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Objective:Currently,patients with pre-exsiting donor-specific antibody(DSA)are prone to antibody-mediated rejection(AMR)after surgery and are at a relatively high risk of postoperative complications and graft failure.The risk of postoperative complications and graft failure is relatively high.This study aims to discuss the clinical outcome of DSA-positive kidney transplantation and analyze the role and safety of preoperative pretreatment in DSA-positive kidney transplantation,providing single-center treatment experience for DSA-positive kidney transplantation. Methods:We retrospectively analyzed the clinical data of 15 DSA-positive kidney transplants in the Department of Renal Transplantation of First Affiliated Hospital of Zhengzhou University from August 2017 to July 2022.Eight cases were organ donation after citizen's death(DCD)kidney transplant recipients,of which 3 cases in the early stage were not treated with preoperative desensitisation therapy(DCD untreated group,n=3),and 5 recipients were treated with preoperative rituximab desensitisation(DCD preprocessing group,n=5).The remaining 7 cases were living related donors recipients(LRD)who received preoperative desensitisation treatment with rituximab and plasma exchange(LRD preprocessing group,n=7).We observed and recorded the incidence of complications with changes in renal function and DSA levels in the recipients and the survival of the recipients and transplanted kidneys at 1,3 and 5 years,and to compare the differences in recovery and postoperative complications between 3 groups. Results:All 15 recipients were positive for preoperative panel reactive antibody(PRA)and DSA and were treated with methylprednisolone+rabbit anti-human thymocyte immunoglobulin induction before kidney transplantation.DCD untreated group all suffered from DSA level rebound,delayed renal graft function(DGF)and rejection reaction after surgery.After the combined treatment,DSA level was reduced and the graft renal function returned to normal.The DCD preprocessing group were all without antibody rebound,1 recipient developed DGF and the renal function returned to normal after plasmapheresis,and the remaining 4 recipients recovered their renal function to normal within 2 weeks after the operation.In the LRD preprocessing group,2 cases had antibody rebound and 1 case had rejection,but all of them recovered to normal after treatment,and DSA was maintained at a low level or even disappeared.The incidence of DGF and rejection in the DCD untreated group were significantly higher than that in the DCD preprocessing group and the LRD preprocessing group;and there were no significant difference in the incidence of postoperative haematuria,proteinuria,bacterial and fungal infections,and BK virus infection between the 3 groups(all P>0.05).A total of 11 of the 15 recipients were followed up for more than 1 year,6 for more than 3 years,and 1 for more than 5 years,and the survival rates of both the recipients and the transplanted kidneys were 100%. Conclusion:Effective preoperative pretreatment with desensitization therapy can effectively prevent antibody rebound in DSA-positive kidney transplantation and reduce perioperative complications.
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Objective:To investigate the role of nuclear transcription factor Gli1/Gli2 of the sonic hedgehog (Shh) signaling pathway in the hepatic epithelial mesenchymal transition (EMT) of biliary atresia mice caused by Rhesus rotavirus (RRV) infection.Methods:The biliary atresia model in mice was generated by RRV infection.Mice were divided into normal group, model group, Gli1 overexpression group, Gli1 shRNA group, Gli2 overexpression group and Gli2 shRNA group.Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the mRNA and protein expressions of regulatory factors for EMT (Snail/Slug) and characteristic cytokines of EMT [Vimentin, α-smooth muscle actin(α-SMA), E-cadherin] in mouse liver tissues.Additionally, hematoxylin-eosin staining and Masson staining were performed to calculate the percentage of liver fibrous tissue expression area.The data were analyzed by One- Way ANOVA and LSD- t test. Results:The relative mRNA expression of Snail, Slug, Vimentin, α-SMA and E-cadherin in Gli2 overexpression group, Gli2 shRNA group and model group were 15.13±3.40, 5.48±0.46, 8.78±1.06, 12.40±2.18 and 3.06±0.53; 3.73±1.16, 5.62±1.75, 3.56±1.06, 3.88±1.16 and 10.51±1.83; 8.13±1.27, 5.32±0.98, 5.05±0.98, 4.02±0.77 and 5.12±1.60.Compared with those of the model group, mRNA levels of Snail, Vimentin and α-SMA were significantly higher in Gli2 overexpression group, while that of E-cadherin was significantly lower( t=4.53, 5.29, 8.12, -2.13; all P<0.05); compared with those of the model group, mRNA levels of Snail and Vimentin in Gli2 shRNA group significantly decreased, while that of E-cadherin significantly increased( t=-2.86, -2.12, 5.62; all P<0.05). In Gli2 overexpression group, Gli2 shRNA group and model group, the protein levels of Snail, Slug, Vimentin, α-SMA and E-cadherin were 2.02±0.39, 0.31±0.08, 0.95±0.17, 1.07±0.17 and 0.42±0.06; 0.53±0.13, 0.40±0.18, 0.20±0.04, 0.28±0.07 and 1.09±0.31; 0.70±0.15, 0.42±0.22, 0.64±0.13, 0.81±0.11 and 0.42±0.09.Compared with those of the model group, protein levels of Snail, Vimentin and α-SMA were significantly higher in Gli2 overexpression group( t=12.71, 4.28, 3.70; all P<0.05); compared with those of the model group, protein levels of Vimentin and α-SMA in Gli2 shRNA group significantly decreased, while that of E-cadherin significantly increased( t=-6.14, -7.57, 5.96; all P<0.05). However, no significant change trend were detected in expression levels of characteristic cytokines of EMT between Gli1 overexpression group and Gli1 shRNA group.The area percentage of liver fiber expression in normal group, model group, Gli1 overexpression group, Gli1 shRNA group, Gli2 overexpression group and Gli2 shRNA group were (1.03±0.58)%, (33.02±11.39)%, (39.81±5.67)%, (26.06±1.29)%, (49.81±8.57)% and (17.55±0.66)%, respectively.Besides, in terms of percentage of area expressed in liver fiber tissue, the Gli2 overexpression group and Gli2 shRNA group were statistically significant compared with the model group( t=3.21, -2.96; all P<0.05), while the Gli1 overexpression group and Gli1 shRNA group were not statistically significant compared with the model group (all P>0.05). Conclusions:The Shh signaling pathway plays an important role in liver fibrosis in mice with biliary atresia.Gli2, a key transcription factor of Shh signaling pathway, can significantly regulate liver EMT process in mice with biliary atresi.
