Palmitate induces apoptosis and endoplasmic reticulum stress in human umbilical cord-derived mesenchymal stem cells / 生理学报

The saturated free fatty acid (FFA), palmitate, could induce apoptosis in various cell types, but little is known about its effects on human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). Here, we investigated whether palmitate induced apoptosis and endoplasmic reticulum (ER) stress in hUC-MSCs. hUC-MSCs were stained by labeled antibodies and identified by flow cytometry. After administration with palmitate, apoptotic cell was assessed by flow cytometry using the Annexin V-FITC/7-AAD apoptosis detection kit. Relative spliced XBP1 levels were analyzed using semi-quantitative RT-PCR. The mRNA of BiP, GRP94, ATF4 and CHOP were analyzed by real-time PCR. Relative BiP and CHOP protein were analyzed using Western blot analysis. The results showed that hUC-MSCs were homogeneously positive for MSC markers; palmitate increased apoptosis of hUC-MSCs and activated XBP1 splicing, BiP, GRP94, ATF4 and CHOP transcription. These findings suggest that palmitate induces apoptosis and ER stress in hUC-MSCs.

Effect of CYP3A4*18B, CYP3A5*3 gene polymorphism on dosage and concentration of tacrolimus in renal transplant patients / 药学学报

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).

Is islet transplantation ready for widespread use in diabetes? / 中华医学杂志(英文版)

Up till 2000 when Edmonton group introduced islet transplant procedure in conjunction with a novel glucocorticoid-free immunosuppressive regimen rendering 100% (n=7) of patients with type 1 diabetes insulin-independent for at least 1 year, islet transplant was taken into the clinic. Although significant progress in clinical islet transplant has occurred during recent years, challenges remain, including shortage of available donor organs, technical aspects of islet preparation and transplantation, immunological rejection post-transplant, unclear long-term outcomes of islet transplantation. Special attention is given to current limitation in islet transplantation together with new possible strategies that raise expectations for the widespread use of islet transplantation in the future.

Alemtuzumab induction therapy in highly sensitized kidney transplant recipients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Immunosuppression for immunologically high-risk kidney transplant patients usually involves antithymocyte globulin induction with triple drug maintenance therapy. Alemtuzumab, a humanized anti-CD52 antibody, was expected to be a promising induction therapy agent for kidney transplantation. However, currently no consensus is available about its efficacy and safety. This study aimed to evaluate the efficacy and safety of alemtuzumab as immune induction therapy in highly sensitized kidney transplant recipients.</p><p><b>METHODS</b>In this prospective, open-label, randomized, controlled trial, we enrolled 23 highly immunological risk patients (panel reactive antibody > 20%). They were divided into two groups: alemtuzumab group (trial group) and anti-thymocyte globulin (ATG) group (control group). Patients in the alemtuzumab group received intravenous alemtuzumab (15 mg) as a single dose before reperfusion. At the 24th hour post-operation, another dosage of alemtuzumab (15 mg) was given. The control group received a bolus of rabbit ATG (9 mg/kg), which was given 2 hours before kidney transplantation and lasted until the removal of vascular clamps when the anastomoses were completed. Maintenance immunosuppression in both groups comprised standard triple therapy consisting of tacrolimus, prednisone, and mycophenolate mofetil (MMF). Acute rejection (AR) and infection episodes were recorded, and kidney function was monitored during a 2-year follow-up. χ(2) test, t test and Kaplan-Meier analysis were performed with SPSS17.0 software.</p><p><b>RESULTS</b>Median follow-up was 338 days. In both the alemtuzumab group and ATG group, creatinine and blood urea nitrogen values in surviving recipients were similar (P > 0.05). White blood cell counts were significantly reduced in the alemtuzumab group for the most time points up to 6 months (P < 0.05). One patient receiving alemtuzumab died for acute myocardial infarction at the 65th day post-operation. Two ATG patients died for severe pulmonary infection or cardiac and pulmonary failure. Cumulative 2-year graft survival rate was 90.9% in the alemtuzumab group and 81.8% in ATG group (P > 0.05) respectively. There was one graft failure in the alemtuzumab group and two graft failures in ATG group, with all graft failures at tributed to rejection episodes. The alemtuzumab group had a 2-year cumulative freedom from rejection rate of 81.8%, compared with 72.7% for the ATG group (P > 0.05).</p><p><b>CONCLUSION</b>Alemtuzumab induction therapy for highly sensitized kidney transplant recipients is an effective and safe protocol yielding an acceptable acute rejection rate.</p>

