OBJECTIVE: To prepare decellularized nerve grafts from alpha-1, 3-galactosyltransferase (GGTA1) gene-edited pigs and explore their biocompatibility for xenotransplantation. METHODS: The sciatic nerves from wild-type pigs and GGTA1 gene-edited pigs were obtained and underwent decellularization. The alpha-galactosidase (α-gal) content in the sciatic nerves of GGTA1 gene-edited pigs was detected by using IB4 fluorescence staining and ELISA method to verify the knockout status of the GGTA1 gene, and using human sciatic nerve as a control. HE staining and scanning electron microscopy observation were used to observe the structure of the nerve samples. Immunofluorescence staining and DNA content determination were used to evaluate the degree of decellularization of the nerve samples. Fourteen nude mice were taken, and subcutaneous capsules were prepared on both sides of the spine. Decellularized nerve samples of wild-type pigs ( n=7) and GGTA1 gene-edited pigs ( n=7) were randomly implanted in the subcutaneous capsules. Blood was drawn at 1, 3, 5, and 7 days after implantation to detect neutrophil counting. RESULTS: IB4 fluorescence staining and ELISA detection showed that GGTA1 gene was successfully knocked out in the nerves of GGTA1 gene-edited pigs. HE staining showed that the structure of the decellularized nerve from GGTA1 gene-edited pigs was well preserved; the nerve basement membrane tube structure was visible under scanning electron microscopy; no cell nuclei was observed, and the extracellular matrix components was retained in the nerve grafts by immunofluorescence staining; and the DNA content was significantly reduced when compared with the normal nerves ( P<0.05). In vivo experiments showed that the number of neutrophils in the two groups were similar at 1, 3, and 7 days after implantation, with no significant difference ( P>0.05); only at 5 days, the number of neutrophils was significantly lower in the GGTA1 gene-edited pigs than in the wild-type pigs ( P<0.05). CONCLUSION The decellularized nerve grafts from GGTA1 gene-edited pigs have well-preserved nerve structure, complete decellularization, retain the natural nerve basement membrane tube structure and components, and low immune response after xenotransplantation through in vitro experiments.
Animals ; Transplantation, Heterologous ; Galactosyltransferases/genetics* ; Sciatic Nerve/immunology* ; Swine ; Tissue Engineering/methods* ; Humans ; Graft Rejection/prevention & control* ; Gene Editing ; Mice ; Mice, Nude ; Heterografts/immunology* ; Animals, Genetically Modified ; Tissue Scaffolds ; Decellularized Extracellular Matrix

BACKGROUND: The interaction between killer immunoglobulin-like receptors (KIRs) and HLA class I regulates natural killer (NK) cell cytotoxicity and function. The impact of NK cell alloreactivity through KIR in liver transplantation remains unelucidated. Since the frequency of HLA-C and KIR genotypes show ethnic differences, we assessed the impact of HLA-C, KIR genotype, or KIR-ligand mismatch on the allograft outcome of Korean liver allografts. METHODS: One hundred eighty-two living donor liver transplant patients were studied. Thirty-five patients (19.2%) had biopsy-confirmed acute rejection (AR), and eighteen (9.9%) had graft failure. The HLA-C compatibility, KIR genotypes, ligand-ligand, and KIR-ligand matching was retrospectively investigated for association with allograft outcomes. RESULTS: Homozygous C1 ligands were predominant in both patients and donors, and frequency of the HLA-C2 allele in Koreans was lower than that in other ethnic groups. Despite the significantly lower frequency of the HLA-C2 genotype in Koreans, donors with at least one HLA-C2 allele showed higher rates of AR than donors with no HLA-C2 alleles (29.2% vs 15.7%, P=0.0423). Although KIR genotypes also showed ethnic differences, KIR genotypes and the number of activating KIR/inhibitory KIR were not associated with the allograft outcome. KIR-ligand mismatch was expected in 31.6% of Korean liver transplants and had no impact on AR or graft survival. CONCLUSIONS: This study could not confirm the clinical impact of KIR genotypes and KIR-ligand mismatch. However, we demonstrated that the presence of HLA-C2 allele in the donor influenced AR of Korean liver allografts.
Adult ; Alleles ; Asian Continental Ancestry Group/*genetics ; Female ; Genotype ; Graft Rejection ; Graft Survival ; HLA-C Antigens/*genetics ; Homozygote ; Humans ; Killer Cells, Natural/cytology/immunology ; Ligands ; *Liver Transplantation ; Male ; Middle Aged ; Proportional Hazards Models ; Receptors, KIR/chemistry/*genetics/metabolism ; Republic of Korea ; Tissue Donors ; Transplantation, Homologous