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Objective:To explore the clinical efficacy of vascular interventional therapy in children with transplantation renal artery stenosis(TRAS).Methods:From January 2013 to September 2021, retrospective analysis was performed for clinical data of 238 TRAS children.Peak systolic velocity(PSV)of transplant renal artery, interlobular artery PSV, transplant renal artery PSV/ interlobular artery PSV(post PSV ratio)and serum creatinine level before and after vascular interventional therapy and at the last follow-up were compared.Results:Six pediatric kidney transplantation recipients were diagnosed as TRAS.The median operative age was 12(9-17)years, the median postoperative time to diagnosing TRAS 4(1.7-18.0)months and the median follow-up period 6.6(2.5-8.0)years.All of them received vascular interventional therapy of percutaneous transluminal angioplasty(PTA, n=5)and stent angioplasty( n=1). The serum creatinine pre-treatment with vascular interventional therapy was significantly higher than baseline serum creatinine level at discharge(200.8±88.5)vs(75.2±27.9)μmol/L, P=0.025 and decreased to(103.8±44.7)μmol/L at Month 1 post-treatment( P=0.196)and(98.7±30.2)μmol/L at the last follow-up( P=0.115). Comparing with internal diameter of grafted renal artery anastomosis site(2.6±0.6 mm)pre-treatment with vascular interventional therapy, significant changes occurred at 24 h post-treatment(3.8±0.5 mm)and at the last follow-up(4.1±0.8 mm)(all P=0.027). In addition, PSV and post PSV ratio of transplanted renal artery at 24 h post-treatment(163±45.0 cm/s, 6.5±2.2)and at the last follow-up(184.7±80.8 cm/s, 5.4±2.0)were significantly lower than that before vascular interventional therapy(356.5±77.9 cm/s, 18.0±5.8)and interlobular artery PSV was significantly higher than that before vascular interventional therapy( P=0.024, P=0.032, respectively). During follow-ups, no restenosis or thrombosis occurred in transplanted renal arteries. Conclusions:PTA or stent angioplasty for TRAS children is technically feasible with low restenosis rate and relatively satisfactory mid/long-term outcomes.
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Objective:To retrospectively analyze clinical data of infant donors with body weight ≤15 kg into children recipients, and to investigate the efficacy and complications under the strategy of pediatric donor to pediatric recipient (PTP) of pediatric kidney transplantation allocation.Methods:Clinical data of kidney transplantation for children with infant donors performed in the First Affiliated Hospital of Zhengzhou University from August 2010 to December 2019 were collected.Clinical data of donors and recipients, postoperative adverse events, postoperative renal recovery, and human and renal survival were analyzed.Results:A total of 50 infant donors and 93 pediatric recipients were enrolled in this study.Recipients included 89 patients with single kidney transplantation (SKT) and 4 with en-bloc kidney transplantation (EBKT). The major perioperative complications were delayed graft function (DGF) (5 cases, 5.4%) and vascular thrombosis (VT) (3 cases, 3.2%), followed by recurrence of primary nephropathy (3 cases, 3.2%), respiratory tract infection (3 cases, 3.2%), and acute rejection (AR) (2 cases, 2.2%). During the follow-up period, the main cause of death was respiratory tract infection (4 cases, 4.3%). Except for the cause of death, the main causes of graft loss were rejection (2 cases, 2.2%) and recurrence of primary kidney disease (2 cases, 2.2%). Serum creatinine decreased progressively from (824.77±150.24) μmol/L preoperatively to (90.73±47.24) μmol/L 1 month postoperatively.In SKT group, the median follow-up time was 31 months (3-74 months), and the survival rates of recipients and transplanted kidneys at 1, 3 and 5 years postoperatively were 97.5%/94.2%, 96%/88.8% and 93.1%/86.1%, respectively.In EBKT group, the median follow-up time was 50 months (13-65 months), and the survival rates of recipients and transplanted kidneys at 1, 3 and 5 years postoperatively were all 100.0%.During the fo-llow-up period, there was no significant difference in the human/kidney survival rate between groups (all P>0.05), and well acceptable transplantation outcomes were obtained. Conclusions:Single/double kidney transplantation for children and adolescent recipients from infant donors in the First Affiliated Hospital of Zhengzhou University has achieved acceptable outcomes.Adopted by the PTP strategy, the incidence of complications after kidney transplantation does not increase, indicating its safety and reliability.
