Proposal to modify the International Union Against Cancer staging system for perihilar cholangiocarcinomas.

BACKGROUND: The International Union Against Cancer (UICC) staging system for perihilar cholangiocarcinoma changed in 2009. The aim of this study was to validate and optimize the UICC system for these tumours. METHODS: This retrospective study was conducted in eight Japanese hospitals between 2001 and 2010. Perihilar cholangiocarcinoma was defined as a cholangiocarcinoma that involves the hilar bile duct, independent of the presence or absence of a liver mass component. The stratification ability of the UICC tumour node metastasis (TNM) system was compared with that of a modified system. RESULTS: Of 1352 patients, 35.9, 44.8 and 12.6 per cent had Bismuth type IV tumours, nodal metastasis (N1) and distant metastasis (M1) respectively. T4 tumours (43.2 per cent) and stage IVA (T4 Nany M0; 36.3 per cent) disease were most common. Survival was not significantly different between patients with T3 versus T4 tumours (P = 0.284). Survival for patients with stage IVA disease was comparable to that for patients with stage IIIB tumours (T1-3 N1 M0) (P = 0.426). Vascular invasion, pancreatic invasion, positive margin, N1 and M1 status were identified as independent predictors of survival. When Bismuth type IV tumours were removed from the T4 determinants and N1 tumours grouped together, the modified grouping had a higher linear trend χ2 and likelihood ratio χ2 compared with the original system (245.6 versus 170.3 respectively and 255.8 versus 209.3 respectively). CONCLUSION: The present data suggest that minimal modification with removal of Bismuth type IV tumours from the T4 determinants and bundling of N1 disease may enhance the prognostic ability of the UICC system. However, this requires validation on an independent data set.

Temporary auxiliary partial orthotopic liver transplantation using a small graft for familial amyloid polyneuropathy.

Donor shortage is a major issue in liver transplantation. We have successfully performed temporary auxiliary partial orthotopic liver transplantation (APOLT) using a small volume graft procured from a living donor for recipients with familial amyloid polyneuropathy (FAP). The aim of this study was to evaluate this procedure by comparing it with standard living donor liver transplantation (LDLT). We compared 13 recipients undergoing this procedure with 23 recipients undergoing a standard LDLT for the treatment of FAP. The estimated donor graft volume and the graft volume/recipient's standard liver volume ratio were significantly smaller in the temporary APOLT group than in the standard LDLT group. Postoperative complications were comparable, although the hospital stay was longer in the temporary APOLT group. All the patients safely underwent a remnant native liver resection about 2 months after their first operation in the temporary APOLT group. No symptoms related to FAP developed before the remnant liver resection, and no significant differences in graft and patient survival were observed between the two groups. We successfully performed temporary APOLT using a small volume liver graft without postoperative liver failure for FAP. Temporary APOLT for FAP might be a useful alternative procedure for expanding the donor pool for LDLT.

Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family.

BACKGROUND: Mutations in TRPV4, a gene that encodes a Ca(2+) permeable non-selective cation channel, have recently been found in a spectrum of skeletal dysplasias that includes brachyolmia, spondylometaphyseal dysplasia, Kozlowski type (SMDK) and metatropic dysplasia (MD). Only a total of seven missense mutations were detected, however. The full spectrum of TRPV4 mutations and their phenotypes remained unclear. OBJECTIVES AND METHODS: To examine TRPV4 mutation spectrum and phenotype-genotype association, we searched for TRPV4 mutations by PCR-direct sequencing from genomic DNA in 22 MD and 20 SMDK probands. RESULTS: TRPV4 mutations were found in all but one MD subject. In total, 19 different heterozygous mutations were identified in 41 subjects; two were recurrent and 17 were novel. In MD, a recurrent P799L mutation was identified in nine subjects, as well as 10 novel mutations including F471del, the first deletion mutation of TRPV4. In SMDK, a recurrent R594H mutation was identified in 12 subjects and seven novel mutations. An association between the position of mutations and the disease phenotype was also observed. Thus, P799 in exon 15 is a hot codon for MD mutations, as four different amino acid substitutions have been observed at this codon; while R594 in exon 11 is a hotspot for SMDK mutations. CONCLUSION: The TRPV4 mutation spectrum in MD and SMDK, which showed genotype-phenotype correlation and potential functional significance of mutations that are non-randomly distributed over the gene, was presented in this study. The results would help diagnostic laboratories establish efficient screening strategies for genetic diagnosis of the TRPV4 dysplasia family diseases.

