Accommodation In Renal Allograft In Presensitized Nonhuman Primates

Transplantation of grafts into presensitized recipients,which may result from multipleblood transfusions,previous pregnancy,or prior transplantation,results in severe humoralrejection,including hyperacute rejection,accelerated rejection and acute vascular rejection.Along with the increase of cases in transplantation,more and more presensitized recipientsare waiting for a second,even third transplantation.The present costly"desensitization"therapy to these presensitized patients has some unmanageable adverse side effects and isonly effective in some patients.In this study,we tried to establish a renalallotransplantation model in skin-presensitized non-human primates and induce long-timerenal allograft survival by an"accommodation"strategy.To know ABO blood types of monkeys exactly is the first step of the reaserch innon-human primate animal transplantation area.Monkeys do not express ABO antigens onred blood cells(RBCs),but on tissues,which is an obstacle of its ABO typing.In this studywe describe a simple and efficient method to blood type monkeys.Rhesus monkeys(n=38)and cynomolgus monkeys(n=26)were blood typed by the direct and reverse gel system,which is widely used in clinics in recent years.Based on the results ofimmunohistochemical staining,we evaluated the feasibility and the interference factors ofthe gel system to blood type monkeys.The results revealed that the direct gel system hadnone positive report,the fibrinogen and non-specific anti-human antibodies in monkeyblood interfered with the reverse gel system in some samples.Being in accord with theresults of immunohistochemical staining,using clear sera,which were pre-absorbed onnomal human type O RBCs,the reverse gel system gave an accurate blood typedetermination of monkeys.We concluded that the reverse gel system could be used forABO typing of monkeys,and the fibrinogen and non-specific anti-human antibodies werethe major interference factors,which could be eliminated by using clear and pre-absorbed sera.To establish an accelerated rejection model of renal allotransplantation in presensitizedmonkeys.Recipient rhesus monkeys were presensitized by grafting of 2 full-thicknessabdominal skin grafts from ABO-compatible donor monkeys and subsequently received akidney from the same donor two weeks later.In comparision with non-presensitised controlgroup,the changes of serum creatinine levels,survival time and the pathology of graftswere observed and studied.The efficiency of a regular clinical immunosuppressive regimenwith CsA+MMF+Prednisone on this presensitized transplant model was also evaluated.The results revealed that,four renal grafts in non-presensitized control group survived for 7,8,9,and 18 days respectively;In contrast,3 presensitized recipients without treatment(untreated presensitized group)rejected allografts in 3,3,4 days respectively;3presensitized recipients with triple therapy(treated presensitized group)rejected allograftsin 2,3,4 days respectively.We concluded that,presensitization by donor skin canacceletate allograft rejection,which can not be reversed by the administration of CsA incombination with MMF and prednisone.We described,for the first time,a non-humanprimate animal model of accelerated humoral rejection,which will enable us to moreextensively study in the case ofpresensitized or hyperimmunized patients.To study the immunologic and pathologic features of an accelerated rejection model ofrenal allotransplantation in presensitized monkeys.The accelerated rejection model of renalallotransplantation was established in presensitized monkeys,which received donor skintransplantation in advance.The changes of donor specific antibody(DSA)levels in therecipient monkeys before/after skin and kidney transplantation were measured.The kidneygrafts were examined for routine pathology,antibody and complement depositions,virouslymphocyte subsets infiltration by HE stainning,immunofluorescence,orimmunohistochemistry.The results revealed that,all renal allografts in presensitizeduntreated monkeys developed accelerated rejetion within 4 days.In 2 presentized monkeys,the levels of DSA and their mediated complement- dependent cytotoxicity(CDC)significantly increased after skin transplantation,and further markedly elevated at the timeof kidney graft rejection.In the rejected renal grafts,massive C3,C4,C5b-9 and IgGdeposits with few lymphocytes infiltration were found.Typical pathologic changes included severe arterionecrosis,thrombosis,interstitial hemorrhage,and infiltration of neutrophils.In the rest one presentized monkey,the levels of DSA and CDC only marginally increased,and the pathological changes of the rejected renal graft characterized mainly by the injuryof renal tubules.To compare with the results of presensitized untreated group,thepathological damage in renal allograft was much slighter in presensitized treated group.Weconcluded that,presensitization by donor skin transplantation could elevate the levels ofDSA and CDC in recipient monkeys,which resulted in severe antibody mediated acutehumoral rejection in most of the following renal transplants;The typical