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Objective:To compare the efficacy of single kidney transplantation for children from pediatric donors between body weight ≤15 kg and >15 kg.Methods:A retrospective review in 156 children with single donor kidney transplantation from August 2010 to December 2019 in the Kidney Transplantation Department of the First Affiliated Hospital of Zhengzhou University was conducted. The patients were classified into the small kidney group (pediatric donor body weight ≤15 kg) and the big kidney group (pediatric donor body weight >15 kg). In this study, 89 cases were concluded in the small kidney group and 67 cases were concluded in the big kidney group. The donor kidneys were obtained from 46 cases of small weight (≤15 kg) pediatric donors and 48 cases of large weight (>15 kg) pediatric donors. There were significant differences in age [1.00 (0.02 - 4.00) years vs. 10.00 (3.00-18.00) years], body weight [10.0 (3.4 - 15.0) kg vs. 35.0 (16.2- 35.0) kg], height [76 (50- 113) cm vs. 144 (67-172) cm], GFR [(31.50±7.46)ml/min vs. (36.79±7.00) ml/min], and renal length to diameter [(5.91±0.48) cm vs. (8.71±1.88) cm] between the small kidney group and the big kidney group ( P < 0.01). There was no significant difference between the two groups of donors in gender, cold/warm ischemia time and cause of death ( P>0.05). There were significant differences in age [(11.28±3.89) years vs. (13.86±3.56) years], body weight [(31.83±10.45)kg vs. (35.13±9.15) kg], and height [(130.02±28.56) cm vs. (143.97±16.59) cm] between recipients of the small kidney group and big kidney group ( P < 0.05). While there were no significant differences in preoperative serum creatinine level [(822.65 ± 135.04) μmol/L vs. (777.31 ± 165.40) μmol/L], HLA mismatch [(3.4 ± 1.4) site vs. (3.2±1.3) site], and primary disease between the two groups ( P > 0.05). The recovery of renal function, postoperative adverse events, postoperative children, and graft survival were compared between the two groups. Results:The renal function of the two groups of recipients returned to normal 3 months after operation. The perioperative complications in the small kidney group and the big kidney group mainly included renal delayed recovery [5.6% (5/89) vs. 7.5% (5/67), P=0.89], renal vascular embolization [3.4% (3/89) vs. 0, P=0.35], and acute rejection [2.2% (2/89) vs. 4.3% (3/67) , P=0.75]. The main cause of recipient death during the follow-up period was pulmonary infection [4.5% (4/89) vs. 6.0% (4/67) , P=0.68]. The postoperative small kidney group was followed up for an average of 30 (3-74) months. The survival rates of children in the small kidney group at the 1, 3 and 5 years after surgery were 96.6% (86/89), 91.0% (81/89) and 91.0%(81/89), while the transplanted renal survival rates were 92.1% (82/89), 86.5% (77/89) and 84.2% (75/89), respectively. The postoperative big kidney group was followed up for an average of 32 (4-89 ) months. The survival rates of children in the big kidney group were 95.5% (64/67), 94.0% (63/67) and 91.0%(61/67) in the first 1, 3 and 5 years postoperatively, while the graft survival rates were 92.5% (62/67), 83.6% (56/67) and 83.6% (56/67), respectively. The postoperative kidneys of two groups were fast-growing, and there was no significant difference between the small kidney group and the big kidney group in graft length to diameter [(9.63±0.31) cm vs. (9.75±0.71) cm] after 1 year ( P>0.05). Conclusions:The effect of single pediatric kidney transplantation for pediatric donor with body weight ≤15 kg is equivalent to that for pediatric donor with body weight >15 kg , which can be carried out clinically.