Disease recurrence patterns after R0 resection of hilar cholangiocarcinoma.

BACKGROUND: There is little information regarding the clinical behaviour of hilar cholangiocarcinoma after curative resection. METHODS: A retrospective study was undertaken of 79 consecutive patients with hilar cholangiocarcinoma who had undergone major hepatectomy (three or more Couinaud segments) concomitant with caudate lobectomy, and had negative resection margins. Sites of initial disease recurrence were classified as locoregional (porta hepatis) or distant (intrahepatic, peritoneal, para-aortic lymph nodal or extra-abdominal). Univariable and multivariable analyses were performed to determine the factors potentially related to recurrence. RESULTS: Disease recurrence was observed in 42 (53 per cent) of the 79 patients. Cumulative recurrence rates at 3 and 4 years after surgery were 52 and 56 per cent respectively. Locoregional recurrence alone was observed in eight (10 per cent) and distant metastasis in 34 (43 per cent) of the 79 patients after R0 resection. Positive nodal involvement and high International Union Against Cancer tumour (T) stage were independent prognostic factors associated with distant metastasis. CONCLUSION: Distant metastases are more common than locoregional recurrence after R0 resection for hilar cholangiocarcinoma, and associated with nodal involvement and high T stage.

Purification method using iodixanol (OptiPrep)-based density gradient significantly reduces cytokine chemokine production from human islet preparations, leading to prolonged beta-cell survival during pretransplantation culture.

Purification is one of the most important steps in human islet isolation. Although Ficoll-based density gradients are widely used, OptiPrep-based density gradients are used in few centers. Cytokine/chemokine production from human islet preparations varies widely. Some cytokines/chemokines have been reported to have adverse effects on human islet preparations. Control of cytokine/chemokine production may be a key to improve islet quality and quantity, leading to better transplantation outcomes. The aim of the present study was to investigate the effects on islet preparations of purification methods using various density gradients on viability, cellular composition, and proinflammatory cytokine/chemokine production. After the digestion phase, the extracts were divided into 2 groups for purification using a semiautomated cell processor with Ficoll-based or OptiPrep-based density gradients. Islet preparations cultured for 2 days were assessed regarding islet cell viability (fluorescein diacetate/propidium iodide [FDA/PI]), fractional beta-cell viability by FACS, and beta-cell content using iCys. Cytokine/chemokine production from islet preparations was also measured by Bio-plex. After purification, the purity, islet equivalents (IEQ), and islet recovery rates were comparable between the 2 groups. Although FDA/PI and fractional beta-cell viability showed no significant difference, survival of beta cells during culture was significantly higher in the OptiPrep compared with the Ficoll-based density gradient group. There were significantly lower tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, interferon (IFN)-gamma, IL-6, and MIP-1beta productions from the OptiPrep-based density gradient group. OptiPrep-based density gradients reduced cytokine/chemokine production by islet preparations. In addition, OptiPrep-based density gradient purification significantly reduced the loss of beta-cell mass during pretransplantation culture.

Prevalence of porcine endogenous retroviruses in domestic, minature, and genetically modified pigs in Japan.

The present study examined the prevalence of porcine endogenous retroviruses (PERV) in pigs available in Japan using polymerase chain reaction (PCR) with primers specific for PERV-A, PERV-B, and PERV-C and for the full-length 5' to 3' long terminal repeat and using PCR-Southern blotting with env A-, env B-, env C-, and pol/pro-specific probes. All 376 pigs tested--Berkshire (B), Landrace (L), Duroc (D), Large White (W), miniature, and genetically modified triple-cross breed (LWD)--harbored both PERV-A and PERV-B genes. However, the prevalence of PERV-C differed among pigs: LWD, miniature, B, D, W, and L pigs were 100% (36/36), 83% (5/6), 68% (129/191), 52% (26/50), 21% (9/43), and 16% (8/50), respectively. These results show that W and L pigs may be preferable as xenotransplantation donors, because they may not produce human-tropic replication-competent hybrids of PERV-A and PERV-C.