BACKGROUND/AIMS: The relationship between patient survival and biopsy-proven acute rejection (BPAR) in liver transplant recipients with hepatitis C remains unclear. The aims of this study were to compare the characteristics of patients with and without BPAR and to identify risk factors for BPAR. METHODS: We retrospectively reviewed the records of 169 HCV-RNA-positive patients who underwent LT at three centers. RESULTS: BPAR occurred in 39 (23.1%) of the HCV-RNA-positive recipients after LT. The 1-, 3-, and 5-year survival rates were 92.1%, 90.3%, and 88.5%, respectively, in patients without BPAR, and 75.7%, 63.4%, and 58.9% in patients with BPAR (P<0.001). Multivariate analyses showed that BPAR was associated with the non-use of basiliximab and tacrolimus and the use of cyclosporin in LT recipients with HCV RNA-positive. CONCLUSION: The results of the present study suggest that the immunosuppression status of HCV-RNA-positive LT recipients should be carefully determined in order to prevent BPAR and to improve patient survival.
Antibodies, Monoclonal/therapeutic use ; Biopsy ; Cyclosporine/therapeutic use ; Drug Therapy, Combination ; Genotype ; Graft Rejection/mortality/*prevention & control ; Hepacivirus/genetics/isolation & purification ; Hepatitis C/drug therapy/*virology ; Humans ; Immunosuppressive Agents/*therapeutic use ; *Liver Transplantation/adverse effects ; Polymerase Chain Reaction ; RNA, Viral/blood ; Recombinant Fusion Proteins/therapeutic use ; Recurrence ; Retrospective Studies ; Survival Rate ; Tacrolimus/therapeutic use

BACKGROUNDOX40/OX40 ligand (OX40/OX40L) and programmed death-1/programmed death ligand-1 (PD-1/PD-L1) costimulatory signals play important roles in T cell-induced immune responses. The aim of this study was to investigate the roles of OX40/OX40L and PD-1/PD-L1 costimulatory pathways in mouse islet allograft rejection.

METHODSLentiviral vectors containing OX40L siRNA sequences and an adenovirus vector containing the PD-L1 gene were constructed. The streptozotocin-induced model of diabetes was established in C57BL/6 (H-2(b)) mice. Diabetic C57BL/6 mice were randomly allocated into five groups: group 1, untreated control; group 2, Ad-EGFP treatment; group 3, Ad-PD-L1 treatment; group 4, OX40L-RNAi-LV treatment; group 5, OX40L-RNAi-LV combined with Ad-PD-L1 treatment. Lentiviral vector and the adenovirus vector were injected, singly or combined, into the caudal vein one day before islet transplantation. The islets of DBA/2 (H-2(d)) mice were transplanted into the renal subcapsular space of the diabetic recipients. Recipient blood glucose and the survival time of the allografts were monitored. Antigen-specific mixed lymphocyte reaction was also evaluated.

RESULTSThe recombinant lentiviral RNA interference vector OX40L-RNAi-LV reduced OX40L protein expression by 70%. The recombinant adenovirus vector Ad-PD-L1 increased PD-L1 protein expression in vivo in C57BL/6 recipient mice. Combined OX40L-RNAi-LV/Ad-PD-L1 treatment induced a synergistic protective effect in pancreatic islet allografts. Allograft survival time in the combined treatment group was (92.27±9.65) days, not only longer than that of the control ((6.51±0.27) days) and Ad-EGFP groups ((7.09±0.13) days) (P < 0.01), but also significantly longer than that of Ad-PD-L1 and OX40L-RNAi-LV single treatment groups ((40.64±3.95) days and (55.14±5.48) days respectively, P < 0.01). The blood glucose concentration of recipient mice in the combined treatment group was also stable and kept within the normal range. Flow cytometry analysis showed that combined OX40L-RNAi-LV/Ad-PD-L1 treatment significantly decreased proliferation in an antigen-specific mixed lymphocyte reaction. After donor DBA/2 lymphocyte stimulation, 89.71% of lymphocytes from recipient combination treatment C57BL/6 mice were not split and proliferated. In contrast, after stimulation with third party Lewis rat lymphocytes, only 45.84% lymphocytes of C57BL/6 mice were not split and proliferated.