Comparison of biologic activity of two anti-PSA/anti-CD3 bispecific single-chain antibodies / 中华男科学杂志

<p><b>OBJECTIVE</b>To study and compare the biologic activity of two anti-PSA/anti-CD3 bispecific single-chain antibodies.</p><p><b>METHODS</b>Flow cytometry (FCM) was used to detect the binding activity of two antibodies to CD3-positive cell line Jurkat and prostate carcinoma cell line LNCaP. The effect of the two antibodies in mediating tumor cell lysis in vitro was determined by using the 51Cr-release test. For in vivo evaluation of the two antibodies activity, a nude mouse model was used. The mice were inoculated with LNCaP prostate cancer cells.</p><p><b>RESULTS</b>FCM showed that both the antibodies could bind Jurkat and LNCaP cells with high specificity. The percentages of the cells bond by the bispecific single-chain antibodies were 56.3% and 55.4%, and those by the multivalent antibodies were 74.0% and 83.0% respectively. Both the antibodies mediated a specific lysis of LNCaP cells in vitro, with activated CTLs as effector cells, and significantly reduced tumor growth of nude mice in vivo as compared with the untreated controls and the group treated with CTLs only (P <0.05). The experiment also showed that the multivalent antibody had a better activity than the bispecific antibody in binding antigens, mediating lysis of LNCaP cells and reducing tumor growth (P < 0.05).</p><p><b>CONCLUSION</b>Both the anti-PSA/anti-CD3 bispecific single-chain antibody and multivalent antibody have good biologic activity, and the formation of the tetramerization of single-chain antibody can improve its biologic activity.</p>

Analysis of HLA-DRB1 allele polymorphism in the Fujian Han nationality population of China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the HLA-DRB1 allele polymorphism in Fujian Han nationality population.</p><p><b>METHODS</b>Six hundred and twenty individual samples collected from unrelated Fujian Han population were subjected to genotyping using oligonucleotide microarray technique. And the allele frequencies of HLA-DRB1 were calculated and compared with other populations.</p><p><b>RESULTS</b>Fourteen HLA-DRB1 alleles of Fujian Han population were detected. The gene frequencies ordered from high to low were HLA-DRB1*9, 12, 15, 4, 8 respectively.</p><p><b>CONCLUSION</b>The HLA-DRB1 distribution of Fujian Han population shares some genetic characters with southern Chinese Han populations, but these characters differ from northern Chinese populations.</p>

Prolongation of survival time of allogenous skin graft after transfection of Molluscum contagiosum virus 148 gene in rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the immunosuppressive effect of local transfection of Molluscum contagiosum virus 148 (MC148) gene to allogenous skin graft against rejection.</p><p><b>METHODS</b>MC148 gene was cloned from molluscum contagiosum virus (MCV), and was employed to construct recombinant adenovirus vector (Ad-MC148). The recombinant Ad-MC148 was then locally transfected into a part of the tail skin of eight Lewis rats, which served as skin donors for grafting. The wounds (1 cm x 1 cm) were produced on the tails of 16 Wistar rats, and they were then randomly divided into control (C, n=8, with grafting of skin from donor rats without transfection), and transfection (T, n=8, with grafting of skin from donor rats with transfection of the recombinant Ad-MC148) groups. The expression of MC148 mRNA gene in T group was detected on 6 post operation hour( POH) and 2, 3, 7 and 10 post operation day (POD), and the results were expressed by the ratio of absorption value (A) between MC148 gene and beta-actin. The survival time of skin grafts in both groups was compared. Gross examination of grafted skin was carried out from 7 POD on in both groups, and the pathomorphological changes were examined in both groups on 7 POD.</p><p><b>RESULTS</b>The MC148 gene expression in rat skin of T group could be identified in 6 POH, and it reached the peak on 3 POD (A(MC148 mRNA) / A(beta_actin) = 0.86), and then subsided thereafter, but it maintained for 10 days. The survival time of the grafts in T group was (15.0 +/- 2.0) days, and it was significantly longer than that in C group (8.5 +/- 3.4) days, (P < 0.01). Gross and microscopic examination showed that the tail skin of T group appeared ruddy on 7 POD, with little leukocytic infiltration in subcutaneous tissue; it began to turn black after 12 to 20 PODs. On the other hand, the tail skin of C group began to turn black and to shed off on 7 POD, with evident leukocytic infiltration in subcutaneous tissue and dermis.</p><p><b>CONCLUSION</b>Local transfection of MC148 gene may promote immunosuppression by inhibiting leukocytic infiltration after allogenous skin transplantation.</p>