Arterial reconstruction in a case of subintimal dissection of celiac arterial tributaries in living donor liver transplantation: a case report.

BACKGROUND: Hepatic arterial reconstruction is one of the critical issues in living donor liver transplantation (LDLT). Herein we have reported an LDLT case whose celiac arterial trunk tributaries were insufficient as host arteries because of extensive subintimal dissection proceeding to all tributaries of the celiac arterial trunk. PATIENTS AND METHODS: A 45-year-old woman with fulminant hepatic failure underwent LDLT. After reperfusion of the hepatic and portal veins, subintimal dissection of the recipient right and left hepatic arteries was found to extend to all tributaries of the celiac arterial trunk, preventing an anastomosis using the more proximal part of these arteries. Therefore, a jejunal arterial arcade of Roux-en-Y limb mobilized for biliary reconstruction was anastomosed to the donor left hepatic artery in end-to-end fashion. RESULTS: Arterial blood flow to the grafted liver was established successfully, and the patient's postoperative recovery was excellent. Postoperative computed tomography demonstrated sufficient hepatic arterial blood flow. The patient is doing well 4 years after transplantation. CONCLUSION: The method of hepatic graft arterialization described herein is an important option for LDLT recipients when tributaries of the celiac arterial trunk are insufficient as host arteries.

Isolation and sequencing of pig Blm and Ubl-1/SUMO-1 genes that relate to the recombination machinery.

BACKGROUND: The gene knockout technique is important in xenotransplantation research. Herein we have described the molecular cloning of two genes that are candidates to overcome the poor rate of homologous recombination. METHODS: Candidate cDNA fragments were amplified by polymerase chain reaction (PCR) with the corresponding primer sets deduced from a multiple alignment analysis of other mammalian genes from a cDNA library prepared from pig spleen tissue. To obtain the full-length cDNA, a 5'- and 3'-RACE PCR experiments was performed. RESULTS: We successfully isolated the cDNA sequences of two pig genes--BLM, a Bloom's syndrome-related gene, and UBL-1/SUMO-1--which are closely related to homologous recombination events. As a result, we verified the sequences of pig BLM and pig UBL-1/SUMO-1. The nucleic acid and amino acid coding sequence homologies of pig BLM gene with the corresponding human gene were 87.3% and 82.9%, respectively. The nucleic acid and amino acid coding sequence homologies of the pig UBL-1/SUMO-1 gene with the human gene were 96.4% and 100%, respectively. CONCLUSION: Current research into homologous recombination provides the possibility for improvement of gene knockout efficiency by regulating the gene expression profiles of recombination-related genes. Transient interference with the expression of pig UBL-1/SUMO-1 and BLM is expected to improve gene targeting. The results of the present study provided important information to design siRNA knockdown vectors. They were also useful for ex ante evaluation of expression profiles of these genes in primary cultures of somatic cells, which may enhance the production of gene knockout pigs.

Molecular cloning of pig Rad51, Rad52, and Rad54 genes, which are involved in homologous recombination machinery.

BACKGROUND: The low rate of homologous recombination in somatic cells is considered to be an urgent issue. Therefore, we molecularly cloned three genes that relate to efficient homologous recombination. METHODS: Polymerase chain reaction (PCR) was performed to isolate candidate cDNA fragments from a pig spleen cDNA library with the corresponding primer sets deduced from multiple alignment analysis of other mammalian genes. A 5'- and 3'-RACE PCR experiment was performed to determine the complete cDNA sequences. RESULTS: The complete cDNA sequences of the pig RAD51, RAD52, and RAD54 genes, which are closely related to homologous recombination events, were identified using molecular cloning technique. The cDNA sequences of three genes were successfully isolated by PCR-based methods. As a result, we determined the sequences of pig RAD51 (1663 bp, 339 aa), RAD52 (1884 bp, 406 aa), and RAD54 (2884 bp, 747 aa). The nucleic acid sequence homologies of the pig RAD51, RAD52, and RAD54 genes compared with the corresponding human genes were 92.9%, 77.3%, and 90.0%; the corresponding amino acid sequence homologies were 98.8%, 71.1%, and 95.0%, respectively. CONCLUSION: The knockout of alpha-1,3-galactosyltransferase in pigs resulted in a drastic reduction in xenoantigenicity. However, other xenoantigens, in particular, the non-Gal antigens, also need to be down-regulated. Gene transfer to alter expression levels of these recombination-related molecules and/or ex ante evaluation of expression profiles of these genes in primary cultures of somatic cells constitute a new approach to enhancing homologous recombination events during the production of gene knockout pigs.

