The effects of diltiazem in renal transplantation patients treated with cyclosporine A

Objective： To investigate the effects of diltiazem and cyclosporine A （CsA） combination therapy on protecting the kidney, promoting graft functioning and improving post-transplanted kidney recovery. Methods： The blood con- centrations of CsA, the condition of the post-transplant kidney, the rate of acute rejection （AR）, as well as hepatic and renal toxicity in 636 cases of renal transplant recipients were determined after being treated by CsA, with or without diltiazem. Results： Compared with the control group which received CsA, mycophenolate mofetil （MMF） and prednisolone （Pred） but lacked diltiazem, the group receiving these agents together with diltiazem required reduced dosage of CsA （P 〈 0.01）, while blood concentrations of CsA were significantly increased （P 〈 0.01）; the recovery time of graft function was reduced from （6.2± 1.5） d to （3.9± 1.4） d （P 〈 0.01）, and the rate of AR was decreased from 13.2% to 7.9% （P 〈 0.01）. Conclusion： In renal transplantation patients treated with CsA and diltiazem, blood concentrations of CsA were increased while the dosage was decreased. This efficient combination therapy reduced patients economic burden, at the same time retained kidney function, promoted graft function recovery and decreased hepatic and renal toxicity and the rate of AR.

The clinical value of enzyme-multiplied immunoassay technique monitoring the plasma concentrations of cyclosporine A after renal transplantation

The feasibility and the clinical value of the enzyme-multiplied immunoassay technique （EMIT） monitoring of blood concentrations of cyclosporine A （CsA） in patients treated with CsA were investigated after kidney transplantation. The validation method was performed to the EMIT determination of CsA blood concentration, the CsA whole blood trough concentrations （Co） of patients in different time periods after renal transplantation were monitored, and combined with the clinical complications, the statistical results were analyzed and compared. EMIT was precise, accurate and stable, also with a high quality control. The mean postoperative blood concentration of CsA was as follows： 〈1 month, （281.4± 57.9）ng/mL; 2 - 3 months, （264.5 ± 41.2） ng/mL; 4 - 5 months, （236.4 ± 38.9） ng/mL; 6 - 12 months, （206.5± 32.6）ng/mL; 〉12 months, （185.6± 28.1）ng/mL. The toxic reaction rate of CsA blood concentration within the recommended therapeutic concentration was 14.1%, significantly lower than that of the none-recommended dose group （37.2%） （P〈0.05）; the transplantation rejection rate was 4.4%, significantly lower than that of the none- recommended dose group （22.5%） （P〈0.05）. Using EMIT to monitor the blood concentration of CsA as the routine laboratory method is feasible, and is able to reduce the CsA toxicity and rejection significantly, leading to achieving the desired therapeutic effect.

To analyze the differentially expressed genes in chronic rejection after renal transplantation by bioinformatics

Objective: To use bioinformatics technology to analyse differentially expressed genes in chronic rejection after renal transplantation, we can screen out potential pathogenic targets associated with the development of this disease, providing a theoretical basis for finding new therapeutic targets. Methods: Gene microarray data were downloaded from the Gene Expression Profiling Integrated Database (GEO) and cross-calculated to identify differentially expressed genes (DEGs). Analysis of differentially expressed genes (DEGs) with gene ontology (GO) is a method used to study the differences in gene expression under different conditions as well as their functions and interrelationships, while Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis is a tool used to explore the functions and pathways of genes in specific biological processes. By calculating the distribution of immune cell infiltration, the result of immune infiltration in the rejection group can be analysed as a trait in Weighted Gene Co-Expression Network Analysis (WGCNA) for genes associated with rejection. Then, protein-protein interaction networks (PPI) were constructed using the STRING database and Cytoscape software to identify hub gene markers. Results: A total of 60 integrated DEGs were obtained from 3 datasets (GSE7392, GSE181757, GSE222889). By GO and KEGG analysis, the GEDs were mainly concentrated in the regulation of immune response, defence response, regulation of immune system processes, and stimulation response. The pathways were mainly enriched in antigen processing and presentation, EBV infection, graft-versus-host, allograft rejection, and natural killer cell-mediated cytotoxicity. After further screening using WGCNA and PPI networks, HLA-A, HLA-B, HLA-F, and TYROBP were identified as hub genes (Hub genes). The data GSE21374 with clinical information was selected to construct the diagnostic efficacy and risk prediction model plots of the four hub genes, and the results concluded that all four Hub genes had good diagnostic value (area under the curve in the range of 0.794-0.819). From the inference, it can be concluded that the four genes, HLA-A, HLA-B, HLA-F and TYROBP, may have an important role in the development and progression of chronic rejection after renal transplantation. Conclusion: DEGs play an important role in the study of the pathogenesis of chronic rejection after renal transplantation, and can provide theoretical support for further research on the pathogenesis of chronic rejection after renal transplantation and the discovery of new therapeutic targets through enrichment analysis and pivotal gene screening, as well as inferential analyses of related diagnostic efficacy and disease risk prediction.