Protection of human islets from induction of apoptosis and improved islet function with HO-1 gene transduction / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Islet transplantation represents an ideal therapeutic approach for treatment of type 1 diabetes but islet function and regeneration may be influenced by necrosis or apoptosis induced by oxidative stress and other insults. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide. It has also been reported to be an antioxidant enzyme which can improve the function of grafted islets by cytoprotection via free radical scavenging and apoptosis prevention. In the present study, we investigated whether transduction of HO-1 genes into human islets with an adenovirus vector has cytoprotective action on islets cultured in vitro and discuss this method of gene therapy for clinical islet transplantation.</p><p><b>METHODS</b>Cadaveric pancreatic islets were isolated and purified in vitro. Transduction efficiency of islets was determined by infecting islets with adenovirus vector containing the enhanced green fluorescent protein gene (Ad-EGFP) at multiplicities of infection (MOI) of 2, 5, 10, or 20. Newly isolated islets were divided into three groups: EGFP group, islets transduced with Ad-EGFP using MOI = 20; HO-1 group, transduced with adenovirus vectors containing the human HO-1 gene using MOI = 20; and control group, mock transduced islets. Insulin release after glucose stimulation of the cell lines was determined by a radioimmunoassay kit and the stimulation index was calculated. Flow cytometry was used to detect apoptotic cells in the HO-1 group and in the control group after induction by recombinant human tumor necrosis factor-alpha (rTNFalpha) and cycloheximide (CHX) for 48 hours.</p><p><b>RESULTS</b>Adenovirus vectors have a high efficiency of gene transduction into adult islet cells. Transduction of islets with the Ad-EGFP was most successful at MOI 20, at which MOI fluorescence was very intense on day 7 after transduction and EGFP was expressed in cultured islet cells for more than four weeks in vitro. The insulin release in the control group was (182.36 +/- 58.96) mIU/L after stimulation by high glucose media (16.7 mmol/L), while insulin release from the HO-1 group and the EGFP group were (270.09 +/- 89.37) mIU/L and (175.95 +/- 75.05) mIU/L respectively. Compared to the control group and the EGFP group, insulin release in the HO-1 group increased significantly (P < 0.05). After treatment with rTNFalpha and CHX the apoptotic ratio of islet cells was (63.09 +/- 10.86)% in the HO-1 group, significantly lower than (90.86 +/- 11.25)% in the control group (P < 0.05).</p><p><b>CONCLUSIONS</b>Transduction of human islets with Ad-HO-1 can protect against TNF-alpha and CHX mediated cytotoxicity. The HO-1 gene also appears to facilitate insulin release from human islets. Transduction of donor islets with the adenovirus vector containing an HO-1 gene might have potential value in clinical islet transplantation.</p>

Preoperative single-bolus high-dose antithymocyte globulin as induction therapy in sensitized renal transplant recipients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Immunological sensitization remains a major problem following renal transplantation. There is no consensus for the management of sensitized renal allograft recipients. The patients become tethered to dialysis while waiting for compatible donors. This study was designed to evaluate the efficacy and safety of preoperative single-bolus high-dose antithymocyte globulin (ATG) as induction therapy in sensitized renal transplant recipients.</p><p><b>METHODS</b>A total of 56 patients were divided into two groups according to the level of panel reactive antibody (PRA): non-sensitized group (PRA < 10%, n = 30) and sensitized group (PRA > or = 10%, n = 26). The characteristics of the recipients and donors were comparable between the two groups. Mycophenolate mofetil (MMF, 1 g) or ATG (iv. 9 mg/kg) were given preoperatively in the two groups as induction therapy. After the transplantation, the patients were treated with standard triple therapy regimen consisting of tacrolimus (FK-506) or cyclosporine A, MMF, and prednisolone. Acute rejection (AR) and infection episodes were recorded and renal function was monitored during a 12-month follow-up. Chi(2) test and t test were used to analyze the data.</p><p><b>RESULTS</b>During the follow-up, 6 patients (20.0%) suffered AR episodes in the non-sensitized group and 4 (15.4%) in the sensitized group (P = 0.737); 8 patients (26.7%) experienced 11 infection episodes (average, 1.4 episodes per infected patient) in the non-sensitized group, and 6 (23.1%) experienced 10 infection episodes (average, 1.7 episodes per infected patient) in the sensitized group (P = 0.757, 0.890). The safety of the drugs, which was assessed by the occurrence of side effects, was comparable between the two groups. The hospital stay was 13 - 25 days (mean, 16.7 +/- 3.3) in the non-sensitized group and 14 - 29 days (mean, 16.2 +/- 3.1) in the sensitized group, respectively (P = 0.563). No delayed graft function (DGF) was observed in all the patients. Both the 12-month actuarial patient and graft survival rates were 100% in the two groups.</p><p><b>CONCLUSION</b>Preoperative single-bolus high-dose ATG is an effective and safe induction therapy yielding acceptable acute rejection rate in sensitized renal transplant recipients.</p>

