Anti-Na+/K+-ATPase DR antibody attenuates UUO-induced renal fibrosis through inhibition of Na+/K+-ATPase α1-dependent HMGB1 release.

Reduced Na+/K+-ATPase (NKA) activity and NKAα1 expression are engaged in the pathologies of renal diseases. NKA-mediated Src activation is not the only reason for NKA-related renal fibrosis. In this study, we found that genetic reduction of NKAα1 exhibited exacerbated tubulointerstitial lesions and fibrosis in the UUO mice model. Activation of NKAα1 with an antibody against the extracellular DR region of the NKAα1 subunit (DRm217) prevented UUO-induced tubulointerstitial lesions, preserved kidney function, and decrease renal fibrosis. Further studies revealed that NKAα1 deficiency mice exhibited high inflammation factors expression when they suffered UUO surgery, compared with NKAα1+/+ (WT) mice. DRm217 alleviated inflammatory cell infiltration, suppress NF-κB phosphorylation, and decreased inflammatory factors expression in the UUO mice model. Released HMGB1 can trigger the inflammatory response and contribute to renal fibrosis. Knockdown of NKA in renal tubular cells or in NKAα1+/- mice was associated with more susceptibility to HMGB1 release in the UUO mice model. DRm217 exerted its antifibrotic effect via inhibiting HMGB1 release. Furthermore, AMPK activation participates in the effect of DRm217 on inhibiting HMGB1 release. Our findings suggest that NKAα1 is a regulator of renal fibrosis and its DR-region is a novel target on it.

BMSCs overexpressed ISL1 reduces the apoptosis of islet cells through ANLN carrying exosome, INHBA, and caffeine.

Early apoptosis of grafted islets is one of the main factors affecting the efficacy of islet transplantation. The combined transplantation of islet cells and bone marrow mesenchymal stem cells (BMSCs) can significantly improve the survival rate of grafted islets. Transcription factor insulin gene enhancer binding protein 1 (ISL1) is shown to promote the angiogenesis of grafted islets and the paracrine function of mesenchymal stem cells during the co-transplantation, yet the regulatory mechanism remains unclear. By using ISL1-overexpressing BMSCs and the subtherapeutic doses of islets for co-transplantation, we managed to reduce the apoptosis and improve the survival rate of the grafts. Our metabolomics and proteomics data suggested that ISL1 upregulates aniline (ANLN) and Inhibin beta A chain (INHBA), and stimulated the release of caffeine in the BMSCs. We then demonstrated that the upregulation of ANLN and INHBA was achieved by the binding of ISL1 to the promoter regions of the two genes. In addition, ISL1 could also promote BMSCs to release exosomes with high expression of ANLN, secrete INHBA and caffeine, and reduce streptozocin (STZ)-induced islets apoptosis. Thus, our study provides mechanical insight into the islet/BMSCs co-transplantation and paves the foundation for using conditioned medium to mimic the ISL1-overexpressing BMSCs co-transplantation.

Donor-specific antibodies, glomerulitis, and human leukocyte antigen B eplet mismatch are risk factors for peritubular capillary C4d deposition in renal allografts.