STUDY ON B7-1 PROTEIN'S EXPRESSION IN BIOPSIES IN RENAL TRANSPLANTATION

Objective To study the expression of B7-1 protein in biopsies in allograft renal transplantation,and explore the expression patern and the functional role of B7-1 molecule. Methods Renal allograft sample tissue by Tru-cut needle aspiration were taken from 64 paients(42 male,22 female) with renal transplantation, aging from 17 to 58 years old; 64 cases were categorized into six groups: ①acute rejection (AR n =22);②accelerated rejection (AAR n =8);③chronic rejection (CR n =10 );④hyperacute rejection (HR n =4);⑤allograft with stable function (ASF n =10); ⑥health donor kidney (HDK n =10);Immunohistochemical assays(ABC) were used, and comparison B7-1 and HLA-DR expression between tubularand interstitial,the number of interstitial infiltrating lymphocytes. Results ①The sequence of the expression of B7-1 molecule in terms of intensity from the strongest to the weakest in different groups is AR group, AAR group, CR group, HR group,ASF group and HDK group; ②During acute rejection response, a large number of CD4 and CD8 T lymphocytes infiltrate kidney interstitium, accompanied by strong expression of HLA-DR in tubular cell(donor MHC-Ⅱantigen),which is the first signal necessary for T lymphocyte activation. Conclusion The results demonstrate that B7-1 expression of tubular cells as APC actively take part in immunological reactions and play an important role in expanding the immunological reactions.

Gastrointestinal tumors in transplantation: Two case reports and review of literature

BACKGROUND Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors of the gastrointestinal tract.As most of them harbor a KIT mutation(75%),selective kinase inhibitors are the therapeutic option and show a sustained objective response among patients with metastatic or unresectable GISTs.A wellknown higher risk of neoplasm has been described among renal transplant recipients(RTRs).Nevertheless,only few cases of GIST onset among transplant patients have been reported in the literature.CASE SUMMARY Here,we describe 2 cases of gastric GIST occurring during the follow-up of RTRs.We also review the existing literature concerning GIST occurrence in transplant patients.In total and in association with our 2 cases,16 patients have been reported.The median age was 59.5 years and 69%were male.With a median tumor size of 45 mm,no patient displayed metastatic dissemination at diagnosis.Time from transplantation to diagnosis was highly variable between 5 mo and 21 years.Histopathological data mostly revealed high risk of progression(43%).Death increased to 29%during follow-up.Surgical treatment was systematically performed when the tumor was operable(94%).The use of adjuvant therapy was uncommon(19%).CONCLUSION GISTs represent rare but potentially severe malignant complication among transplant patients.

What is the purpose of launching the World Journal of Transplantation?

Congratulations to the publisher,members of the editorial board of the journal,all the authors and readers for launching the World Journal of Transplantation(WJT)as a new member of the World series journal family.Transplantations are rapidly evolving and share knowledge with a number of basic and clinical sciences:molecular biology,stem cell investigators,immune system,pharmacology,biotechnology,surgery and physicians of different organs such as the kidneys,liver,heart,lung,bone marrow and so on.The WJT is a peer reviewed open access journal centered on the different fields involved in transplant activity.If you want to share your experiences and new findings in the field of transplantation with your peers you will find the WJT a good media to publish your papers.