CONCLUSIONSThis study demonstrates the successful construction of the recombinant lentivirus vector OX40L-RNAi-LV and adenovirus vector Ad-PD-L1 for the blockade of OX40/OX40L and activation of PD-1/PD-L1 costimulatory pathways simultaneously in pancreatic islet allografts in diabetic mice. Combination therapy with these two vectors resulted in inhibition of T cell activation, synergistically prolonging the survival time of pancreatic islet allografts.

Animals ; B7-H1 Antigen ; genetics ; metabolism ; Graft Rejection ; genetics ; prevention & control ; Islets of Langerhans Transplantation ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; OX40 Ligand ; genetics ; metabolism ; Receptors, OX40 ; genetics ; metabolism ; Transplantation, Homologous

OBJECTIVETo establish a calculated panel reactive antibody (CPRA) method to analyze the donor-recipient incompatibility rate in PRA-positive kidney recipients and estimate the probability of these recipients to receive kidney transplantation.

METHODSBased on the database of HLA-A, -B, -DR genes and A-B, A-DR, B-DR, A-B-DR haplotype frequencies collected from 2004 donors from Jan 2000 to Dec 2012, we analyzed CPRA in 202 PRA-positive recipients and evaluated the consistency between PRA and CPRA assessments using a CPRA-Java calculator software, which returned a percentage of CPRA (representing the probability of unacceptable HLA in the donor group) after input of HLA-specific antibodies of a PRA-positive recipient.

RESULTSThe mean PRA intensity of the 202 PRA-positive recipients was (23.12∓17.83)% with a mean CPRA% of (46.07∓23.30)%. A significant difference was found between the mean PRA% and CPRA% in low sensitized recipients (PRA 0-10%) [(6.87∓2.41)% vs (21.63∓11.75)%, P<0.05) and in moderately sensitized recipients (PRA 10%-30%) [(20.15∓5.70)% vs (50.56∓16.86)%, P<0.05), but not in highly sensitized recipients (PRA>30%); The concordance rates between PRA% and CPRA% in the 3 groups were 19.35% (P<0.05), 10.99% (P<0.05), and 100% (P>0.05), respectively.

CONCLUSIONSLowly sensitized kidney recipients might have a lower probability of actually receiving a transplant than PRA% shows. A PRA%>30% is a risk factor for kidney transplantation. PRA reflects the sensitized level of a renal recipient, and reliable detection of HLA antibody specificity is of critical importance. CPRA accurately reflects the probability of a recipient to receive a transplant to assist clinicians in predicting the waiting time and selecting the transplant approach.

Antibodies ; Antibody Specificity ; Graft Rejection ; immunology ; Graft Survival ; immunology ; HLA Antigens ; genetics ; Haploidy ; Histocompatibility Testing ; methods ; Humans ; Kidney Transplantation

The effect of CYP3A4*18B and CYP3A5*3 on concentration/dosage x body surface area ratios (C/D'), adverse effects and acute rejection of tacrolimus in renal transplant patients were investigated. The CYP3A4*18B genotypes of 227 renal transplant patients were determined by PCR-RFLP method. The differences of C/D' ratios, adverse reactions and acute rejection were compared among all of the genotype groups treated with tacrolimus. The frequencies of CYP3A4*18 and CYP3A5*3 alleles in renal transplant patients were 30.8% and 74.2%, respectively. No significant association was found between the C/D's of tacrolimus and CYP3A4*18B genotypes when they were classified by two CYP3A5 genotypes (P > 0.05). While after the effects of CYP3A4*18B genotype were eliminated, the C/D' ratio of tacrolimus in patients with CYP3A5*1/*1 and *1/*3 genotype group was significantly lower than those with CYP3A5*3/*3 genotype groups (P < 0.01). There is no significant difference in adverse effects and acute rejection among different genotypes (P > 0.05).
Adult ; Alleles ; Cytochrome P-450 CYP3A ; genetics ; Digestive System Diseases ; chemically induced ; Dose-Response Relationship, Drug ; Female ; Genotype ; Graft Rejection ; genetics ; Humans ; Immunosuppressive Agents ; administration & dosage ; adverse effects ; blood ; therapeutic use ; Kidney Transplantation ; Male ; Middle Aged ; Polymorphism, Genetic ; Retrospective Studies ; Tacrolimus ; administration & dosage ; adverse effects ; blood ; therapeutic use

