primate lentiviral vector derived from feline immunodeficiency virus (FIV) and pseudotyped with a GP64 envelope confers gene transfer to porcine airway epithelia both in well-differentiated primary cultures in vitro and in wild-type pig lungs in vivo. The CF pig model recapitulates many features of human disease and provides a new platform for preclinical gene therapy studies. Here, we show that lentiviral-mediated delivery of CFTR to nasal and lung epithelia corrects the anion channel defect in the CF pig model. We delivered GP64-FIV-pigCFTR to the nose and lung of 2-week old gut-corrected CF pigs using a MADgic atomizer. Two weeks post-transduction, we harvested sinus, trachea, and bronchus tissue to assay for gene correction. In freshly excised trachea and bronchus tissue explants, we observed a short-circuit current response to forskolin and IBMX (F&I) and inhibition by GlyH-101, consistent with CFTR function. Compared to non-transduced animals, cultured ethmoid sinus epithelia also showed evidence of correction by short-circuit current measurements. Loss of CFTR results in reduced bicarbonate transport and a lower ASL pH. A reduction in ASL pH impairs bacterial killing. Following gene transfer, we observed an increase in nasal and tracheal pH and rescue of bacterial killing. Together, these exciting results demonstrate the first evidence of functional in vivo correction of CFTR by a lentivirus in the CF pig model. These findings support the utility of a lentivirus for CF gene therapy.

Xenotransplantation literature update, January-February 2015.

Xenotransplantation literature update, January-February 2015.

Dominant-negative inhibition of glucose-dependent insulinotropic polypeptide impairs function of β cells in transgenic pigs.