Cloning and in vitro antiapoptotic effects of pig FLIPs.

INTRODUCTION: Cellular FLICE-like protein (cFLIP) inhibits death receptor-mediated apoptosis signal transduction, such as that induced by Fas and TNFR. The present study examined the role of antiapoptotic molecules to protect pig cells from human natural killer (NK) cells in vitro, as a model of delayed-type xenograft rejection. METHODS: Pig FLIPs were cloned using the TBLASTIN program to search for cDNA fragments of pig FLIPs. The sequence was identified using the dideoxy chain termination method and an ABI PRISM3100 genetic analyzer. The cDNA of pig FLIPs was inserted into the cloning site of the chicken beta-actin promoter (pCXN2). The cDNA was then transfected into pig endothelial cells (PEC), to establish several stable PEC clones containing the cDNA. Expression of the pig FLIP gene was evaluated by reverse-transcriptase polymerase chain reaction, and NK cell-mediated cytolysis assessed, using YT cells (an NK-like cell line). RESULTS: The full-length pig FLIP encoding sequence, total 5'-region to 3'-region, was defined for the first time. PEC transfectants with the FLIP showed moderate expression of FLIPs. Transfection of PEC with plasmids encoding FLIPs inhibited NK cell-mediated PEC lysis. While approximately half of parental PEC were injured by the human NK-like YT cells, the injury rate was relatively lower in the transfectants. CONCLUSION: Overexpression of the antiapoptotic molecules, pig FLIPs, has the potential for use in protecting graft cells from human NK cells.

Investigation of cynomolgus monkey complement.

BACKGROUND: The cynomolgus monkey is one of the most popular recipient animals in xenotransplantation experiments. However, studies of the cynomolgus monkey complement are rare. In the present study, based on the study that compared the hemolytic complement titer in cynomolgus monkeys with that in humans, the complement regulatory function of human decay accelerating factor (CD55) in both human and cynomolgus monkey sera was studied. METHODS: Hemolytic complement titers in cynomolgus monkeys were calculated using the same methods as are used in humans. Next, the complement regulatory function of human DAF (CD55) in cynomolgus monkey serum was studied using porcine endothelial cells (PECs) and human DAF. RESULTS: Of the complement titers tested, such as CH50, ACH50, C4, C2, and C3, the values were relatively high, except for the C4 titer. Human DAF on the surface of PEC resulted in nearly identical complement regulatory function in the human and cynomolgus monkey sera. CONCLUSIONS: Human DAF showed nearly the same complement regulatory function in both human and cynomolgus monkey sera.

Successful Heart Transplantation After Desensitization in a Patient With Extremely High Panel-Reactive Antibody Levels and Pretransplant Donor-Specific Antibody: A Case Report.

Elevated panel-reactive antibody (PRA) levels serve as a significant risk factor for allograft survival and episodes of rejection after heart transplantation (HTX). Patients with high PRA levels tend to show expressions of donor-specific human leukocyte antigen antibodies (DSA), which can cause catastrophic hyperacute rejection after HTX. Therefore, such highly sensitized patients are required to undergo strategic perioperative desensitization therapy. We describe a successful HTX after desensitization in a patient with extremely high PRA levels and pretransplant DSA positivity.

Immunological Response of Pigs to Human Cells, Including Issues Such as the Production of Natural Antibodies in Newborns.