Risk stratification of renal transplant recipients using routine parameters: Implication of learning from SARS-CoV-2 into transplant follow-up program

BACKGROUND Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)infection is a global pandemic that is associated with a high risk of morbidity and mortality among recipients of solid organ transplantation.In the course of acute SARS-CoV-2 infection,various laboratory markers have been identified as predictors for high risk of mortality.AIM To risk stratify renal transplant recipients(RTxR)using general demographic parameters,comorbidities and routine laboratory markers for the severity of the disease and its outcomes.We believe that learning about these routinely monitored parameters can help us plan better strategies for the RTxR follow-up program.METHODS This present study includes RTxR who acquired SARS-CoV-2 infection from March 2020 to February 2021.We recorded the basic demographics,comorbidities and routine laboratory markers.We investigated the impact of SARS-CoV-2 infection on RTxRs and risk-stratified the progression of disease severity and outcomes in terms of recovery or mortality.RESULTS From 505 RTxRs in our renal transplant follow-up program,29(7.75%)RTxRs had PCR-positive SARS-CoV-2 infection.We recorded 8 deaths from SARS-CoV-2 infection giving an overall mortality rate of 1.6%but a significant 27.6%mortality in SARS-CoV-2 positive recipients.Age more than 68 years,non-Caucasian ethnicity and male gender were associated with a significant drop in survival probability;P≤0.001.<0.001 and<0.0001 respectively.87.5%of the deceased were diabetic;P≤0.0.0001.Estimated glomerular filtration rate of less than 26 mL/min/1.73 m2,serum albumin less than 20 g/L,Hemoglobin less than 9.6 g/L and serum calcium less than 1.70 mmol/L were all associated with significantly increased risk of mortality;P=0.0128,<0.001,<0.0001 and 0.0061 respectively.CONCLUSION This study has identified some routinely used modifiable parameters in predicting a higher risk of mortality and morbidity.This knowledge can be used in RTxR follow-up programs by addressing these parameters early to help reduce the morbidity and mortality in RTxRs.

Update on the reciprocal interference between immunosuppressive therapy and gut microbiota after kidney transplantation

Gut microbiota is often modified after kidney transplantation.This principally happens in the first period after transplantation.Antibiotics and,most of all,immunosuppressive drugs are the main responsible.The relationship between immunosuppressive drugs and the gut microbiota is bilateral.From one side immunosuppressive drugs modify the gut microbiota,often generating dysbiosis;from the other side microbiota may interfere with the immunosuppressant pharmacokinetics,producing products more or less active with respect to the original drug.These phenomena have influence over the graft outcomes and clinical consequences as rejections,infections,diarrhea may be caused by the dysbiotic condition.Corticosteroids,calcineurin inhibitors such as tacrolimus and cyclosporine,mycophenolate mofetil and mTOR inhibitors are the immunosuppressive drugs whose effect on the gut microbiota is better known.In contrast is well known how the gut microbiota may interfere with glucocorticoids,which may be transformed into androgens.Tacrolimus may be transformed by microbiota into a product called M1 that is 15-fold less active with respect to tacrolimus.The pro-drug mycophenolate mofetil is normally transformed in mycophenolic acid that according the presence or not of microbes producing the enzyme glucuronidase,may be transformed into the inactive product.

What is new in the pathogenesis and treatment of IgA glomerulonephritis

Recently,new findings have been clarified concerning both pathogenesis and treatment of IgA nephritis.The four hits theory has been confirmed but several genetic wide association studies have allowed finding several genes connected with the pathogenesis of the disease.All these new genes apply to each of the four hits.Additionally,new discoveries concerning the microbiota and its connection with immune system and IgA generation have allowed finding out the role of the mucosa in IgA nephropathy pathogenesis.The IgA treatment is also changed included the future possibilities.The treatment of the chronic kidney disease,associated with the nephropathy,is mandatory,since the beginning of the disease.The classical immunosuppressive agents have poor effect.The corticosteroids remain an important cornerstone in any phase of the disease.More effect is related to the treatment of B cells and plasma cells.In particular,in very recent studies have been documented the efficacy of anti B cell-activating factor and anti A proliferation-inducing ligand agents.Most of these studies are to date in phase II/III.Finally,new agents targeting complement are arising.These agents also are still in randomized trials and act principally in hit 4 where the immunocomplexes in the mesangium activate the different pathways of the complement cascade.