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Objective:To evaluate the time-zero biopsy of donor kidney with acute kidney injury(AKI)in organ donation donors and examine the clinical effect after transplantation.Methods:From May 2019 to May 2020, clinical data were retrospectively reviewed for 104 donors assessed by time-zero biopsy at First Affiliated Hospital, Zhengzhou University.According to the definition of AKI and Banff2016 criteria, the kidneys of 104 donors were grouped and evaluated for transplantation.And the post-transplantation effects of donor kidneys with different degrees of pathological changes were analyzed.Results:AKI occurred in 32/104 donors.Compared with non-AKI donors, statistically significant differences existed in degrees of renal interstitial fibrosis and acute renal tubular injury ( P<0.05). However, there were no significant differences in other pathological manifestations ( P>0.05). In AKI group, kidneys of 2 donors with Banff score>3 were abandoned; in non-AKI group, among 12 donors with Banff score>3, 1 donor kidney was abandoned due to a high degree of chronic diseases.No significant inter-group difference existed in creatinine value or estimated glomerular filtration rate(eGFR)( P>0.05). AKI group had a higher incidence of postoperative delayed graft function(DGF)and longer duration.There was no statistical significance in other complications ( P>0.05). Conclusions:AKI donor kidneys with pathological manifestations below moderate renal tubular injury and Banff score<3 are feasible for transplantation.Although renal function recovery is slow after transplantation, safe outcomes may be obtained.
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Objective:To explore the efficacies of single-center pediatric transplantations and discuss the current problems.Methods:From July 2007 to September 2019, the clinical data of 202 children (aged ≤17 years) undergoing renal transplantation were reviewed. And their perioperative complications, transplantation outcomes and patient/kidney survival were analyzed.Results:The most common perioperative complication was delayed graft function (DGF)( n=24, 11.9%), recurrence of renopathy ( n=8, 4.0%) and acute rejection ( n=21, 10.4%). The major causes of death and graft failure were lung infection ( n=9, 4.5%) and rejection ( n=11, 5.4%). Perioperative serum creatinine decreased progressively from (816.1±303.1) μmol/L preoperatively to (62.7±20.6) μmol/L at Month 3 post-operation. The value of eGFR were (166.8±37.3), (135.1±29.0) and (109.9±31.1) ml/(min·1.73 m 2) at Year 1/3/5 post-operation respectively. The survival rates were 96.7%, 96.3%, 94.1%, 93.5%, 94.1% and 90.7% at Year 1/3/5 post-operation respectively. No difference existed in human/kidney survival rate between LD and DD groups at Year 1/3/5 post-operation ( P>0.05) and transplantation outcomes were excellent. Conclusions:Effective and successful outcomes have been achieved at our center. And further optimizations are required for resolving various problems.
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Objective To investigate the clinical significance of serum and urinary levels of neutrophil gelatinase-associated lipocalin (NGAL ) for evaluating changes of residual renal function after living donor kidney resection under different operation model in young versus elderly patients. Methods The clinical data of renal transplants were retrospectively analyzed by successfully using 66 living-related donors at the First Affiliated Hospital of Zhengzhou University from September 2016 to October 2017. According to the operation model and age ,renal donors were divided into 4 groups :group A (young/open) ,group B (young/laparoscopic) ,group C (aged/open) ,and group D (aged/laparoscopic).Blood and urinary NGAL and serum levels of creatinine ,cystatin C ,and other indices of renal function were assayed and collected before and at 1 ,3 ,7 days after operation. Results Both blood NGAL levels and urinary NGAL levels showed no statistically significant difference (all P>0.05) among four groups both before and after operation ,except that urinary NGAL was higher in group C (aged/open) than other groups ,at 1 day after operation ,(P = 0.03).The post-vs.pre-operation level dynamic changes of renal function were four or three times higher in urine or serum NGAL level than in serum creatinine or cystatin C level at 1 day after operation ,which showed an important role for predicting an early residual renal damage and relative treatment. Conclusions NGAL can be used as indices in evaluating changes of residual renal function after living donor kidney resection ,especially in the elderly receiving open kidney resection.
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Objective To explore the long-term clinical effect of kidney transplantation in children.Methods The clinical data of 53 children with kidney transplantation from March 2008 to September 2014 were retrospectively analyzed.The influence of the dependent factors on the estimated glomerular filtration rate (eGFR) (greater than 90 mL/min/1.73 m2 or ＜90 mL/min/1.73 m2) was estimated in the three years after the operation,and the influencing factors were analyzed by the dual logistic regression equation.Results There were 19 cases of living donors,17 cases of organ donors after death,and 6 others.The 53 patients were followed up for 3-9 years.The level of blood creatinine was decreased from the preoperative (820.1 ± 323.1) μmol/L to (51.6 ± 24.9) μmol/L 3 years after the operation (P＜0.05).eGFR was increased to (103.5 ± 11.4) mL/min/1.73 m2at 3rd year after the operation from the preoperative (17.1 ± 7.8) mL/min/1.73 m2 (P＜0.05).The age of recipients,preoperative dialysis time,number of HLA mismatching and postoperative delayed graft function healing (DGF),rejection and infection were the influencing factors of eGFR at 3rd year postoperation (P＜0.05).The multi-factor binary logistic regression equation analysis showed that only rejection was the risk factor for eGFR at 3rd year p0ostoperation.Eight cases of DGF (8/53,15.1％) recovered rapidly.There were 6 cases of acute rejection (6/47,12.8 ％) and 1 case of chronic rejection (1/47,2.1％).There were 9 cases of infection (9/47,19.1％).There were 6 cases of recurrence after surgery.The 3-year recipient and kidney survival rate was 94.3％ (50/53) and 88.7％ (47/53) respectively.The average height of the patients in the first,second and third year after the surgery was increased by (4.6 ± 1.9) cm (0.5-19.1 cm),(3.7 ± 1.8) cm (0.7-14.3 cm) and (2.8± 1.2) cm (0.3-8.7 cm) respectively.Conclusion The long-term effect of children kidney transplantation is satisfactory.