BACKGROUND: The complement system plays an important role in the immune response to transplantation, and the diagnostic significance of peritubular capillary (PTC) C4d deposition (C4d+) in grafts is controversial. The study aimed to fully investigate the risk factors for PTC C4d+ and analyze its significance in biopsy pathology of kidney transplantation. METHODS: This retrospective study included 124 cases of kidney transplant with graft biopsy and donor-specific antibody (DSA) testing from January 2017 to December 2019 in a single center. The effects of recipient pathological indicators, eplet mismatch (MM), and DSAs on PTC C4d+ were examined using univariate and multivariate logistic regression analyses. RESULTS: In total, 35/124 (28%) were PTC C4d+, including 21 with antibody-mediated rejection (AMR), eight with renal tubular injury, three with T cell-mediated rejection, one with glomerular disease, and two others. Univariate analysis revealed that DSAs (P < 0.001), glomerulitis (P < 0.001), peritubular capillaritis (P < 0.001), and human leukocyte antigen (HLA) B eplet MM (P = 0.010) were the influencing factors of PTC C4d+. According to multivariate analysis, DSAs (odds ratio [OR]: 9.608, 95% confidence interval [CI]: 2.742-33.668, P < 0.001), glomerulitis (OR: 3.581, 95%CI: 1.246-10.289, P = 0.018), and HLA B eplet MM (OR: 1.166, 95%CI: 1.005-1.353, P = 0.042) were the independent risk factors for PTC C4d+. In receiver operating characteristic curve analysis, the area under the curve was increased to 0.831 for predicting PTC C4d+ when considering glomerulitis, DSAs, and HLA B eplet MM. The proportions of HLA I DSAs and PTC C4d+ in active antibody-mediated rejection were 12/17 and 15/17, respectively; the proportions of HLA class II DSAs and PTC C4d+ in chronic AMR were 8/12 and 7/12, respectively. Furthermore, the higher the PTC C4d+ score was, the more serious the urinary occult blood and proteinuria of recipients at the time of biopsy. CONCLUSIONS: PTC C4d+ was mainly observed in AMR cases. DSAs, glomerulitis, and HLA B eplet MM are the independent risk factors for PTC C4d+.

Bone marrow-derived mesenchymal stem cells improve rat islet graft revascularization by upregulating ISL1.

Revascularization of the islet transplant is a crucial step that defines the success rate of patient recovery. Bone marrow-derived mesenchymal stem cells (BMSCs) have been reported to promote revascularization; however, the underlying cellular mechanism remains unclear. Moreover, our liquid chromatography-tandem mass spectrometry results showed that BMSCs could promote the expression of insulin gene enhancer binding protein-1 (ISL1) in islets. ISL1 is involved in islets proliferation and plays a potential regulatory role in the revascularization of islets. This study identifies the ISL1 protein as a potential modulator in BMSCs-mediated revascularization of islet grafts. We demonstrated that the survival rate and insulin secretion of islets were increased in the presence of BMSCs, indicating that BMSCs promote islet revascularization in a coculture system and rat diabetes model. Interestingly, we also observed that the presence of BMSCs led to an increase in ISL1 and vascular endothelial growth factor A (VEGFA) expression in both islets and the INS-1 rat insulinoma cell line. In silico protein structure modeling indicated that ISL1 is a transcription factor that has four binding sites with VEGFA mRNA. Further results showed that overexpression of ISL1 increased both the abundance of VEGFA transcripts and protein accumulation, while inhibition of ISL1 decreased the abundance of VEGFA. Using a ChIP-qPCR assay, we demonstrated that direct molecular interactions between ISL1 and VEGFA occur in INS-1 cells. Together, these findings reveal that BMSCs promote the expression of ISL1 in islets and lead to an increase in VEGFA in islet grafts. Hence, ISL1 is a potential target to induce early revascularization in islet transplantation.

Different roles of bortezomib and ONX 0914 in acute kidney injury.

Proteasome inhibitor bortezomib offers one more option for acute or chronic antibody-mediated rejection after kidney transplantation, but aggravated acute kidney injury (AKI) in some cases early after surgery using bortezomib bring new problem. Here, we evaluated the effects of bortezomib and ONX-0914 on renal tubule injury in a mouse model of ischemia-reperfusion injury. After treated with bortezomib, serum creatinine, usea nitrogen and tubular necrosis significantly increased compared with vehicle-treated mice, but decreased in ONX-0914 group mildly. Infiltration of neutrophil and macrophage were less in bortezomib and ONX-0914-treated mice than vehicle-treated group, and the same was observed on oxidative stress in the kidneys. Furthermore, the apoptosis of renal tubular epithelial cells increased in bortezomib-treated mice' kidneys compared with ONX-0914 and vehicle-treated controls. In vitro HK2 cell experiments also demonstrated the proapoptotic effect of bortezomib. The mRNA expression of several proapoptotic factors increased in kidneys of bortezomib-treated mice. In brief, bortezomib, as a proteasome inhibitor, shows a certain cytotoxicity to renal tubular epithelial cell during ischemia/reperfusion injury (IRI) through increased apoptosis. ONX-0914, as an immunoproteasome inhibitor, showed equal potency on anti-inflammation and oxidative stress relieving compared with bortezomib, while less cytotoxicity. The results render the immunoproteasome is a better target for anti-rejection and protecting kidney function in the field of organ transplantation.