The effect of HLA matching on patient/graft survival rate of renal transplant recipients positive for PRA / 中华器官移植杂志

Objective To elucidate the effect of human leukocyte antigen(HLA)cross-reactive groups(CREGs)matching on the patient/graft survival rate of renal transplant recipients positive for penal-reactive antibody(PRA).Methods The PRA level and the HLA antibody specificity of renal transplant candidates were assayed using the LAT1240,LM720R,and SSP2LB kits.The appropriate donors were selected according to the CREGs matching standard.Results 112 recipients included 43 positive cases of HLA classⅠantibody,39 positive cases of HLA classⅡantibody,and 30 positive cases of bothⅠandⅡ.The HLA mismatching cases of 0,1,2,3,4 and 5 allele(s)were 6,39,38, 21,7 and 1,respectively.Hyper-acute rejection(2 cases),acute rejection(18 cases),chronic rejec- tion(5 cases),delayed graft function(4 cases),graft excision(one case)due to rejection and deaths (13 cases,5 with a functioning graft)occurred after transplant.Ninety-nine recipients and 96 grafts survived.The 1-,3-,and 5-year graft survival rate was 91.96%,86.96%,and 86.21%,respec- tively.Conclusion The HLA matching rate of patient/graft was elevated significantly and the effect of high PRA level was decreased according to the principle of CREGs,which increased the patient/graft survival rate remarkably in sensitized recipients.

Relationship between cytokine gene polymorphisms and chronic rejection in kidney allograft recipients and donors / 中华器官移植杂志

Objective To study the relationship between cytokine gene polymorphisms and chronic rejection in kidney allograft recipients and their donors.Methods The cytokine genotypes in- cluding TNF-?,IL-6,IL-10,TGF-?_1 and IFN-?in 144 consecutive first cadaveric kidney allograft re- cipients and 65 corresponding donors were detected by PCR-SSP method.The recipients were followed up for 5 years.The relationship between the TGF-?_1 genotypes of the recipients and donors and the chronic renal allograft rejection was analyzed.Results The incidence of chronic rejection in the recipi- ents with TGF-?_1 high producer genotype(39/91,42.9%)was significantly higher than in those with TGF-?_1 middle or low producer genotype(9/53,17.0%,P0.05).The incidence of chronic rejection in the recipients and donors with TGF-?_1 high producer genotype(4/4)was significantly higher than in those with other genotypes(31/110,28.2%,P