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Objective To evaluate the clinical efficacy of en-bloc kidney transplantation from pediatric organ donation after death. Methods Clinical data of donors and recipients undergoing en-bloc kidney transplantation from pediatric donor kidneys were retrospectively analyzed. The 1-year survival rates of the recipient and grafted kidney were calculated. The recovery of renal function at postoperative 1 year was observed. The changes in the length of grafted kidney and incidence of postoperative adverse events were monitored. Results The 1-year survival rate of the recipients was 8/9, and 72% for the grafted kidney. During 1-year follow-up, the serum creatinine (Scr) level was down-regulated from (747± 170) μmol/L before transplantation to (83±27) μmol/L post-transplantation, the blood urea nitrogen concentration was decreased from (24.5±4.9) mmol/L to (6.8±2.0) mmol/L, and the length of transplanted kidney was increased from (61.1±9.8) mm to (100.3±1.7) mm. Two recipients suffered from delayed graft function(DGF) and restored after hemodialysis. Two cases developed acute rejection and healed after methylprednisolone shock therapy. One recipient presented with lung fungal infection at postoperative 2 weeks after transplantation, and was treated by the withdrawal of immunosuppressive agents and antibacterial treatment with poor clinical efficacy. Then the recipient died at 3rd month. One case had renal arterial thrombosis at postoperative 7 d, underwent nephrectomy at postoperative 10 d and returned to hemodialysis. At postoperative 1st month, one recipient suffered from thrombosis of unilateral renal artery. The grafted kidney in other side normally functioned and significantly grew in size at postoperative 6 months. In addition, two cases had ureterostenosis of the transplanted kidney, albuminuria in 2, abdominal aortic stenosis in 1 and urinary fistula in 1. All these symptoms were cured or alleviated after corresponding treatment. Conclusions The incidence of perioperative complications is relatively high in en-bloc kidney transplantation from pediatric organ donation after death, whereas the clinical efficacy of such kidney transplantation can be gradually increased along with the accumulation of clinical experience.
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Objective To detect the expression of microRNA(miRNAs)in the late stage terminal hindgut de-velopment in fetal rats.Methods Twenty -four female rats were randomly divided into 2 groups.They were matched in proportion of male and female 21 .The experimental rats(n =1 2)received 1 0 g/L ethylenethiourea (1 25 mg/kg)by gavage on gestational day 1 1 ,and the control rats (n =1 2)received same amount of distilled water.The fetal rats were obtained by caesarean section on the gestational day 1 6 in each group.One centimeter rectum was taken out from 2 similar weight fetal rats for extracting total RNA by Trizol method.There were 24 fetal rats in each group.Chip hybri-dization was conducted after Poly(A)and biotin added to the RNA.Then,the chip was washed and dyed,and scanned thereafter.According to the differences in the expression profile of miRNAs and target gene analysis results,the miRNAs probably regulating the key gene of hindgut development was selected for target genes expression analysis.Results Compared with control group,expressions of 1 1 1 miRNAs in terminal hindgut of experimental group were up -regulated on the gestational day 1 6,and 1 1 7 miRNAs were down -regulated.Ten miRNAs of biggest differential expression be-tween them were selected for target genes prediction,pathway analysis,and Gene Ontology analysis.The results showed that some genes were closely related to rat fetus terminal hindgut growth and development,such as Shh,Hoxd13,and so on.According to the differential expression of miRNAs and target genes analysis,miR -1 93 might have an important role in the Hoxd13 gene for anorectal development.Real -Time PCR and Western blot showed that the expression of Hoxd13 and its protein level were significantly decreased when miR -1 93 highly expressed in rat intestinal epithelial cells,and the difference was statistically significant compared with the control group(1 .00 ±0.1 2 vs 0.71 ±0.1 0) (0.88 ±0.06 vs 0.75 ±0.08)(t =3.329,3.1 30;P =0.029,0.01 1 ).Conclusions miRNAs probably have an im-portant regulatory role in their target gene expression in terminal hindgut development of fetal rats,and miR -1 93 can significantly inhibit the expression of Hoxd13 gene in rat intestinal epithelial cells.