Prediction of kidney transplant outcome based on different DGF definitions in Chinese deceased donation.

BACKGROUND: Delayed graft function (DGF) is an important complication of kidney transplantation and can be diagnosed according to different definitions. DGF has been suggested to be associated with the long-term outcome of kidney transplantation surgery. However, the best DGF definition for predicting renal transplant outcomes in Chinese donations after cardiac death (DCDs) remains to be determined. METHOD: A total of 372 DCD kidney transplant recipients from June 2013 to July 2017 in the First Affiliated Hospital of Xi'an Jiaotong University were included in this retrospective study to compare 6 different DGF definitions. The relationships of the DGF definitions with transplant outcome were analyzed, including graft loss (GL) and death-censored graft loss (death-censored GL). Renal function indicators, including one-year estimated glomerular filtration rate (eGFR) and three-year eGFR, and were compared between different DGF groups. RESULTS: The incidence of DGF varied from 4.19 to 35.22% according to the different DGF diagnoses. All DGF definitions were significantly associated with three-year GL as well as death-censored GL. DGF based on requirement of hemodialysis within the first week had the best predictive value for GL (AUC 0.77), and DGF based on sCr variation during the first 3 days post-transplant had the best predictive value for three-year death-censored GL (AUC 0.79). Combination of the 48-h sCr reduction ratio and classical DGF can improve the AUC for GL (AUC 0.85) as well as the predictive accuracy for death-censored GL (83.3%). CONCLUSION: DGF was an independent risk factor for poor transplant outcome. The combination of need for hemodialysis within the first week and the 48-h serum creatinine reduction rate has a better predictive value for patient and poor graft outcome.

B-Cell Activating Factor Predicts Acute Rejection Risk in Kidney Transplant Recipients: A 6-Month Follow-Up Study.

B cell activating factor (BAFF) belonging to TNF family is a cytokine that enhances B-cell proliferation and differentiation. Recently, It has been suggested that BAFF might be a potential therapeutic target for treating autoimmune disease. However, the relationship between BAFF and allograft rejection is controversial, and the clinical significance of BAFF in predicting allograft rejection need to be further explored. We conducted 6-month follow-up study to confirm the hypothesis that BAFF might be a risk factor for predicting acute rejection in kidney transplant recipients. At the end of the study, a total of 155 kidney transplant recipients were recruited from October 2015 to October 2017, and classified into acute rejection group (n = 34) and stable renal function group (n = 121) according to their clinical course. We demonstrate that the serum BAFF levels when acute rejection occurred was significantly higher than that in the stable renal function group (2426.19 ± 892.19 vs. 988.17 ± 485.63 pg/mL, P < 0.05). BAFF expression was significantly enhanced in the membrane and cytoplasm of renal tubule epithelial cells in the transplant kidney tissue with acute rejection, and a positive correlation between BAFF and C4d expression was also observed (r = 0.880, P = 0.001). ROC analyses highlight the superiority of serum BAFF level before transplant over those on other post-transplant days in prediction of acute rejection episodes. The sensitivity, specificity and AUC (area under curve) were 83.3, 89.5, and 0.886%, respectively. Kaplan-Meier survival analysis showed that recipients with higher pretransplant BAFF levels had higher acute rejection incidence (P = 0.003). In conclusion, we have identified that BAFF levels are associated with the acute rejection and could be a promising biomarker to predict kidney transplant rejection risks.

Early Immunosuppressive Exposure of Enteric-Coated-Mycophenolate Sodium Plus Tacrolimus Associated with Acute Rejection in Expanded Criteria Donor Kidney Transplantation.