Role of artificial intelligence in gastrointestinal surgery

Artificial intelligence is rapidly evolving and its application is increasing day-byday in the medical field.The application of artificial intelligence is also valuable in gastrointestinal diseases,by calculating various scoring systems,evaluating radiological images,preoperative and intraoperative assistance,processing pathological slides,prognosticating,and in treatment responses.This field has a promising future and can have an impact on many management algorithms.In this minireview,we aimed to determine the basics of artificial intelligence,the role that artificial intelligence may play in gastrointestinal surgeries and malignancies,and the limitations thereof.

Update on ischemia-reperfusion injury in kidney transplantation: Pathogenesis and treatment

Ischemia/reperfusion injury is an unavoidable relevant consequence after kidney transplantation and influences short term as well as long-term graft outcome. Clinically ischemia/reperfusion injury is associated with delayed graft function, graft rejection, chronic rejection and chronic graft dysfunction. Ischemia/reperfusion affects many regulatory systems at the cellular level as well as in the renal tissue that result in a distinct inflammatory reaction of the kidney graft. Underlying factors of ischemia reperfusion include energy metabolism, cellular changes of the mitochondria and cellular membranes, initiation of different forms of cell death-like apoptosis and necrosis together with a recently discovered mixed form termed necroptosis. Chemokines and cytokines together with other factors promote the inflammatory response leading to activation of the innate immune system as well as the adaptive immune system. If the inflammatory reaction continues within the graft tissue, a progressive interstitial fibrosis develops that impacts long-term graft outcome. It is of particular importance in kidney transplantation to understand the underlying mechanisms and effects of ischemia/reperfusion on the graft as this knowledge also opens strategies to prevent or treat ischemia/reperfusion injury after transplantation in order to improve graft outcome.

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

AIM. To investigate the influence of heme oxygenase-1 （HO-1） gene transfer on the viability and function of cultured rat islets in vitro. METHODS： Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene （Ad- HO-1） or enhanced green fluorescent protein gene （Ad- EGFP）, and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index （SI） was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS： After seven days culture, the viability of cultured rat islets decreased significantly （92% ± 6% vs 52% ± 13%, P 〈 0.05）, and glucose-stimulated insulin release also decreased significantly （6.47 ± 0.55 mIU/ L/30IEO vs 4.57 ± 0.40 mIU/L/3OIEO., 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. Transfection of rat islets with adenoviral vectors at an 1±10 of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets（71% ± 15% vs 52% ± 13%, P 〈 0.05）. There was no significant difference in insulin release upon low glucose stimulation （2.8 mmol/L） among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group （P 〉 0.05）, while when stimulated by high glucose （16.7 mmol/L） solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively （12.50 ±2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ, 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively （2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P 〈 0.05）. CONCLUSION： The viability and function of rat islets decrease over time in in vitro culture, and heine oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.

Impact of donor-specific antibodies on the outcomes of kidney graft:Pathophysiology, clinical, therapy

Allo-antibodies, particularly when donor specific, are one of the most important factors that cause both early and late graft dysfunction. The authors review the current state of the art concerning this important issue in renal transplantation. Many antibodies have been recognized as mediators of renal injury. In particular donorspecific-Human Leukocyte Antigens antibodies appear to play a major role. New techniques, such as solid phase techniques and Luminex, have revealed these antibodies from patient sera. Other new techniques have uncovered alloantibodies and signs of complement activation in renal biopsy specimens. It has been acknowledged that the old concept of chronic renal injury caused by calcineurine inhibitors toxicity should be replaced in many cases by alloantibodies acting against the graft. In addition, the number of patients on waiting lists with preformed anti-human leukocyte antigens(HLA) antibodies is increasing, primarily from patients with a history of renal transplant failure already been sensitized. We should distinguish early and late acute antibody-mediated rejection from chronic antibody-mediated rejection. The latter often manifets late during the course of the posttransplant period and may be difficult to recognize if specific techniques are not applied. Different therapeutic strategies are used to control antibody-induced damage.These strategies may be applied prior to transplantation or, in the case of acute antibody-mediated rejection, after transplantation. Many new drugs are appearing at the horizon; however, these drugs are far from the clinic because they are in phase Ⅰ-Ⅱ of clinical trials. Thus the pipeline for the near future appears almost empty.

