Studies of Pig Complement: Measurement of Pig CH50, ACH50, and Components.

BACKGROUND: On the basis of a comparison of the hemolytic complement titer in pigs with that in humans, the complement system of pigs was investigated. The response of innate immunity, such as the natural antibodies, against humans was also examined. METHODS: Hemolytic complement activity of pig serum was measured with the use of a microtitration technique. CH50 was determined according to the method of Mayer. ACH50 was assayed according to the methods of Platts-Milles and Ishizaka. Hemolytic activities of C1, C4, C2, C3, C5, C8, and C9 were estimated through the use of intermediate cells and reagents, as described previously. In addition, the pig natural anti-human antibody was studied with the use of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were stained with 5% pig serum, followed by staining with fluorescein isothiocyanate-labeled goat anti-pig IgG and IgM. The resulting stained cells were quantified by use of a FACScalibur system. The alternative pathway of pig complement was also measured with the use of human erythrocytes and normal pooled pig serum with or without Mg(++)EGTA. RESULTS: Both the CH50 and ACH50 titers were lower than those of humans. Concerning the components, except for C3, each component, that is, C1, C4, C2, C5, C8, and C9, was also lower than that of humans, based on measured values for human complement components. Pig serum clearly contains natural antibodies, IgG and IgM, to human PBMCs. The alternative pathway of pig complement reacted with human erythrocytes. CONCLUSIONS: As a whole, pig innate immunity, the complement system and natural antibody, recognizes the surfaces of human cells.

An Effective New Cryopreservation Procedure for Pancreatic Islets Using Hollow Fiber Vitrification.

The present study aimed at establishing a new cryopreservation method for mouse pancreatic islets by vitrification using hollow fibers as a container. A unique feature of the hollow fiber vitrification (HFV) method is that this method achieves stable vitrification using a minimum volume of cryoprotectant (CPA) solution, thereby ensuring high viability of the islets. The cytotoxicity, optimum composition, and concentration of the CPAs for vitrifying islets were examined. The viability, functional-integrity of vitrified islets were evaluated in comparison with those vitrified by conventional methods. Insulin secretion was measured in vitro by a static incubation assay and the metabolic functions was tested after transplantation into Streptozotocin-induced diabetic mice. The combination of 15% dimethyl sulfoxide+15% ethylene glycol resulted in the best CPA solution for the HFV of islets. HFV showed the highest viability in comparison to 2 vitrification methods, open pulled straws and vitrification with EDT324 solution. The vitrified islets stably expressed ß-cells markers NeuroD, Pancreatic and duodenal homeobox-1, and MafA. Transplantation of the vitrified islets achieved euglycemia of the host diabetic mice and response to an intraperitoneal glucose tolerance test to a similar extent as non-vitrified transplanted islets. The HFV method allows for efficient long-term cryopreservation of islets.

Transgenic pig expressing the red fluorescent protein kusabira-orange as a novel tool for preclinical studies on hepatocyte transplantation.

INTRODUCTION: Research on hepatocyte transplantation as an alternative or supplementary treatment for liver transplantation is progressing. However, to advance to clinical trials, confidence in the technique must be established and its safety must be validated by conducting experiments using animals of comparable sizes to humans, such as pigs. We used transgenic pigs expressing red fluorescence protein for investigating the distribution and survival of transplanted cells. MATERIALS AND METHODS: Donor hepatocytes were isolated from transgenic Kusabira-Orange (KO)-expressing pigs (age, 41 days; weight, 10 kg) created by in vitro fertilization using sperm from a transgenic-cloned KO pig by Matsunari et al. and ova from a domestic pig. The hepatocyte transplant recipients were the nontransgenic, KO-negative littermates. In these recipient pigs, double lumen cannulae were inserted into the supramesenteric veins to access the hepatic portal region. KO-positive donor hepatocytes from the transgenic male pig were isolated using collagenase perfusion. Hepatocytes (1 × 10(9) cells) were transplanted through the cannula. For estimating allogeneic immunogenicity, full-thickness skin (3 × 3 cm) from the same donor was grafted orthotopically on the neck region of the recipients. Immunosuppressive treatment was not implemented. The recipient pigs were humanely killed at 7 and 39 days after transplantation, and the organs were harvested, including the lungs, heart, liver, pancreas, and kidneys. RESULTS: Strong red fluorescence was detected in both the parenchymal and nonparenchymal hepatocytes of the transgenic male donor pig by fluorescent microscopy. Transplanted cells were detected in the liver and lung of the recipient pigs at 7 days after perfusion. Hepatocytes remained in the liver and lung of recipients on day 39, with lower numbers than that on day 7. CONCLUSION: Transgenic pigs expressing the fluorescent protein KO serve as a useful model of cell transplantation in preclinical studies.