BACKGROUND: Immunosuppressive agents are still inefficient in preventing biopsy-proven acute rejection (BPAR) after expanded criteria donor (ECD) kidney transplantation. The aim of this study was to investigate the relationships between early immunosuppressive exposure and the development of BPAR. METHODS: We performed a retrospective study of 58 recipients of ECD kidney transplantation treated with enteric-coated-mycophenolate sodium, tacrolimus (Tac), and prednisone. The levels of mycophenolic acid-area under the curve (MPA-AUC)0-12h and Tac C0were measured at the 1st week and the 1st month posttransplant, respectively. The correlation was assessed by multivariate logistic regression. RESULTS: The occurrence rates of BPAR and antibody-mediated rejection were 24.1% and 10.3%, respectively. A low level of MPA-AUC0-12h at the 1st week posttransplant was found in BPAR recipients (38.42 ± 8.37 vs. 50.64 ± 13.22, P < 0.01). In addition, the incidence of BPAR was significantly high (P < 0.05) when the MPA-AUC0-12hlevel was <30 mg·h-1·L-1 at the 1st week (15.0% vs. 44.4%) or the Tac C0was <4 ng/ml at the 1st month posttransplant (33.3% vs. 21.6%). Multivariable logistic regression analysis showed that the MPA-AUC0-12h at the 1st week (OR: 0.842, 95% CI: 0.784-0.903) and the Tac C0at the 1st month (OR: 0.904, 95% CI: 0.822-0.986) had significant inverse correlation with BPAR (P < 0.05). CONCLUSIONS: Low-level exposure of MPA and Tac C0in the early weeks posttransplant reflects an increased acute rejection risk, which suggested that MPA-AUC0-12h <30 mg·h-1·L-1 and Tac C0 <4 ng/ml should be avoided in the first few weeks after transplantation.

Arctigenin: A two-edged sword in ischemia/reperfusion induced acute kidney injury.

Arctigenin (ATG) is one of the main active substances in fruit derived from Arctium lappa L. Previous studies have reported that ATG have antitumor, neuroprotective, antioxidant, antifibrosis and anti-inflammatory functions. However, the actions of ATG in kidney with acute injury following ischemia/ reperfusion (I/R) is still uncertain. In our study, mice were subjected to kidney I/R by having the kidney pedicles clamped and administered with vehicle or ATG (1, 3 or 9 mg/kg/d) via oral gavage for 7 consecutive days prior to I/R. Notably, ATG aggravated kidney I/R injury with the concentration increases. Multiple biochemical assays and histological examination showed ATG significantly alleviated the inflammatory response as reflected by a decreased expression of proinflammatory cytokine, TLR4/MyD88, and NF-κB, along with the infiltration of CD68+ macrophage and CD11b+Gr1+ neutrophil in the kidneys. Meanwhile, ATG alleviated I/R-induced oxidative stress proved by increasing kidney manganese superoxide dismutase and glutathione peroxidase activity but reducing levels of malonaldehyde and inducible nitric oxide synthase. On the contrary, apoptosis was significantly increased in kidneys of ATG-treated mice compared with vehicle-treated controls, especially in tubular cells. There were increased numbers of TUNEL positive cells and increased Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9 expression. The current study demonstrates that pretreatment of ATG aggravates I/R induced acute kidney injury by increasing apoptosis of tubular cells despite reducing infiltrating inflammatory cells and proinflammatory cytokine.

Knockdown of NLRC5 attenuates renal I/R injury in vitro through the activation of PI3K/Akt signaling pathway.

NLRC5, as the largest member of nucleotide-binding domain and leucine-rich repeat (NLR) family, was involved in various physiological processes, such as inflammation, fibrosis, innate immunity and diabetic nephropathy. However, the role of NLRC5 in acute kidney injury remains unclear. The aim of this study was to investigate the role of NLRC5 in human renal proximal tubular epithelial cells (HK-2) exposed to hypoxia/reoxygenation (H/R). Our results demonstrated that the expression of NLRC5 was significantly up-regulated in HK-2 cells exposed to H/R. Knockdown of NLRC5 significantly improved the viability of HK-2 cells exposed to H/R. In addition, knockdown of NLRC5 efficiently inhibited H/R-induced oxidative stress and apoptosis in HK-2 cells. Mechanistically, knockdown of NLRC5 markedly enhanced the activation of PIK3/Akt signaling pathway in H/R-stimulated HK-2 cells. In summary, our findings indicate that knockdown of NLRC5 attenuates renal I/R injury in vitro through the activation of PI3K/Akt signaling pathway.

Comparison of the characteristics of macrophages derived from murine spleen, peritoneal cavity, and bone marrow.