pathologic featureswere characterized by evidence of acute tissue injury,such as hemorrhagic lesions,necrosis,thrombosis and polynuclear cell infiltratioin;CsA+MMF+Pred administration did notprolonged graft survival,but could mitigate the pathological damage,which gave us a clue.The immunologically sensitized recipients with donor-specific antibody(DSA)areconsidered to be at greater risk for the development of acute humoral rejection followingtransplantation.Induction of self-protection against DSA-mediated humoral injury knownas accommodation may allow an allograft survives continuously in presensitizated recipient.The present study was undertaken to determine whether renal allograft accommodationcould be induced in donor skin-presensitized monkeys by short-term inhibition ofcirculating complement,and to investigate its possible mechanisms via analysis of certainintragraft protective genes expression.ABO-compatible and MHC-mismatched rhesusmonkeys were paired as donor and recipient.Donor skin allografts were transplanted topresensitize recipients 14 days prior to renal transplantation.Based on the CsA+MMF+Predtherapy,we added CVF administration(CVF+CsA+MMF+Pred group)(CVF,0.05 mg/kgiv.on day -2 and -1,then 0.02 mg/kg iv.every other day from POD 0-13,n=5).Serum DSAand its mediated CDC were detected by FACS using donor lymphocytes as targets.Intragraft expression of the antiapoptotic and complement regulatory proteins at differenttime-points was determined by immunohistochemical stainning and western blot.Theresults revealed that,triple therapy with CsA+MMF+Pred adding a short-term CVF therapyin the early period(2 weeks)significantly prolonged graft survival to a median survival of145 days(41,140,＞250,＞500,＞700 days;P＜0.01 vs.other groups).CVF treatment led tovery dramatic fall in serum C3 level and CH50 for at least 2 weeks,and 3 of 5 recipient monkeys received this therapy survived continuously with normal renal function andhistology even after the serum C3 levels returned to normal following the cessation of CVFtherapy.The expression of Bcl-2,CD46 and CD59 were significantly elevated in long-termsurvival grafts via analysis of immunohistochemistry at different time-points(especially onPOD 50 and 100).We concluded that,additionally using CVF in the early critical periodallowed long-term renal allograft survival in most presensitized recipients and most likelyinduced successful accommodation.The establishment of accommodation in this modelmay be associated with up-regulation of certain protective genes and complementregulatory proteins in grafts.These encouraging results indicate that the complementinhibition-based strategy may be valuable in future clinical cross-match positive orABO-incompatible transplantation.The preceding results presented an exciting possible application of Cobra venomfactor(CVF)in clinic.So a deeper research of its property and immunogenicity innon-human primates was necessary.In comparision with various CVF reported previously,Yunnan-cobra venom factor(Y-CVF)has higher anticomplement activity and lowertherapeutic dosage.To investigate whether Y-CVF could induce the specific neutralizedanti-Y-CVF antibody and xenoantibodies in non-human primates,two cynomolgusmonkeys were intravenous injected with Y-CVF(0.05mg/kg)every 2 weeks for 4 times.The changes of serum C3,CH50,anti-Y-CVF antibody and xenoantibody levels weremeasured.The results revealed that the first two injection of Y-CVF resulted in effectivedepletion of complement C3,the third injection had only incomplete effect and the fourthinjection was almost ineffective.The results of Western Blot and ELISA confirmed theproduction of anti-Y-CVF antibody,and its titre was increasing progressively along with theinjection times of Y-CVF.Additionally no significant changes of anti porcine endotheliacell xenoantibodies were found measured by flow cytometry.In addition,we also measuredthe level of anti Y-CVF antibody in the sera of two longtime survival rhesus monkeys,which accepted a short-term Y-CVF therapy in the early period(2 weeks)in combinationwith CsA+MMF+Pred.We found the level of the specific antibody was not high,and thelevel of C3 was steady low during the period of Y-CVF injection.We concluded that,Y-CVF was a more effective;Y-CVF had its immunogenicity,and multiple injection of Y-CVF stimulated anti-Y-CVF antibody production in monkeys,which resulted in theinvalid of Y-CVF;The induction of anti-alpha -Gal xenoantibody by Y-CVF in primateswas not observed;In combination with other immunosupressive agents,a short-termY-CVF therapy in the early period could markedly reduce the production of neutralizingantibody and had a good application prospect in clinic.In sum,we described,for the first time,a simple and reliable method for ABO typingof monkeys and a non-human primate animal model of accelerated humorat rejection as apreclinical model.And we successfully induced a long-time allograft survival andaccommodation in these presensitized recipient monkeys by early inhibition of complementin combination with CsA triple maintenane therapy.The accommodation may be associatedwith up-regulation of intragraft Bcl-2,CD46 and CD59.