OBJECTIVETo evaluate the influence of major histocompatibility complex class I chain-related gene A (MICA) antibodies on acute rejection (AR) and renal function in early stage after renal transplantation.

METHODSA total of 197 renal transplant candidates admitted in Nanfang Hospital in 2009-2010 were enrolled in this study. MICA antibodies and their specificity were detected in all the patients, and 139 patients were followed up for early acute rejection (AR) and graft function after transplantation.

RESULTSMICA antibodies were positive before transplantation in 45 candidates (22.84%). Eleven specific MICA antibodies were identified, among which the frequency of MICA019 antibody (65.7%) was significantly higher than that of MICA015 (8.6%) and MICA017 (8.6%) (P<0.01). Eighteen patients with positive MICA antibodies were single-specific and 17 were polyspecific (51.4% vs 48.6% ). Of the 139 patients undergoing renal transplantation, 39 developed early AR (28.1%). Of the 45 candidates positive for MICA antibodies, 38 received renal transplantation and early AR occurred in 14 of them (36.8%); 101 of 152 candidates negative for MICA antibodies underwent renal transplantation, and 25 experienced early AR (24.8%).

CONCLUSIONMICA019 antibody is a frequent MICA antibody possibly due to the high frequency MICA019 gene in Chinese population.

Adult ; Antibodies ; immunology ; Antibody Specificity ; Female ; Graft Rejection ; immunology ; Graft Survival ; immunology ; Histocompatibility Antigens Class I ; genetics ; immunology ; Humans ; Kidney Transplantation ; Male ; Middle Aged

BACKGROUNDAcute rejection remains an important cause of renal allograft dysfunction and the need for accurate diagnosis is essential to successfully treat transplant recipients. The purpose of this study was to determine the costimulatory molecules OX40 and OX40L messenger RNA (mRNA) levels in peripheral blood mononuclear cells (PBMCs) to predict acute renal transplant rejection.

METHODSThe whole blood samples from 20 recipients with biopsy-confirmed acute rejection (rejection group), 20 recipients with stable graft function and normal biopsy results (stable group) after kidney transplantation, and 20 healthy volunteers (control group) were collected. The mRNA levels of OX40 and OX40L were analyzed with TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR). The association of OX40 and OX40L mRNA levels with disease severity was investigated.

RESULTSThere was no significant difference of OX40, OX40L mRNA levels in PBMCs between the stable group and control group (P > 0.05). The levels of OX40 and OX40L mRNA were significantly higher in the rejection group than in the control group (P < 0.01 and P < 0.05, respectively). Non-significantly higher OX40L mRNA and significantly higher OX40 mRNA in PBMCs were observed in subjects in the rejection group compared with the stable group (P > 0.05 and P < 0.01, respectively). Receiver operating characteristic (ROC) curve analysis demonstrated that OX40 mRNA levels could discriminate recipients who subsequently suffered acute allograft rejection (area under the curve, 0.908). OX40 and OX40L mRNA levels did not significantly correlate with serum creatinine levels in the rejection group (P > 0.05). Levels of OX40 mRNA after anti-rejection therapy were lower than those at the time of protocol biopsy in the rejection group (P < 0.05).

CONCLUSIONOur data suggest that measurement of OX40 mRNA levels after transplant might offer a noninvasive means for recognizing recipients at risk of acute renal allograft rejection.

