The Impact Of Anti-mouse CD52 Monoclonal Antibody To The Intra-epithelial Lymphocytes And Tight Junctions Of Graft After Small Intestinal Transplantation In Mice

An innovative immunosuppressive strategy utilizing induction treatment with Alemtuzumab(CAMPATH-1H)and minimization of post-transplant immunosuppression with tacrolimus(FK506)has been introduced in recent years,which improves the outcomes of clinical small intestinal transplantation obviously.In order to control GVHD(graft-versus-host disease)after bone marrow transplantation,a novel anti-human lymphocyte antibody named CAMPATH-1 was developed by Hale et al.from University of Cambridge in early eighties of last century.This antibody reacts specifically with CD52 antigen expressed on all lymphocytes,and it was turned to CAMPATH-1 H(a humanized anti-CD52 monoclonal antibody)after a series of modifications.It is capable of inducing lymphocytopenia in several ways including complement-dependent cytotoxicity(CDC),antibody-dependent cell-mediated cytotoxicity(ADCC)and apoptosis induction with protracted biological effect lasting for 6-12 months.Considering of the effect of ablating lymphocyte,CAMPATH-1 H was introduced into organ transplantation to induce tolerance.Though the lymphocyte ablating effect is familiar,the impact of anti-CD52 monoclonal antibody to peripheral lymphoid tissue is elusive.More than 60%lymphocytes reside in the intestine.Intestinal intra-epithelial lymphocytes(iIELs)which maintain the homeostasis of the intestinal mucosa make up a special cluster of gut associated lymphoid tissue(GALT).Therefore,the influence of anti-CD52 monoclonal antibody on gut and iIELs is worth concerning.CD52 antigen has been discovered in human and other animals,while only two kinds of anti-CD52 antibody are commercially available:anti-mouse CD52 monoclonal antibody besides CAMPATH-1H to human and Primates.Thus,we chose mice as animal model.In this research,we established both heterotopic and orthotopic small intestinal transplantation models in mice.The impact of anti-CD52 antibody to IELs and tight junction of grafts were investigated after orthotopic transplantation.It could provide theoretical support to refine the utilization of this antibody to minimize the side effects.Part ?Small Intestinal Transplantation Model in MiceObjectives:To establish both heterotopic and orthotopic small intestinal transplantation models in mice,and choose a reasonable model for the following research after comparison between two models.Methods:Syngeneic transplantation was performed between C57BL/6 mice.The superior mesenteric artery and portal vein of grafts were anastomosed to the recipient infrarenal aorta and inferior vena cava in end-to-side way respectively.In heterotopic model the native bowel was saved and stoma-free strategy was carried out in which the distal ileum of graft was end-to-side anastomosed to native ileum after closing proximal jejunum without stoma.While the native small intestine was cut off and replaced by graft in orthotopic transplantation.The change of body weight and complications after surgery were recorded.The recipients were anatomized and the grafts were obtained for histological examination on POD 14.Results:The success rate was 90.9%(30/33)in heterotopic model,while it was 78.4%(29/37)in orthotopic transplantation.Irreversible hypovolemic shock was the primary complication that resulted mortality.A larger extent and a longer duration of the body weight decrease were observed in orthotopic model.The thinner intestine and mucosal atrophy due to graft exclusion were only found in heterotopic model.Conclusions:The success rates of two models were satisfying.However,the orthotopic model was more reasonable for the research of graft functions.Part ?The Impact of Anti-mouse CD52 Monoclonal Antibody to the Graft Intra-epithelial Lymphocytes after Small Bowel Transplantation in MiceObjectives:To discover the impact of anti-mouse CD52 monoclonal antibody to the iIELs after small intestinal transplantation in mice.Methods:Orthotopic small intestinal transplantation was performed in mice using C57BL/6 mice as recipients,and C57BL/6 and BALB/c mice were applied as donors in syngeneic and allogeneic groups respectively.Tacromilus(FK506)was administrated intraperitoneally daily after surgery to control rejection in allogeneic transplantation.There were six groups:(1)syngeneic control group(C57BL/6);(2)-CD52 syngeneic group;(3)+CD52 syngeneic group;(4)allogeneic control group(BALB/c);(5)-CD52 allogeneic group;(6)+CD52 allogeneic group.Recipient mice were sacrificed on either POD7 or POD14.Grafts were examined by histology,flow cytometry for IELs and real-time RT-PCR for keratinocyte growth factor(KGF)mRNA.Results:No rejection was found in allogeneic transplantation.On POD7,the villi of graft were shorter after anti-mouse CD52 antibody administration.At the same time,IELs,especially TCR??+ subtype were reduced.The donor-derived TCRl??+ iIELs were replaced by recipient-derived TCRy??+ iIELs gradually after surgery,and were diminished in +CD52 syngeneic and allogeneic groups on POD7.The antibody also decreased IEL-derived KGF mRNA expression.Conclusions:Anti-mouse CD52 monoclonal antibody eliminated IELs,especially TCR??+ subtype of graft,and decreased IEL-derived KGF mRNA expression,which would have negative effects on transplanted intestine.Part ?The Impact of Anti-mouse CD52 Monoclonal Antibody to the Tight Junctions of Graft after Small Bowel Transplantation in MiceObjectives:To observe the impact of anti-mouse CD52 monoclonal antibody to the tight junctions after small intestinal transplantation in mice.Methods:Orthotopic small intestinal transplantation was performed in mice using C57BL/6 mice as recipients,and C57BL/6 and BALB/c mice were applied as donors in syngeneic and allogeneic groups respectively.Tacromilus(FK506)was administrated intraperitoneally daily after surgery to control rejection in allogeneic transplantation.There were four groups:(1)-CD52 syngeneic group;(2)+CD52 syngeneic group;(3)-CD52 allogeneic group;(4)+CD52 allogeneic group.Recipient mice were sacrificed on POD7.Transmission electron microscope was carried for tight junctions,and both immunofluorescence staining and western blot were applied for tight junction proteins expression.Results:After syngeneic transplantation,the ultrastructure analysis revealed that the tight junctions were disrupted after Anti-mouse CD52 monoclonal antibody administration.Immunofluorescence staining of tight junction proteins showed the decline of Occludin and ZO-1 in the graft villi in +CD52 syngeneic and allogeneic groups.Western blot analysis demonstrated the expression of Occludin and ZO-1 was decreased after anti-CD52 antibody application.Conclusions:Anti-mouse CD52 monoclonal antibody disrupted tight junctions and decline tight junction proteins in transplanted intestine.