Macrophages have a diverse set of functions based upon their activation states. The activation states, including resting (M0) and polarizing (M1 and M2) states, of macrophages derived from the mouse bone marrow, spleen, and peritoneal cavity (BMs, SPMs, and PCMs, respectively) were compared. We evaluated the macrophage yield per mouse and compared the surface markers major histocompatibility complex (MHC) II and CD86 by flow cytometry. The relative mRNA levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, mannose receptor (MR), and Ym1 in the M0, M1, and M2 states were also compared using real-time polymerase chain reaction (PCR) analysis. Bone marrow yielded the most macrophages with the best homogeneity, but they were polarized toward the M2 phenotype. All three types of macrophages had the capacity to polarize into the M1 and M2 states, but SPMs had a stronger capacity to polarize into M1. The three types of macrophages showed no differences in their capacity to polarize into the M2 state. Therefore, the three types of macrophages have distinct characteristics regardless of their resting or polarizing states. Although bone marrow can get large amounts of homogeneous macrophages, the macrophages cannot replace tissue-derived macrophages.

Induction of tolerance and prolongation of islet allograft survival by syngeneic hematopoietic stem cell transplantation in mice.

BACKGROUNDS: Syngeneic or autologous hematopoietic stem cells transplantation (HSCT) has been proposed to treat autoimmune diseases because of its immunosuppressive and immunomodulatory effects, which can also contribute to posttransplant antirejection therapy. In this study, we explored the tolerogenic effect of syngeneic HSCT on prolonging islet allograft survival. METHODS: C57BL/6 mice received syngeneic HSCT plus preconditioning with sublethal irradiation. Then islets of BALB/c mice were transplanted into the renal subcapsular of C57BL/6 mice after chemically induced into diabetes. RESULTS: HSCT mice exhibited improved islet allograft survival and increased serum insulin compared to control mice. Islet allografts of HSCT mice displayed lower level lymphocyte infiltration and stronger insulin staining than control mice. T cells of HSCT mice proliferated poorly in response to allogeneic splenocytes compared to control mice. Mice appeared reversed interferon-γ (IFN-γ)/interleukin-4 (IL-4) ratio to a Th2 immune deviation after syngeneic HSCT. The percentage of CD8(+) T cells was lower, while percentage of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Tregs) was higher in HSCT mice than control mice. HSCT mice showed higher percentage of CTLA-4(+) T cells and expression of CTLA-4 mRNA than control mice. Targeting of CTLA-4 by intraperitoneal injection of anti-CTLA-4 mAb abrogated the effect of syngeneic HSCT on prolonging islet allograft survival, inhibiting activity of T cells in response to alloantigen, promoting Th1 to Th2 immune deviation and up regulating CD4(+)CD25(+)Foxp3(+) Tregs. CONCLUSIONS: Syngeneic HSCT plus preconditioning of sublethal irradiation induces tolerance and improves islet allograft survival in fully mismatched mice model. Th1 to Th2 immune deviation, increased CD4(+)CD25(+)Foxp3(+) Tregs and up-regulation of CTLA-4 maybe contribute to the tolerogenic effect induced by syngeneic HSCT.

Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients.

BACKGROUND: Peripheral blood CD4+ T cell adenosine triphosphate (ATP) release has been reported to be an adjunct tool to evaluate global cellular immune response in solid-organ transplant recipients. However, the correlation between the ATP level and rejection was controversial. The aim of this prospective clinical study was to explore the association between the intracellular ATP level and the occurrence, progression, and treatment of acute rejection (AR) episodes, determine the predicting value of intracellular ATP level for AR in kidney transplant (KT) recipients. PATIENTS AND METHODS: In the period of October 2011 to October 2012, 140 KT recipients were recruited and followed for six months after transplantation. Patients were categorized into stable group and AR group according to their clinical course. Whole blood samples were collected pretransplantation, and at 7, 14, 21, and 28days, and at 2, 3, 4, 5 and 6months post-transplantation. Additional blood samples were obtained from AR patients on the day AR occurred, on the day before and 3 and 7days after intravenous anti-rejection therapy started, and on the day when AR reversed. The intracellular ATP in CD4+ T cells was detected by ImmuKnow Immune Cell Function Assay according to the manufacturer's instruction. The absolute number of CD4+ T cells and the trough levels of tacrolimus and cyclosporine were also measured. RESULTS: The ATP level detected on the day AR occurred (627.07±149.85ng/ml) was obviously higher than that of the stable group (320.48±149.11ng/ml, P<0.05). ATP value decreased to 265.35±84.33ng/m at the end of anti-rejection therapy, which was obviously lower than that measured on the day before the anti-rejection therapy started (665.87±162.85ng/ml, P<0.05). ROC analysis revealed that increased intracellular adenosine triphosphate level showed better sensitivity and specificity than those obtained using single time point detection (89.5% vs 85.0%;95.0% vs 88.9%). The best cutoff value was 172.55ng/ml. A positive correlation between the intracellular ATP level and absolute CD4+ T cell number (r=0.656, P<0.001) was found in the patients with CD4+ T cell counts <200/µl.