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Objective To explore the differentiation of allogeneic naive T cells to regulatory T cells (Tregs) and T helper (TH) 1/2/17 cells by coculture with bone marrow-derivedimmature dendritic cells (irnDC).Method Bone marrow-derived imDC were cultivated from Balb/c mice.Lipopolysaccharide-stimulated DC were harvested as mature dendritic cells (mDC) and unstimulated cells were collected as imDC.Then irnDC or mDC were cocultured with allogeniec naive T cells,respectively.TH1 cytokines [interferon-γ (IFN-γ) and interleukin (IL)-2],TH2 cytokines (IL-4 and IL-10),and TH17 cytokine (IL-17) of co-cultured cells were detected by enzyme linked immunospot assay.CD4+ Forkhead box p3 (FoxP3) + Treg proportion in CD4+ cells in the co-cultured system with IL-2 and transforming growth factor-β1 (TGF-β1) was analyzed by flow cytometry.Result As compared with mDC,na(i)ve T cells cocultured with imDC secreted much less IFN-γ (11.67 ± 2.18 vs.182.00±23.71,P＜0.01),IL-2 (26.67±2.96 vs.318.30± 18.62,P＜0.01),IL-4 (17.00±3.78 vs.45.33±3.48,P＜0.01),IL-10(7.00±1.00vs.158.70±10.90,P＜0.001) and IL-17 (0.66 ± 0.33 vs.238.30 ± 24.39,P＜0.001).Furthermore,imDC induced more CD4+ FoxP3+ Tregs than mDC after adding IL-2 and TGF-β1 in the coculture system for 7 days (22.70 ± 1.53 ％ vs.5.42 ± 1.27％,P＜0.01).Conclusion imDC are more effective to induce na ve T cells to Tregs,but not differentiate to TH 1/TH 2/TH 17 cells.These findings provide in vitro experimental evidence for induction of transplant tolerance by adoptive transfer of imDC.
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Objective To investigate the possibility of measuring ABO blood group antibody levels using renal cortical tubular epithelial cells (RCTECs) of cynomolgus monkey.Method The primary RCTECs were isolated from cynomolgus monkey kidneys and identified by Western blotting and flow cytometry (FACS).FACS was applied to detect the expression of ABO histo-blood group antigens on RCTECs,the binding of blood group antibodies in human serum to RCTECs,and to compare the difference of measuring ABO blood group antibody levels between using human erythrocytes and RCTECs as target cells.Result The majority of cells derived from the kidney cortex were renal tubular epithelial cells.39.90％-73.80％ of RCTECs were found to express the ABO histoblood group antigens with the capability to bind the human blood group antibodies specifically.The expression level of ABO histo-blood group antigen on RCTECs was not influenced by long-term cryopreservation,and the percentage of B antigen positive cells was 39.90％ before frozen storage and 39.65％ after a 10-month cryopreservation respectively.The normal serum and allergic serum presensitized to synthetic A antigen-keyhole limpet hemocyanin (KLH-A) of cynomolgus monkey were used to compare the abilities of RCTECs and human erythrocytes to detect antibody titers.No agglutination was observed in RCTECs test group.The mean fluorescence intensity (MFI) values of antibody were highest when the dilution fold was lowest (1 ∶ 16) and gradually decreased with increased serum dilution in both serum groups.MFI fell towards baseline value at 1 ∶ 128 dilution in normal serum group while at 1∶8 192 dilution in allergic serum group.Between 1 ∶ 16 and 1∶8 192 dilutions,MFIs of normal serum group were all lower than those of allergic serum.In human red cells test group,obvious agglutination appeared at high concentrations of antibodies,and MFIs reached the peak at 1∶64 dilution in normal serum group and at 1∶32 dilution in allergic serum group,but fluctuated irregularly thereafter.Between 1∶64 and 1∶512 dilutions,MFIs of normal serum group were all higher than those of allergic serum.Data above showed that MFI values in RCTECs group could reflect the levels of blood group antibodies more exactly.Conclusion The RCTECs of cynomolgus monkey express ABO tissue-blood group antigens and would be more suitable to be used to measure ABO blood group antibody levels by FACS.