Adult ; Biomarkers ; blood ; Female ; Graft Rejection ; blood ; diagnosis ; Humans ; Kidney Transplantation ; adverse effects ; Male ; Middle Aged ; OX40 Ligand ; genetics ; RNA, Messenger ; blood ; ROC Curve ; Receptors, OX40 ; genetics ; Transplantation, Homologous

BACKGROUNDInvasive kidney biopsy is a priority diagnostic method for the acute rejection after renal transplantation for the past decades. However, no effective and noninvasive assay for predicting the severity of acute rejection is in wide use at present. This study was designed to investigate the predictive value of programmed death 1 (PD-1) mRNA for acute rejection after renal transplantation with real-time reverse transcriptase polymerase chain reaction (RT-PCR). A noninvasive diagnostic method has been expected to replace the traditional kidney biopsy for the diagnosis of acute rejection and prediction of the outcome after kidney transplantation.

METHODSThe whole blood samples from 19 subjects with acute rejection, 20 subjects with delayed graft function (DGF) and 21 subjects with stable recipients after kidney transplantation in a single kidney transplantation center between 2006 and 2009 were collected. The messenger RNA (mRNA) of PD-1 was analyzed with real-time RT-PCR. The associations of PD-1 mRNA levels with acute rejection and disease severity were investigated.

RESULTSThe log-transformed ratio of PD-1 mRNA to GAPDH mRNA was higher in peripheral blood mononuclear cell (PBMC) from the group with acute rejection (4.52 ± 1.1) than that from the group with DGF (1.12 ± 0.6) or the group with normal biopsy results (0.7 ± 0.4) (P < 0.01, by the Kruskal-Wallis test). PD-1 mRNA levels were correlated with serum creatinine levels measured at the time of biopsy in the acute rejection group (Spearman's correlation coefficient, r = 0.81, P = 0.03), but not in the group with DGF or the group with normal biopsy results. PD-1 mRNA levels identified subjects at risk for graft failure within six months after the incident episode of acute rejection.

CONCLUSIONSOur data suggest that PD-1 status may be a new predictor of acute rejection and the levels of PD-1 mRNA in whole blood cells may positively correlate with the severity of acute rejection after renal transplantation. Meanwhile, the data provide the rational for interfering into the PD-1/PD-L1 as a novel therapy against the acute rejection after renal transplantation in clinical settings.

Adult ; Antigens, CD ; genetics ; Apoptosis Regulatory Proteins ; genetics ; Biomarkers ; blood ; Delayed Graft Function ; blood ; Female ; Graft Rejection ; blood ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Programmed Cell Death 1 Receptor ; RNA, Messenger ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

BACKGROUNDDonor organ rejection continues to be a significant problem for patients receiving transplants. We therefore tested whether transferring a donor's major histocompatibility complex (MHC) gene to the recipient would mitigate the rejection of transplanted hearts in mice.

METHODSH-2K(k) gene from donor mice was amplified using nested polymerase chain reaction (PCR) and ligated into a mammalian expression vector, which was then transfected into thymus ground mass cells collected from the recipients. Clones stably expressing the transgene were then injected into the recipients' thymus visualized using ultrasound. Control mice were administered cells previously transfected with empty vector. Following heart transplantation, cardiac activity was monitored electrocardiographically. Recipient thymus cells were tested for MHC antigenicity using flow cytometry and spleen cells were subjected to mixed lymphocyte culture tests. Finally, the transplanted hearts were sectioned, stained and examined under light microscopy.

RESULTSSouthern analysis following nested PCR revealed clear expression of H-2K(k) gene. Following transplantation, electrocardiosignals were detectable highly significantly longer in recipients administered thymal cells expressing donor H-2K(k) than in those receiving control cells. Flow cytometric analysis using an anti-H-2K(k) antibody confirmed its expression in H-2K(k) treated recipients but not in control mice. Mixed lymphocyte cultures containing H-2K(k) treated cells showed significantly less proliferation than those containing control cells. Hearts from control mice showed substantially greater lymphocyte infiltration than those from H-2K(k) treated mice and large areas of necrosis.

CONCLUSIONRejection of transplanted hearts can be mitigated substantially by introducing the donor's MHC into the recipient.

Animals ; Blotting, Southern ; Electrocardiography ; Female ; Flow Cytometry ; Graft Rejection ; genetics ; immunology ; Heart Transplantation ; immunology ; methods ; Major Histocompatibility Complex ; genetics ; immunology ; Male ; Mice ; Polymerase Chain Reaction