Simultaneous blockade of the CD40/CD40L and NF-κB pathways prolonged islet allograft survival.

Activation of NF-κB pathway and co-stimulatory system CD40/CD40L promotes the inflammation, which plays a key role in the failure of islet graft. Therefore, the purpose of this study was to determine if simultaneous blockade of CD40/CD40L and IκB/NF-κB pathways could protect islet graft. Streptozocin-induced diabetic Wistar rats were transplanted intraportally with 2000 IEQ islets isolated from Sprague-Dawley rats. The rats were divided into five groups: nontreatment group, AdGFP-treated group, Ad-IκBα-treated group, Ad-sCD40LIg-treated group, and Ad-IκBα-IRES(2) -sCD40L-treated group. The islet graft mean survival time (MST), insulin expression of islet grafts, and the levels of cytokines in peripheral blood, were measured for the animals in each group. Our study confirmed that islet cells transfected with low doses of adenovirus could achieve high transfection efficiency, and would not affect the function of islet cells (P > 0.05). Splenocytes cultured with Ad-IκBα-IRES2-CD40L-transfected islets resulted in homospecific hyporesponsiveness. The islet graft MST (>100 d) in the Ad-IκBα-IRES2-sCD40L-treated group was dramatically prolonged compared with that in the nontreatment group (7.1 ± 1.16 d). In addition, TNF-α, IL-1ß, and IFN-γ were diminished in the Ad-IκBα-IRES2-sCD40L-treated group, which was commensurate with the reduced cellular infiltration (P < 0.01). Simultaneous blockade of the CD40/CD40L and IκB/NF-κB pathways could effectively extend the survival of islet grafts.

Tea polyphenol-induced neuron-like differentiation of mouse mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) can be induced to differentiate into neuron-like cells under appropriate conditions often involving toxic reagents that are not applicable for clinical transplantation. The present study investigated whether tea polyphenol (TP), a native nontoxic antioxidant, could induce mouse neuron-like cell differentiation of BMSCs in vitro. BMSCs, dissected from mouse femur bone marrow, were amplified in culture and treated with TP or beta-mercaptoethanol (BME, control). Morphological changes were observed under light microscopy. After 12 h treatment with 50 microg/ml TP or 5 mM BME, most cells differentiated into neuron-like cells exhibiting neuronal morphological characteristics, cellular shrinkage and neurite growth. Immunocytochemistry and reverse transcription (RT)-PCR results demonstrated neuronal marker expression in the induced cells with no glial fibrillary acidic protein expression. Taken together, TP induced mouse BMSCs to differentiate into neuron-like cells in vitro. These findings provide a potential source for the treatment of various neurological diseases.

[Evaluation of immune status of kidney transplant recipients by combined HLA-G5 and sCD30].