Pigs have recently become very popular for use not only in xenotransplantation field, but in regeneration studies as well, sometimes with pigs being used as the scaffold. We have already presented our findings related to the pig immune system against human cells, including the complement systems, natural antibodies (NAs), and NK cells. In this study, we investigated the pig innate immunological reaction against human cells further. Our investigations included issues such as the production of NAs in newborns, day 0 and day 1, and sow colostrum. The alternative pathway for pig complement reacted with human cells, and pig NK cells and macrophages directly injured human aortic endothelial cells. Pig serum clearly contains the natural antibodies IgG and IgM to human peripheral blood mononuclear cells (PBMCs). Pig plasma from day 1 newborns contained almost the same levels of these natural antibodies to human PBMCs as those of sow plasma. On the other hand, pig plasma from day 0 newborns did not contain IgG and IgM to human PBMCs. In addition, sow colostrum clearly contained both IgG and IgM to human PBMCs. As expected, the pig innate immunity system reacted to human cells, including natural antibodies. However, the NAs of pigs, both IgM and IgG, against human cells do not exist in pig serum at day 0, but at day 1 and in mother's milk, indicating that NAs in newborns did not come from the placenta but from sow colostrum.

Cross-species function of the pig C1 esterase inhibitor.

BACKGROUND: The use of a bioartificial liver with pig cells for the treatment of fulminant hepatic failure will require research on the plasma complement regulatory proteins of the pig, because the liver produces most of the complement components and plasma complement regulatory proteins. In our previous study, the pig C1 esterase inhibitor (C1-INH), which functions as an inhibitor of the complement reaction in the first step of the classical pathway in the fluid phase, was cloned and some relevant features of the molecule were characterized, especially its cross-species regulation, in comparison with human C1-INH. In a further analysis, the species specificity of C1-INH was examined, using pig endothelial cells (PEC) and several types of sera. MATERIALS AND METHODS: The cDNA of pig C1-INH was used to produce the membrane type pC1-INH, pC1-INH-PI, and inserted into the cloning site of pCXN2 (chicken beta actin promoter). The pCX/pCl-INH-PI plasmid was then transfected into PEC to establish stable PEC with pCl-INH-PI. The expression of the pCl-INH-PI was evaluated by a FACS analysis, and complement-dependent cell lysis with human, dog, rabbit, and mouse sera was then assessed. RESULTS: The transfectant with pig Cl-INH-PI showed a high level of expression on PEC. The PEC transfectants showed an inhibitory effect on complement-dependent PEC lysis. Pig Cl-INH did not show the same suppressive effect for each serum. However, considering the alternative pathway activation of each serum on the pig cell membrane, it can be concluded that pCl-INH has a relatively small species restriction. CONCLUSION: Pig Cl-INH, having a similar structure to human Cl-INH, shows a strong complement regulatory function on other species sera.

Expression of a Synthetic Gene of CTDM by Transgenic Animals.

BACKGROUND: The purpose of this study was to produce molecules that can precisely regulate the complement and coagulation system and to assess the expression of such molecules in transgenic animals. METHODS: The CTDM gene, which is composed of the delta-1-99 amino acid (aa) C1-INH, EGF domain 4-6 of thrombomoduline (TM), short consensus repeat (SCR) 2-4 of DAF(CD55), and SCR 2-4 of MCP(CD46) was established. The codon usage for expression in mammals was adopted. The cDNA of CTDM was subcloned into the pCPI site (the human insulin promoter and a cytomegalovirus enhancer). pCPI-CTDM was transfected into pig endothelial cells (PEC). The expression of the molecule was clearly assessed by means of flow cytometry. RESULTS: BD3F1 female mice were induced to superovulate and were then crossed with BD3F1 males. Micro-injection and embryo transfer were performed by standard methods, thus generating transgenic mice that express CTDM. The mice carried the CTDM plasmid, as verified by PCR. Tissue expression levels in transgenic mouse lines generated with the constructs were follows: pancreas, 1.0; brain, 5.4; thymus, 0.3; heart, 0.2; lung, 1.2; liver, 0.1; kidney, 0.1; intestine, 0.4; and spleen, 1.6. A naive control mouse was also analyzed in the exact manner as for the transgenic mice. CONCLUSIONS: A synthetic CTDM gene with codon usage optimized to the mammalian system represents a critical factor in the development of transgenic animals.

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.

Supplemental Analysis for N-linked Sugars in Adult Pig Islets.