Dear Editor: Glucose-dependent insulinotropic polypeptide (GIP) and proglucagon product glucagon-like peptide-1 (GLP-1) and their corresponding receptors promote secretion of glucose-dependent insulin[1] and may be responsible for up to 70% of postprandial insulin secretions[2]. A diminished incretin effect is typical for type 2 diabetes mellitus (T2DM) patients and their normoglycemic first-degree relatives due to reduced insulinotropic action of GIP[3]. Desensitization of the GIP/GIPR axis[4], down-regulation of GIP receptor (GIPR) on β cells[5], and variation in the GIPR gene[6] all contribute to reduced insulinotropic activity of GIP. GIPRdn encodes human GIPR that is modified in the region of the third intracellular loop by a single mutation (Ala340→Glu340) and deletion of eight amino acids (residues 319–326) essential for signal transduction[8]. In this study, we produced porcine transgenic fibroblasts that overexpressed a GIPRdn under the control of rat Ins2 promoter in the pancreas. The study protocol was approved by the local institutional review board and animal study was done in accordance with established national and institutional guidelines. Oral glucose tolerance tests (OGTTs) in 2-month-old GIPRdn pigs and controls revealed no differences in area under the glucose response curve (AUCglucose) between the two groups (P = 0.07, Fig. 1A). However, the postprandial blood glucose declined slowly and the area under the insulin response curve (AUCinsulin) in GIPRdn pigs increased 2.3-fold versus the controls, implying impaired glucose tolerance (P = 0.02; Fig. 1B). OGTTs in 5-month-old GIPRdn pigs and controls showed no significant differences in glucose levels or insulin content between the groups (Fig. 1C, 1D). Additionally, we observed no significant differences in the insulin response profiles between 2-month-old and 5-month-old controls. However, AUCinsulin was reduced by 44% when the transgenic pigs grew from 2 months to 5 months of ages (Fig. 1E, 1F). To clarify the effect of GIPRdn expression on the proliferation of pancreatic cells, we measured insulin and Ki67 expression by immunohistochemistry in 5-month-old GIPRdn pigs and age-matched controls. Compared with the controls, the number and volume of islets in transgenic pigs decreased. Insulin expression in the islets was reduced by 83% (P<0.01) in 5-month-old GIPRdn pigs (Fig. 2A-2C). In addition, Ki67 expression in GIPRdn pigs decreased to 36% of controls, suggesting that GIPRdn expression impaired islet development and reduced insulin secretion (Fig. 2D-2F). Fig. 1 Oral glucose tolerance test in GIPRdn transgenic (Tg) and wild type (wt) pigs. Fig. 2 Insulin and Ki67 expression in the pancreas. Transgenic mice overexpressing a dominant-negative GIPR were found to develop diabetes accompanied by a marked fasting hypoinsulinemia and severe reduction of β-cell mass by 10 days of age[7]. Similarly, transgenic pigs overexpressing a dominant-negative GIPR exhibited a pre-diabetic phenotype of hyperglycemia and hyperinsulinemia in OGTT[8]. However, no transgenic pigs had developed fasting hyperglycemia. In this study, 2-month-old GIPRdn pigs exhibited a slow decline in postprandial blood glucose and an increase in insulin secretion following an oral glucose load. This increase was approximately 2.3 times higher than in the control, thus imitating insulin secretion in the prodromal stage of T2DM. As the pigs grew up, pancreas development was significantly impaired due to the compromised GIP/GIPR axis, which resulted in reduced insulin secretion and islet dysfunction. Studies indicated that GIP and GLP-1 induced the transcription of beta cell proliferation, differentiation, survival and transcription of some genes[9]. GIP was believed to be the mitogen of beta cells, resulting in insulin secretion and inhibiting the apoptosis of beta cells. In this study, there were no significant differences in insulin content between the 5-month-old groups. However, the oral glucose load of 5-month-old transgenic pigs resulted in a 44% decrease in insulin secretion compared with the 2-month-old transgenic pigs. This is consistent with previous studies and implies progression of T2DM. Defective pancreatic proliferation led to fewer and smaller islets, which were excessively activated to produce more insulin by compensatory mechanism. Overload pressure from the compensation mechanism and impaired proliferation for inactivated GIP/GIPR axis further deteriorated islet function. Similar to the previous study[9], the transgenic pigs did not develop diabetes until 2 years of age. Diabetes is caused by many factors; a single gene change would not lead to the occurrence of the disease. In summary, GIPRdn pigs exhibit impaired glucose metabolism and beta cell function and simulated the phenotype of the prodromal stage in diabetes and progression of the disease.

Abstract 17809: In Vivo High Resolution Isotropic 3D MRI of Coronary Atherosclerosis in Hypertensive Hypercholesterolemic Minipigs

Introduction: Accurate non-invasive visualization and quantification of coronary atherosclerosis may greatly facilitate clinical research and practice, but the lack of suitable human-sized atherosclerosis models with progressive coronary lesions has hampered development and validation of reliable imaging techniques. Methods: D374Y-PCSK9 transgenic minipigs with (n=7) or without (n=8) hypertension, induced by suprarenal aortic banding, were placed on a cholesterol-rich diet for 16 months. Wildtype minipigs (n=4) on normal diet served as disease-free controls. A T1-weighted inversion recovery fast gradient echo magnetic resonance imaging (MRI) sequence was used to acquire 1x1x1mm images of the coronary arteries. MRI scans were conducted following injection of a gadolinium labeled elastin specific contrast agent (LMI1174). Pigs were subsequently euthanized and the coronary arteries were processed for pathological analysis. LMI1174 was provided by Lantheus Medical Imaging under a material transfer agreement Results: Hypertension significantly augmented coronary fibroatheromas by more than 5-fold compared to normotensive transgenic pigs. No signal enhancement was observed in lesion-free coronaries of wildtype pigs. In hypertensive pigs, there was a strong correlation between plaque area measured by MRI and plaque area measured postmortem r2=0.84, p=0.02 (Fig 1). Conclusions: Hypertensive transgenic minipigs with accelerated coronary atherosclerosis provide a useful model for coronary imaging. Using this model we were able to develop a high-resolution isotropic LMI1174-enhanced scanning technique that accurately depicts coronary atherosclerotic lesion size and distribution. Figure 1: A) Digitally unfolded and color coded MRI of the LAD (left) and corresponding sudan IV stained segment opened and viewed en face (right). B) Cross-sectional MRI of another LAD and corresponding histological sections showing fibroatheromas.