Advancing pig cloning technologies towards application in regenerative medicine.

Regenerative medicine is expected to make a significant contribution by development of novel therapeutic treatments for intractable diseases and for improving the quality of life of patients. Many advances in regenerative medicine, including basic and translational research, have been developed and tested in experimental animals; pigs have played an important role in various aspects of this work. The value of pigs as a model species is being enhanced by the generation of specially designed animals through cloning and genetic modifications, enabling more sophisticated research to be performed and thus accelerating the clinical application of regenerative medicine. This article reviews the significant aspects of the creation and application of cloned and genetically modified pigs in regenerative medicine research and considers the possible future directions of the technology. We also discuss the importance of reproductive biology as an interface between basic science and clinical medicine.

A cloning of cytidine monophospho-N-acetylneuraminic acid hydroxylase from porcine endothelial cells.

INTRODUCTION: The Hanganutziu-Deicher (H-D) antigen with terminal N-glycolyl neuraminic acid-(NeuGc) is widely distributed in mammalian species including monkeys and apes, but is not found in humans and birds. After the knock out of α1, 3galactosyltransfease, the H-D antigen became a major antigen of the "non-Gal antigen." The expression of NeuGc is controlled by the activity of cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). In this study, molecular cloning of pig CMAH was performed, as the first step in producing H-D knockout pigs. METHODS: A pig endothelial cell line, MYP30, was used. The DNA sequence of pig CMAH was queried in dbEST (NCBI) using the BLAST program to search for cDNA fragments of pig CMAH, based on an alignment analysis of the mouse CMAH sequence. A polymerase chain reaction experiment was performed and candidate cDNA clones were isolated. To obtain the 5'-end and 3'-end of the open reading frame sequence, a 5'-full RACE Core Set and 3'-full RACE Core Set were used. RESULTS: We cloned and characterized the pig CMAH gene. The ATG is located in exon 4, which corresponds to the mouse gene, and the stop codon is in exon 17. In the case of the 5' site of the gene, exon 3 was identified but exons 1 and 2 are still being investigated. On the other hand, exon 18 was newly identified in the 3' site of the gene. CONCLUSION: The results represent useful information for future clinical xenotransplantation studies.

Comparison of taurine biosynthesis ability between juveniles of Japanese flounder and common carp.

This study was conducted to investigate taurine deficiency and the ability of taurine biosynthesis in both juvenile Japanese flounder (JF) and juvenile common carp (CC) in vivo using low taurine level diets. Three different taurine level diets were prepared by the supplementation of taurine to the basal composition (JF--0, 0.5 and 1.5% in JF; CC--0, 1, 3% in CC). The final average body weight and feed efficiency of JF fed the JF - 1.5% was significantly higher than those of fish fed on the JF--0%. On the other hand, no significant difference was observed in CC fed with CC--0, 1, and 3% diets. The taurine retention rate was negative in the case of JF-fed with the taurine-free supplement (JF--0%). On the other hand, the taurine retention rate was about 280% in the case of CC-fed with the taurine-free supplement (CC--0%). These findings indicate that while taurine is essential for growth of JF, it is not essential for the growth of CC.