OBJECTIVE: to study the relationship between the expression of serum human leucocyte antigen-G5 (HLA-G5)/soluble CD30 (sCD30) and the function of renal graft in kidney transplant recipients and investigate the immune status of recipients with combined HLA-G5 and sCD30. METHODS: from January 2002 to November 2008, a total of 66 kidney transplant recipients in our centre were selected as subjects and divided into three groups: stable function of renal graft (n = 38), acute rejection (n = 15) and chronic rejection (n = 13). The expressions of serum HLA-G5 and sCD30 were detected. There were two different immune conditions with acute/chronic allograft rejection and normal renal graft in kidney transplant recipients as evaluated by combined HLA-G5 and sCD30. The sensitivity, specificity and critical value of the method were analyzed by the curve of receiver operating characteristic. RESULTS: the levels of HLA-G5 and sCD30 were significantly correlated with serum creatinine (r = -0.493, 0.691, both P < 0.01). Within the first year post-transplantation, the sensitivity was 78.6% and the specificity 85.7% when HLA-G5 critical value 82 microg/L and sCD30 critical value 12.2 microg/L. After one year post-transplantation: the sensitivity was 92.3% and the specificity 84.6% when HLA-G5 critical value 141 microg/L and sCD30 critical value 10.3 microg/L. CONCLUSION: the immune state of recipients are evaluated by combine HLA-G5 and sCD30 which may be a simple and valid method.

[The study of the inducing donor specific immunological tolerance by GM-CSF and TGF-ßtreated donor's marrow cell].

AIM: to observe whether the donor's marrow (BM) cells treated by GM-CSF and TGF-ß in vitro were beneficial in inducing specific hypoergia to donor's lymphocyte in recipient. METHODS: donor's marrow cell were cultured with GM-CSF and TGF-ß in vitro and its maturities were tested by treating with LPS. BALB/c mice were divided into three groups and received treatment as following: (1) BM pretreatment group: donor's BM cell mentioned above were injected into caudal vein with 10(5); cell each mouse; (2) donor's splenocyte group: C57BL/6 mouse's splenocyte were injected through caudal vein with 10(5); cell each mouse; (3) Negative control group: PBS with the same volume were injected through caudal vein. The treatment were performed twice with one week internal in each group. One week after the second treatment, all of the mice were challenged by splenocyte of C57BL/6 mouse in abdominal cavity with 10(5); cell each mouse. Three days later, the allo-reactivities were tested: the proliferation of recipient mouse lymphocyte to donor's lymphocyte were test by single mix lymphocyte reaction, the level of serum IFN-γ and IL-10 were tested with ELISA methods. CD4(+);CD25(high); Treg cell and expression of killer cell lectin-like receptor G1 (KLRG1) on NK cell were tested with flow cytometric technique and the NK cytotoxicity were measured by LDH release method. RESULTS: the donor's marrow cell treated by GM-CSF and TGF-ß in vitro could resist the maturation promotion by LPS. Compared with donor's lymphocyte pretreatment, pre-treatment with BM cell induced decreased IL-10, increased CD4(+);CD25(high); Treg cell in spleen, decreased proliferation to donor's lymphocyte in vitro. Additionally, NK cell cytotoxicity was also decreased. CONCLUSION: the donor's marrow cell treated by GM-CSF and TGF-ßcan induce the donor specific tolerance in some degree, and modulation of NK cell maybe participate in inducing immunological tolerance.

Study on systemic immune tolerance induction in rat islet transplantation by intravenous infusion of Sertoli cells.

BACKGROUND: Sertoli cells are usually co-transplanted with pancreatic islets to induce local immune tolerance. In this report, we used infusion with Sertoli cells in islet transplantation to induce systemic immune tolerance and studied the mechanism of the tolerance induction. METHODS: Streptozotocin-induced diabetic rats were divided into four groups before islet transplantation: group A as control; group B with intravenous infusion of Sertoli cells; group C with Sertoli cell infusion and Fas ligand antibody treatment; and group D with Sertoli cell infusion and transforming growth factor-beta1 antibody treatment. The mean survival time (MST) and insulin expression of islet grafts were measured. The number of lymphocytes and the levels of cytokines in peripheral blood were also measured. RESULTS: Group B had the longest MST of islet allografts (41.6+/-4.20 days) followed by groups C, D, and A (P<0.05). Immunohistochemistry showed similar results with MST. The rats in group B had the least CD4 T cells (only 15.6%+/-6.4%) compared with other groups (P<0.05). The numbers of CD8 T cells in rats of groups B (11.2%+/-4.3%) and D (14.5%+/-5.6%) were significantly lower than those of groups A and C (P<0.05). After transplantation, group B's interleukin (IL)-2 level (1.92+/-0.68 ng/mL) was found to be significantly lower than that of other groups (P<0.05). Interferon-gamma showed similar pattern of change as IL-2 (P<0.05). Groups A and D had significantly lower levels of IL-4 (4.31+/-1.97 pg/mL 4.69+/-1.33 pg/mL, respectively) than groups B and C (P<0.05). CONCLUSION: Infusion of Sertoli cells could effectively prolong the survival of islet grafts and reduce peripheral blood lymphocyte and cytokine levels. In this process, transforming growth factor-beta1 played a major role and Fas ligand played a smaller additional role.