Perilymphatic Gentamicin Trafficking through Basolateral Channels May Underlie Enhanced Ototoxicity by Noise‐Induced Threshold Shifts

Objectives: (1) Demonstrate gentamicin uptake from perilymph into hair cells via basolateral channels such as the postsynaptic acetylcholine receptor (nAChR) and the TRPA1 in neonatal mice in vivo and in vitro. (2) Recognize alternative gentamicin trafficking pathways to the well established endolymphatic mechanoelectrotransducer channel underlying aminoglycoside ototoxicity. Methods: Neonatal mice were intraperitoneally injected with fluorescently-conjugated Gentamicin-Texas Red (GTTR), prior to fixation 30 minutes later. Cochlear explants were treated with GTTR 1 µg/mL, with or without 100 µM acetylcholine or cinnamaldehyde then fixed, and analyzed. Fluorescence intensities were measured by calculating the mean gray value of each cell with Image J. Student t test was used to determine any significant differences in fluorescence values between treatment groups. Results: Application of acetylcholine increased GTTR uptake by wildtype outer hair cells (OHC) in vivo and in vitro. These results were also replicated in Myo7a8J/8J mice, which lack functional mechanoelectrotransducer channels. Application of cinnamaldehyde (TRPA1 agonist) also increased GTTR uptake in OHCs in wildtype, myo7a8J/8J mice and guinea pigs. Conclusions: Endolymphatic gentamicin trafficking via the mechanoelectrotransducer (MET) channel is a well established mechanism of ototoxicity. Noise-induced threshold shifts (NITS) have been shown to damage the mechanically gated tip link, thus closing MET channel. However, aminoglycoside hair cell uptake is enhanced by NITS. This suggests one or more aminoglycoside entry route(s) into hair cells in addition to, and independent of, the MET channel. The results of this study support perilymphatic trafficking of gentamicin via basolateral channels including the nAChR Ca2+ channel, and TRPA1.

A comparison of three methods of decellularization of pig corneas to reduce immunogenicity.

To investigate whether decellularization using different techniques can reduce immunogenicity of the cornea, and to explore the decellularized cornea as a scaffold for cultured corneal endothelial cells (CECs). Transplantation of decellularized porcine corneas increases graft transparency and survival for longer periods compared with fresh grafts. Six-month-old wild-type pig corneas were cut into 100-200 µm thickness, and then decellularized by three different methods: 1) 0.1% sodium dodecyl sulfate (SDS); 2) hypoxic nitrogen (N2); and 3) hypertonic NaCl. Thickness and transparency were assessed visually. Fresh and decellularized corneas were stained with hematoxylin/eosin (H&E), and for the presence of galactose-α1,3-galactose (Gal) and N-glycolylneuraminic acid (NeuGc, a nonGal antigen). Also, a human IgM/IgG binding assay was performed. Cultured porcine CECs were seeded on the surface of the decellularized cornea and examined after H&E staining. All three methods of decellularization reduced the number of keratocytes in the stromal tissue by >80% while the collagen structure remained preserved. No remaining nuclei stained positive for Gal or NeuGc, and expression of these oligosaccharides on collagen was also greatly decreased compared to expression on fresh corneas. Human IgM/IgG binding to decellularized corneal tissue was considerably reduced compared to fresh corneal tissue. The cultured CECs formed a confluent monolayer on the surface of decellularized tissue. Though incomplete, the significant reduction in the cellular component of the decellularized cornea should be associated with a significantly reduced in vivo immune response compared to fresh corneas.