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Objective To study the receptors signaling of prostaglandin E2 on the differentiation of regulatory T (Treg) cells and Th17 cells.Methods The expression of prostaglandin E2 receptors (EP1/EP2/EP3/EP4) on the MACS-purified CD4+CD62L+ T (Th0) cells was analyzed by flow cytometry and reverse transcription polymerase chain reaction (RT-PCR).The quantity of CD25+Foxp3+ cells was examined by flow cytometry,the expression of FoxP3 mRNA and RORγt mRNA were detected using real-time RT-PCR,the level of IL-17 in the culture supernatants was detected by enzyme-linked immunosorbent assay (ELISA).ANOVA,LSD-t,Dunnett T3 were used for statistical analysis.Results EP1,EP2,EP3,EP4 were expressed on Th0 cells at different levels,and EP2 [(89.7±9.1)％] had the strongest expression.PGE2 [(3.0± 2.2) ％],EP2 agonist [(4.5± 1.0) ％] and EP4 agonist [(8.8 ±2.5) ％] decreased the quantity of CD25 +Foxp3 + cells compared with the control group [(28.6±6.8)％] (t=7.156,P=0.021; t=6.958,P=0.032; t=5.359,P=0.044).PGE2(0.210±0.020),EP1 agonist (0.833±0.045),EP2 agonist (0.227±0.025) and EP4 agonist (0.450±0.060) decreased the expression of Foxp3 mRNA compared with the control group (1.000) (t=23.817,t=5.026,t=23.313,t=16.581; all P=0.000).PGE2 [(22±6)pg/ml],EP2 agonist [(24±5)pg/ml]and EP4 agonist [(207±19) pg/ml] decreased the secretion of IL-17 compared with the control group [(678±87) pg/ml] (t=14.925,P=0.004; t=14.873,P=0.004; t=10.480,P=0.008).PGE2 (0.141±0.027),EP1 agonist (0.869±0.033),EP2 agonist (0.176±0.029) and EP4 agonist(0.371±0.042) decreased the expression of RORγt mRNA compared with the control group (1.000) (t=34.046,t=5.184,t=32.673,t=24.962,all P=0.000).Conclusion EP1,EP2,EP3,EP4 receptors are expressed on CD4+CD62L+ T (Th0) cells at different levels.Prostaglandin E2 inhibits the differentiation of Treg cells and Th17 cells via the EP2 and EP4 receptors signaling.
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Objective To establish a hyperacute rejection model in ABO-incompatible renal allotransplantation in nonhuman primates.Method ABO-incompatible renal transplantation was performed using blood group B cynomolgus monkeys as recipients and blood group A cynomolgus monkeys as donors.The transplants were distributed into 2 groups according to whether the recipient monkey was presensitized or not:(1) non-presensitized control group (n =1),not receiving any pretreatment; (2) KLH-conjugated blood group antigen A (KLH-A) presensitized group (n =3),being presensitized by subcutaneous injection of KLH-A 2 weeks prior to ABO-incompatible renal transplantation.The serum anti-blood group A antibody levels were measured using a FACS method.The graft survival time was observed and the pathologic studies were performed using the endpoint renal graft tissue samples.Result In non-presensitized control group,no hperacute rejection was observed during the surgery.With the traditional CsA triple therapy,the renal allograft survived was more than 30 days without obvious rejection,and the serum creatinine level was 263 μmol/L at day 30.After the presentization with KLH-A,recipient monkeys of KLH-A presensitized group had a markedly increased anti-A antibody levels and rapidly rejected the renal allografts from blood group A donors within 1 h after the reperfusion,which was demonstrated to be a hyperacute rejection with the pathologic studies.Conclusion The strategy of presensitization with KLH-conjugated blood group antigen significantly increases the corresponding blood group antibodies and allows the establishment of a hyperacute rejection model in ABO-incompatible renal allotransplantation in nonhuman primates.
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Objective To study the HPLC fingerprint methodology of Rupikang Capsules. Methods The precision, stability and repeatability of the fingerprint of Rupikang Capsules were researched by HPLC. The HPLC system:Agilent ZORBAX SB-C18 column (4.6 mm×250 mm, 5μm);the mobile phase:water-0.1% phosphoric acid water-acetonitrile, with linear gradient elution;the flow rate:1 mL/min;the detection wavelength:280 nm.Results The precision, stability and repeatability of Rupikang Capsules met the requirements of HPLC fingerprint technology. Conclusion This method is simple, feasible and can be used as the whole quality control method of Rupikang capsules.
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Objective To study the effects of EP4 and EP2 antagonists on the differentiation of Treg/ Th17 cells and disease progression in mice of collagen-induced arthritis (CIA) model.Methods DBA/1 mice wereimmunized subcutaneously twice at the root of the tail with type Ⅱ collagen emulsified in Freund's complete adjuvant.EP2 and EP4 antagonist therapies were intraperitoneally administrated for 14 consecutive days after the second immunization.Clinical signs,histological manifestation,serum interleukin (IL)-17 and quantity of CD4+CD25+Foxp3+ Treg cells were determined.ANOVA and t-test were used for statistical analysis.Results Clinical signs of the disease appeared on day 27 and peaked on day 35 after the first immunization.The quantity of CD4+CD25+Foxp3+ Treg cells in spleens [(1.67±0.15)％] and draining inguinal lymph nodes [(3.30±0.36)％] isolated from CIA mice were significantly lower than those of normal DBA/1 mice [(2.77±0.45)％ and (4.73 ±0.45)％ respectively,P＜0.05].Serum IL-17 level of CIA mice [(27±7) pg/ml] was significantly higher than that of normal DBA/1 mice [(14±4) pg/ml,P＜0.05].Intra-peritoneal injection of EP4 but not EP2 antagonist to CIA mice decreased paw edema and swelling,and alleviated the histological manifestations (1.8±1.0 vs 3.5±0.6,P＜0.05) on day 35 after the first immunization.The percentages of CD4+CD25+Foxp3+ Treg cells in both inguinal lymph nodes [(4.20±0.32)％] and spleens [(2.63±0.40)％] were significantly higher in EP4 antagonist-treated but not EP2 antagonist-treated CIA mice compared with CIA mice group [(3.30±0.36)％ and (1.67±0.15)％ respectively,P＜0.05].The level of serum IL-17 was significantly lower in EP4 antagonist-treated [(15±7) pg/ml] but not EP2 antagonist-treated CIA mice compared with CIA mice group [(27±7) pg/ml,P＜0.05].Conclusion EP4 antagonist therapy alleviates clinical symptoms of CIA,improves the histological manifestations,decreases the serum IL-17 level and increases the percentages of CD4+CD25+Foxp3+ Treg cells in both spleens and draining inguinal lymph nodes,so targeting EP4 receptor may be a new possible therapeutic possibility in the prevention and treatment of rheumatoid arthritis.