[Comparison of different isolation methods on adult Sertoli cells co-transplanted with islets].

AIM: To establish a more convenient and effective method for isolating adult Sertoli cells and apply the method to islet transplantation. METHODS: Trypsin, DNase, collagenase and hyaluronidase were used for a one-step digestion in group A. A two-step digestion was used in group B, in which testis tissues were first digested by trypsin and DNase and then were digested by collagenase and hyaluronidase. In group C, trypsin and DNase were used in the first step of digestion, hyaluronidase was used in the second step and collagenase was used in the third step. Sertoli cells were identified by morphology and immunohistochemistry and the viability and purity of Sertoli cells were detected by MTT and flow cytometry (FCM). Expression of Fas-L was detected by Western blot and the effects of the co-transplantation of islets and Sertoli cells were compared between the three groups. RESULTS: Typical Sertoli cells were seen after isolation using the three different methods. Sertoli cells isolated by method A and method B were large in number while those isolated by method B and method C were pure. The results of MTT showed that the viability of Sertoli cells in group B was significantly higher than that in group A and group C (P<0.05) and Western blot results showed that expression of Fas-L on Sertoli cells in group B was significantly stronger than that in group A and group C (P<0.05). The purity of Sertoli cells detected by FCM in group B and group C were significantly higher than that in group A (P<0.05). The survival of islets co-transplanted with Sertoli cells to renal capsule of diabetic mice was significantly longer in group B compared with that in control group as well as in group A and group C (P<0.05). CONCLUSION: Sertoli cells obtained by the two-step digestion method are of higher purity and viability, which significantly prolong the survival of islet graft by co-transplantation.

[Construction and identification of recombinant adenovirus vector expressing IkappaBalpha-IRES2-shCD40L].

AIM: To construct adenovirus expressing vector secretory human CD40L extracellular domain (shCD40L) and IkappaBalpha. METHODS: The gene of the shCD40L and the secreting signal peptide was amplified with PCR respectively. Then the CD40L and signal peptide was colligated to get shCD40L segment in vitro. The gene which had cuted from PODB7I kappaBalpha and IRES2 were amplified with PCR respectively. After colligated successfully, the gene shCD40L, IRES2, IkappaBalpha were inserted into pGEMT-easy to amplify. The gene shCD40L was linked with EGFP and the gene IkappaBalpha was linked with IRES2. The two pieces of recombinant gene were inseeted into pshuttle-cmv vector. The pshuttle-cmv-IkappaBalpha-EGFP plasmid and pshuttle-cmv-shCD40L-IRES2 plasmid were transdcut into AdEasy adenovirus vector. System and acquired the recombinant plasmid pAdvIkappaBalpha-IRES2-shCD40L. The pAdvIkappaBalpha-IRES2-shCD40L plasmid harboring was constructed by homologous recombination in E.coil AdEasy-1-BJ5183. Then recombinant vector was propagated in 293 cells and obtain the recombination replication-deficient adenovirus AdvIkappaBalpha-IRES2-shCD40L. PCR method was used in identification of recombinant adenovirus vector harboring IkappaBalpha-IRES2-shCD40L gene. Expressing products in supernatant adenovirus was identified by PCR method. The safety was evaluated by morphology of PK15 and 293 cells after adenovirus infection. RESULTS: The recombinant IkappaBalpha-IRES2-shCD40L adenovirus was generated by homologous and identified by PCR methods. The adenovirus titre reached 6.561 x 10(12) pfu/L. The adenovirus didn't replicate in PK15 cells. CONCLUSION: The IkappaBalpha-IRES2-shCD40L adenovirus is prepared successfully and its biotic safety is confirmed.