| ObjectivesABO-incompatible liver transplantation (ABO-I LT) is an alternative treatment for critical patients with acute liver failure (ALF). The traditional immunosupperessive regimens are too complicated and unsuitable for emergent ABO-I LT. Herein, we explored a new experience of patients with ALF treated by emergent ABO-I LT, using innovative and simple immunosuppressive protocol consisting of rituximab and intravenous immunoglobulin (IVIG), and analyzed the results compared with ABO-compatible liver transplant (ABO-C LT) cases, to evalute its efficiency and safety.We also described a type of animal model (PBMC-NOD/SCID chimeric mice, immunized by donor blood type erythrocytes) to provide evidence of immune tolerance to donor blood group antigens after ABO-I LT, and preliminary studied the role played by CD19+CD24hiCD38hi regulatory B lymphocytes in specific immune tolerance to blood group antigens after ABO-I LT.. Materials and MethodsPart oneA modified protocol with rituximab and intravenous immunoglobulin in emergent ABO-incompatible liver transplantation for acute liver failure1. Patients:101adult patients with ALF receiving LT in emergency conditions from were studied. All patients were divided into ABO-C LT group (n=66) and ABO-I LT group (n=35);2. Protocol of immunosuppression:All ABO-I LT patients received a single dose of rituximab (375mg/m2) and IVIG (0.4g/kg) at the beginning of operation. IVIG (0.4g/kg) for induction therapy were administered last for10days after LT. Quadruple regimen composed of basiliximab, tapering steroid, tacrolimus and mycophenolatemofetil was adopted as the basic immunosuppression in both group;3. Postoperative prophylaxis for infection;4. Tacrolimus trough level was monitored after transplantation:enzyme linked immunosorbent assay (ELISA);5. Detection for anti-donor blood group antibodies IgM in a perioperative period in ABO-I LT patients:saline dilution method;6. Analysis of peripheral blood CD20+B cells counts in a perioperative period in ABO-I LT:flow cytometry;7. Statistical analysis:the software program SPSS version17.0was used and p<0.05was considered statistically significant. Part twoImmune tolerance to donor ABO blood group antigens induced by ABO-incompatible liver transplantation1. Patients data:6patients underwent liver transplantation were defined according to blood type of donor and recipients:1A (ABO-I,ABâ†'A), positive control1B (ABO-C,Aâ†'A),negative control1C(ABO-C, ABâ†'AB);2A(ABO-I, ABâ†'O), positive control2B (ABO-C, Oâ†'O), negative control2C (ABO-C,ABâ†'AB);2. Reagents and formula;3. Devices and equipments;4. Detection of anti-blood group antibodies (IgM) in patients(1A,2A):saline dilution method;5. Peripheral blood mononuclear cells(PBMCs) from patients:ficoll-hypaque density gradient centrifugation,(1A,1B,1C,2A,2B,2C);6. Breeding NOD/SCID mice;7. Establishment of human PBMC-NOD/SCID chimeric mice and sensitization with blood group-AB erythrocytes;8. Detection of anti-blood group antibodies (IgM) in chimeric mice:ELISA;9. Analysis of CD19+CD24hiCD38hi regulatory B lymphocyte counts:flow cytometry;10. Statistical analysis:the software program SPSS version17.0was used and p<0.05was considered statistically significant. ResultsPart one1. No significant difference of pre-operative clinical characteristics between the two groups;2. No significant difference of post-operative tacrolimus concentration between the two groups;3. Combination of ABO blood-type between donors and recipients in the ABO-I group was observed,34%of which was AB to O;4. The anti-donor blood type IgM titers were reduced immediately after LT in the ABO-I group. The lower titers level maintained for several months. Rebound elevation of the titers was seen in two cases within one month after LT and they developed into AMR. The count of peripheral CD20+B cells decreased to <1%in all cases of ABO-I group and last for3months.5. AMR was happened on2cases (5.72%) with rebound elevation of the titers, clinical manifestations were intrahepatic biliary complication and hepatic necrosis. Casel recovered after high dose IVIG administration and plasma exchange; Case2had no response to any methods, and received ABO-C LT at6months post first LT.6. The3-year cumulative patient survival rates in ABO-I group and ABO-C group were83%and86%respectively (lok-rank, p=0.596), the3-year cumulative graft survival rates were80%and86%respectively (lok-rank, p=0.417);7. Two groups showed a similar frequency of biliary complication, infection, acute cellular rejection, vascular complication, renal dysfunction, and GVHD。 Part two1. Anti-blood group antibodies (IgM) in patients:anti-B IgM of patient1A, anti-A and anti-B IgM of patient2A were persistently low or undetectable after ABO-I LT;2. Anti-blood group antibodies (IgM) in chimeric mice:after sensitization with blood group-AB erythrocytes, anti-B IgM of1A PBMC-NOD/SCED chimeric mice was significantly lower than1B mice, and similar with1C mice; anti-A/B IgM of2A chimeric mice was significantly lower than2B mice, and similar with2C mice;3. Counts of CD19+CD24hiCD38hi regulatory B lymphocytes in patient1A and2A4years after ABO-I LT were5.95%and4.51%respectively, which were higher than the normal population and ABO-I LT recipients in early post-operative period.Conclusions1. The simplified regeimen consisting of rituximab and IVIG could prevent AMR in ABO-I LT. With this treatment, the patients did not need plasma exchange, splenectomy and graft local infusion. This treatment was safe and efficient for emergent ABO-incompatible liver transplantation of the patients with ALF;2. Immune tolerance to ABO blood group antigens induced by ABO-I LT could be observed, and CD19+CD24hiCD38hi regulatory B lymphocytes maybe play part role in this immunological mechanism. |
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