Bilateral renal cortical necrosis associated with smoking synthetic cannabinoids

Synthetic cannabinoids have become a common drug of abuse in recent years and their toxicities have come to light as well. They are known to be notorious for the kidneys, with acute tubular necrosis, acute interstitial nephritis and rhabdomyolysis induced renal injury being the frequent nephrotoxic outcomes in users. We report a case of bilateral renal cortical necrosis, leading to irreversible renal damage and lifelong dialysis dependency.

Reclassification of membranoproliferative glomerulonephritis:Identification of a new GN:C3GN

This review revises the reclassification of the mem-branoproliferative glomerulonephritis （MPGN） after the consensus conference that by 2015 reclassified all the glomerulonephritis basing on etiology and patho-genesis, instead of the histomorphological aspects. After reclassification, two types of MPGN are to date recognized： The immunocomplexes mediated MPGN and the complement mediated MPGN. The latter type is more extensively described in the review either because several of these entities are completely new or because the improved knowledge of the complement cascade allowed for new diagnostic and therapeutic approaches. Overall the complement mediated MPGN are related to acquired or genetic cause. The presence of circulating auto antibodies is the principal acquired cause. Genetic wide association studies and family studies allowed to recognize genetic mutations of different types as causes of the complement dysregulation. The complement cascade is a complex phenomenon and activating factors and regulating factors should be distinguished. Genetic mutations causing abnormalities either in activating or in regulating factors have been described. The diagnosis of the complement mediated MPGN requires a complete study of all these different complement factors. As a consequence, new therapeutic approaches are becoming available. Indeed, in addition to a nonspecifc treatment and to the immunosuppression that has the aim to block the auto antibodies production, the specific inhibition of complement activation is relatively new and may act either blocking the C5 convertase or the C3 convertase. The drugs acting on C3 convertase are still in different phases of clinical development and might represent drugs for the future. Overall the authors consider that one of the principal problems in fnding new types of drugs are both the rarity of the disease and the consequent poor interest in the marketing and the lack of large international cooperative studies.

Complement related kidney diseases:Recurrence after transplantation

The recurrence of renal disease after renal transplantation is becoming one of the main causes of graft loss afterkidney transplantation. This principally concerns some of the original diseases as the atypical hemolytic uremic syndrome(HUS), the membranoproliferative glomerulonephritis(MPGN), in particular the MPGN now called C3 glomerulopathy. Both this groups of renal diseases are characterized by congenital(genetic) or acquired(autoantibodies) modifications of the alternative pathway of complement. These abnormalities often remain after transplantation because they are constitutional and poorly influenced by the immunosuppression. This fact justifies the high recurrence rate of these diseases. Early diagnosis of recurrence is essential for an optimal therapeutically approach, whenever possible. Patients affected by end stage renal disease due to C3 glomerulopathies or to atypical HUS, may be transplanted with extreme caution. Living donor donation from relatives is not recommended because members of the same family may be affected by the same gene mutation. Different therapeutically approaches have been attempted either for recurrence prevention and treatment. The most promising approach is represented by complement inhibitors. Eculizumab, a monoclonal antibody against C5 convertase is the most promising drug, even if to date is not known how long the therapy should be continued and which are the best dosing. These facts face the high costs of the treatment. Eculizumab resistant patients have been described. They could benefit by a C3 convertase inhibitor, but this class of drugs is by now the object of randomized controlled trials.