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Objective To establish the quality standard for Rupikang Capsules. Methods Lumbricus were identified by TLC. Tanshinone IIA was determined by HPLC. HPLC analysis was carried out on Hitachi L-2000 C18 column (250 mm×4.6 mm, 5 μm) and the mixture of methanol-water (75∶25) was used as the mobile phase, the flow rate was 1.0 mL/min and the detection wavelength was at 270 nm. Results TLC spots developed were fairly clear and the blank test showed no interference. The linear range was 0.16-0.4 μg for tanshinone ⅡA, r=0.999 9. The average recovery was 99.1%, RSD=2.20%. Conclusion The methods is easy to operate, with good specificity and reproducibility. It can be used for the quality control of Rupikang Capsules.
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Objective To investigate if the administration of CORM-2 can provide protection against renal ischemia-reperfusion injury (IRI).Method Murine renal ischemia was induced by clamping left renal pedicles for 40 min with vascular micro damps at 32 C,then the contralateral kidney was removed.CORM-2 or vehicle was administered via intravenous infusion 1 h before the onset of ischemia.The blood plasma and renal samples were obtained at 24 h after reperfusion to assess renal function and cellular injury.Plasma Cr and BUN levels,HE and TUNEL were performed to estimate the magnitude of renal damage.Kidneys were retrieved from indicated animals at various time points after renal IRI,and the sections were prepared for histological evaluation.MPO staining procedures were performed to assess the neutrophils infiltration in the renal IRI.Besides,Immunofluorescent stain of TNF-α was performed on the kidneys which were retrieved from indicated animals to determine the production of inflammatory mediators in renal I/R.Results The plasma Cr and BUN were significantly increased at 24 h after reperfusion in IRI control mice,and CORM-2 treatment could markedly diminish the increase of plasma Cr and BUN in mice subjected to I/R.In parallel,histological analysis demonstrated that CORM2 treatment markedly reduced apoptosis of the renal tubular epithelium cells and hemorrhage.IRI caused marked infiltration and accumulation of the MPO-positive neutrophils in renal interstitium.Administration of CORM-2 before ischemia dramatically inhibited neutrophils infiltration as compared with IRI or iCORM-2 group.Furthermore,we confirmed that CORM-2 markedly decreased production of TNF-α.Conclusion Carbon monoxidereleasing molecule CORM-2 could ameliorate inflammation to protect against the renal IRI in mice.
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Objective The goal of this study was to investigate the correlation between the level of serum vitamin D and bone metabolism and disease activity in ankylosing spondylitis(AS) patients,and thus to explore the role of vitamin D in bone metabolism in AS patients.Methods The serum levels of BALP,TRACP-5b,25-(OH)D3 and blood lymphocytes VDR of 80 AS patients were measured by enzyme-linked immunosorbent assay (ELISA) and compared with those of the control group.Bone mineral density (BMD)was measured by dual-energy X-ray absorptiometry (DEXA).The ESR and CRP level of AS patients were also measured.The correlation between those parameters was analyzed and evaluated.Patients were divided into normal,insufficient and deficient subgroups according to the serum 25-(OH)D3 levels for further comparison.Indepondent saimple t test,t'test andx2 test were used for statistical analysis.Results The 25-(OH)D3 of AS patients [(11.9±2.7) μg/L] was significantly lower than that of the control groups [(22.3±7.9) μg/L] (P＜0.05),while the serum levels of BALP [(3.9±2.7) μg/L] and TRACP-5b [(46±25) ng/L] of AS patients were significantly higher than those of the control group [(2.4±1.0) μg/L] (P＜0.05).According to linear correlation analysis,25-(OH)D3 was negatively correlated with CRP (r=0.324,P=0.003).The ESR,BALP,TRACP-5b in the deficient subgroup were higher than those in the normal and insufficient subgroups(P＜0.05).Conclusion The plasma 25-(OH)D3 may decrease in AS patients,and this may activate bone metabolism,results in increased morbidity of osteoporo-sis,and negatively affect disease activity.