The pig pancreas is considered to be one of the most suitable sources of islets for clinical xenotransplantation. However, after producing α1-3galactosyltransferase knockout pigs, most of the organs of these pigs showed less antigenicity to the human body. Wild-type adult pig islets (APIs) that originally produced negligible levels of α-Gal, different from neonatal porcine islet-like cell clusters, showed a clear antigenicity to human serum. Concerning the so-called non-Gal epitopes, many studies related to glycoproteins and glycolipids are ongoing in efforts to identify them. However, our knowledge of non-Gal glycoantigens remains incomplete. In our previous study, N-glycans were isolated from APIs, and the structures of 28 of the N-glycans were detected. In this study, to identify additional structures, further analyses were performed by liquid chromatography-mass spectrometry (LC-MS). N-glycans were isolated from APIs by the method described by O'Neil et al with minor modifications and LC-MS-based structural analyses were then performed. The detected N-glycan peaks in the LC-MS spectra were selected using the FLexAnalysis software program and the structures of the glycans were predicted using the GlyocoMod Tool. The API preparation contained 11 peaks and 16 structures were then nominated as containing N-linked sugars. Among them, 5 sulfated glycans were estimated, confirming the existence of sulfate structures in N-glycans in API. In addition, these data may supplement several N-glycan structures that contain two deoxyhexose units, such as fucose, to our previous report. The data herein will be helpful for future studies of antigenicity associated with API.

Knockout of Cytidine Monophospho-N-Acetylneuraminic Acid (CMP-NeuAc) Hydroxylase From Porcine Endothelial Cells by a CRISPR System.

BACKGROUND: We attempted to knock out the expression of Hanganutziu-Deicher (H-D) antigens through the use of a CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9 system for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). METHODS: Plasmids expressing hCas9 and sgRNA for pCMAH were prepared by ligating oligos into the BbsI site of pX330. The N-terminal and C-terminal EGFP coding regions overlapping 482 bp were PCR-amplified and placed under a ubiquitous CAG promoter. The approximately 400-bp genomic fragments containing the sgRNA target sequence of pCMAH were placed into the multi-cloning sites flanked by the EGFP fragments. The pCAG-EGxxFP-target was mixed with pX330 with/without the sgRNA sequences and then introduced into HEK293T cells. RESULTS: Four oligos and primers, gSO1, gSO3, gSO4, and gSO8, were nominated from 8 candidates. Among them, gSO1 showed the best efficiency. Pig endothelial cells (PECs) from an α-Gal knockout pig were then used to examine the changes in the expression of the H-D antigen by the knockout of the CMAH genome by the pX330-gS01. CONCLUSIONS: Changes in the expression of the H-D antigen in the PECs with the CRISPR (gS01) were clear in comparison with those in the parental cells, on the basis of FACS analysis data. The expression of the H-D antigen can be knocked out by use of the CRISPR system for pCMAH, thus confirming that this system is a very convenient system for producing knockout pigs.

HLA-G1, but Not HLA-G3, Suppresses Human Monocyte/Macrophage-mediated Swine Endothelial Cell Lysis.

The inhibitory function of HLA-G1, a class Ib molecule, on monocyte/macrophage-mediated cytotoxicity was examined. The expression of inhibitory receptors that interact with HLA-G, immunoglobulin-like transcript 2 (ILT2), ILT4, and KIR2DL4 (CD158d) on in vitro-generated macrophages obtained from peripheral blood mononuclear cells and the phorbol 12-myristate 13-acetate (PMA)-activated THP-1 cells were examined by flow cytometry. cDNAs of HLA-G1, HLA-G3, HLA-E, and human ß2-microglobulin were prepared, transfected into pig endothelial cells (PECs), and macrophage- and the THP-1 cell-mediated PEC cytolysis was then assessed. In vitro-generated macrophages expressed not only ILT2 and ILT4 but CD158d as well. The transgenic HLA-G1 on PEC indicated a significant suppression in macrophage-mediated cytotoxicity, which was equivalent to that of transgenic HLA-E. HLA-G1 was clearly expressed on the cell surface of PEC, whereas the levels of HLA-G3 were much lower and remained in the intracellular space. On the other hand, the PMA-activated THP-1 cell was less expressed these inhibitory molecules than in vitro-generated macrophages. Therefore, the HLA-G1 on PECs showed a significant but relatively smaller suppression to THP-1 cell-mediated cytotoxicity compared to in vitro-generated macrophages. These results indicate that by generating HLA-G1, but not HLA-G3, transgenic pigs can protect porcine grafts from monocyte/macrophage-mediated cytotoxicity.