DETECTION OF CYTOMEGALOVIRUS(CMV) IMMEDIATE EARLY ANTIGEN IN KIDNEY BIOPSIES AND TRANSPLANT NEPHRECTOMIES

Objective To investigate the relationship between CMV infection and renal allograft rejection. Methods 39 kidney biopsies and transplant nephrectomies were collected and investigated for CMV immediate early antigen by immunohistochemistry. Results In 14 out of 39 tissue specimens CMV immediate early antigen were found. 8 biopsies from normal donor kidneys were negative; only 1 (10%) in 10 tissue specimens with early stage acute rejection was positive; 5(55.6%) in 9 biopsies with late stage acute rejection and 8 (66.7%) in 12 tissue blocks with chronic rejection were positive. Compared with normal kidney tissues, the infections in tissues with early stage acute rejection didn't increase obviously, but increased obviously in kidney tissue specimens with late stage rejection and with chronic rejection (P< 0.05). Conclusion CMV infection appears to contribute to late stage acute rejection and chronic rejection after renal transplantation.

Is it time to give up with calcineurin inhibitors in kidney transplantation?

Calcineurin inhibitors(CNIs) represent today a cornerstone for the maintenance immunosuppressive treatment in solid organ transplantation. Nevertheless, several attempts have been made either to minimize their dosage or to avoid CNIs at all because these drugs have the severe side effect of chronic nephrotoxicity. This issue represents a frontier for renal transplantation. The principal problem is to understanding whether the poor outcome over the long-term may be ascribed to CNIs nephrotoxicity or to the inability of these drugs to control the acute and chronic rejection B cells mediated. The authors analyze extensively all the international trials attempting to withdraw, minimize or avoid the use of CNIs. Few trials undertaken in low risk patients with an early conversion from CNIs to proliferation signal inhibitors were successful, but the vast majority of trials failed to improve CNIs side effects. To date the use of a new drug, a co-stimulation blocker, seems promising in avoiding CNIs with similar efficacy, better glomerular filtration rate and an improved metabolic profile. Moreover the use of this drug is not associated with the development of donorspecific anti-human leukocyte antigen antibodies. Thispoint has a particular relevance, because the failure of CNIs to realize good outcomes in renal transplantation has recently ascribed to their inability to control the acute and chronic rejections B-cell mediated. This paper analyzes all the recent studies that have been done on this issue that represents the real frontier that should be overcome to realize better results over the long-term after transplantation.

Co-expression of sCD40LIg and CTLA4Ig mediated by adenovirus prolonged mouse skin allograft survival

Objective： To investigate the role of simultaneous blockade of CD40/CD40L and B7/CD28 pathways in the immune tolerance via co-expression of sCD40LIg and CTLA4Ig mediated by replication-defective adenovirus. Methods： Ad-sCD40LIg- IRES2-CTLA4Ig, replication-defective adenovirus co-expressing sCD40LIg and CTLA4Ig, was constructed and identified. The co-expression of sCD40LIg and CTLA4Ig was evaluated with confocal laser scanning microscope and Western blotting. Skin transplantations of C57BL/6 to BALB/c mice were performed. PBS, Ad-Shuttle-CMV and Ad-sCD40LIg-IRES2-CTLA4Ig were administered. Skin graft survival was monitored and the mRNA expression of both genes was evaluated in the skin allografts. Results： Ad-sCD40LIg-IRESE-CTLA4Ig was constructed successfully and identified. The co-expression of sCD40LIg and CTLA4Ig was identified with confocal laser scanning microscopy and Western blotting. Compared to the skin graft mean survival time （MST） of non-treated group （（5.75±0.71） d） or Ad-Shuttle-CMV-treated group （（5.50±0.53） d）, the skin graft MST was dramatically prolonged in the Ad-sCD40LIg-IRESE-CTLA4Ig-treated group （（ 16.38± 1.19） d, P〈0.001）. The mRNA expression of both genes was detected. Conclusion： Ad-sCD40LIg-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding sCD40LIg and CTLA4Ig, was constructed. Simultaneous blockade of CD40/CD40L and B7/CD28 costimulatory pathway mediated by replication-defective adenovirus significantly prolonged skin allografi survival in mice.