Renal transplantation in children, report of 21 cases / 中华儿科杂志

<p><b>OBJECTIVE</b>The survival rate of cadaveric renal transplant in children has been improved following the development of transplantation technology and the application of immunosuppressive agents. In this study, the prognosis of renal transplantation, operative procedure and immunosuppressive agents administration in 21 children with end-stage renal disease (ESRD) were analyzed.</p><p><b>METHODS</b>From January 1985 to December 2001, 21 patients (9 males and 12 females with a mean age of 14 +/- 2 yr, mean body weight of 33.4 kg and mean height of 136.5 cm) received renal transplantation because of ESRD were enrolled in the study. The patients with an average GFR of 8.28 ml/min were managed with dialysis for 13.4 months in average pro-transplantation. All cadaveric kidneys were from adults, which included 1 donor with one HLA mismatch, 3 with two mismatches, 5 with three mismatches and 4 with four mismatches. All transplantations were performed with anastomoses of the adults' renal artery and vein to the children's iliac externa artery and iliac externa vein. Biological inducement therapy was given in 4 cases. At the first 3 - 5 days post-transplantation, methylprednisolone was administered [7 mg/(kg.d)]. All patients received baseline diploid or triple immunosuppression therapy of cyclosporin A [6 - 8 mg/(kg.d)] or FK506 [0.18 - 0.25 mg/(kg.d)], mycophenolate mofetil [MMF 10 - 15 mg/(kg.d)] or azathioprine [1 - 3 mg/(kg.d)] and prednisone [0.4 - 0.6 mg/(kg.d)]. High-dose methylprednisolone [10 mg/(kg.d)] was administered to control the acute rejection.</p><p><b>RESULTS</b>The renal function of patients was restored 5.6 days in average after transplantations. The 1st, 3rd and 5th year survival rates of recipient/graft were 95.2%/95.2%, 86.7%/73.3% and 72.7%/63.6%, respectively. One case had super-acute renal rejection, 5 cases had acute rejection, 3 cases had delayed graft function and 3 patients died. The longest survival time was 12 years. The major complications included hypertension (47.6%), diabetes (19.4%), infection (19.4%) and drug-induced hepatic injury (14.2%). Catch-up growth was seen in most of the pediatric recipients.</p><p><b>CONCLUSION</b>Renal transplantation is the most ideal method to treat children with ESRD, and the growth of the pediatric patients will be improved after transplantation. Adult donor kidneys adapt to the school age patient. And the protocol of immunosuppressive therapy (prednisone plus MMF and FK506) should be applied.</p>

Development of oligoneucleotide arrays for HLA-DR53-associated group genotyping / 中华外科杂志

<p><b>OBJECTIVE</b>To develop a method performed on an oligonucleotide array for HLA-DR53 group genotyping.</p><p><b>METHODS</b>According to the specific allelic frequency and sequence of HLA-DRB loci in Chinese Han population, HLA-DR53 group typing probes which were immobilized on a glass supports were synthesized. A pair of group-special primers labeled by the Cy5-dCTP were designed, and the primers were used in the PCR, thus the PCR products were labeled with Cy5. The labeled PCR products were hybridized with array. The signals were scanned by scanner and analyzed by image software. The typing results were confirmed by standard DNA and PCR-SSO. One hundred and eleven samples were typed by this array.</p><p><b>RESULTS</b>There were 72 HLA-DR53 group loci typed by oligonucleotide array. Among them, 34 loci were DR9, 25 were DR4, and 13 were DR7. No false positive or false negative typing results were observed. The specificity and reproducibility were 100% and the overall time of genotyping was 5 hours.</p><p><b>CONCLUSION</b>The oligonucleotide array technique is a precise, rapid molecular method for HLA-DR53 genotyping, suited for clinical practice.</p>

Expression of fractalkine and its receptor in acute cardiac allografts rejection / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of fractalkine (FKN) and its receptor CX3CR1 in cardiac allografts and the effect of Cyclosporin A (CsA).</p><p><b>METHODS</b>Three groups of rats underwent heterotopic cardiac transplantation, 45 cases in each group and 5 cases in control group: SD to SD regarded as isograft group (group A), Wistar to SD divided into CsA untreated allograft group (group B) and CsA treated allograft group (group C), normal SD rats as control group. The FKN mRNA expression was detected by one-step RT-PCR method and the expression of FKN and CX3CR1 protein was detected by standard ABC immunohistochemical technique.</p><p><b>RESULTS</b>The expression of FKN mRNA and protein was weak in both isografts and normal heart specimens. The changes of FKN mRNA expression were correlated with the process of acute allograft rejection. The peak of FKN mRNA expression (0.8 +/- 0.26) appeared on the seventh day after transplantation, which could be down-regulated by CsA significantly (t = 2.390, P < 0.05). FKN protein was located in endothelia cells and its receptor CX3CR1 was located in infiltrating mononuclear cells in allografts.</p><p><b>CONCLUSIONS</b>Upregulation of FKN and its receptor was significantly correlated with the trafficking of mononuclear cells which play an important role in acute allograft rejection. It may be one of the important mechanisms of CsA to intervene the acute rejection by inhibiting the activation of the FKN-CX3CR1 pathway.</p>