1,25(OH)2D3 prolongs islet graft survival by inflammatory inhibition.

OBJECTIVE: This study aimed to determine the protective effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) against islet graft loss. METHODS: Proliferation of tumor necrosis factor (TNF)-α-induced macrophages was determined in vitro after treatment with different concentrations of 1,25-(OH)2D3. Intraportal islet transplantation (IPIT) was performed with islets harvested from the Sprague-Dawley rats and transplanted to the diabetic rats. The transplanted rats were assigned to receive 1,25-(OH)2D3 or propylene glycol (control). Islet graft survival; inflammatory cytokine (TNF-α and interleukin [IL]-1); numbers and percentages of macrophages, CD4(+), and CD8(+) T cells in bloods; and expression of nuclear factor (NF)-κB and TNF-α were analyzed. Hematoxylin and eosin staining was performed. RESULTS: We found 100 mg/mL 1,25-(OH)2D3 per day to have the strongest inhibitory effect on macrophages. Survival time of islet grafts significantly increased in the rats receiving 1,25-(OH)2D3. There were fewer infiltrated inflammatory cells in both islet graft and adjacent tissue in the drug-treated rats with lower serum IL-1 and TNF-α. Furthermore, percentage of macrophages and expression of p-NF-κB p65 and TNF-α in graft sites were significantly lower in the treated rats. CONCLUSION: Our results demonstrated that 1,25(OH)2D3 prolongs islet graft survival by decreasing nonspecific inflammation in syngeneic IPIT through inhibiting TNF-α/NF-κB pathway and macrophage infiltration.

Comparative study of one-stitch versus Lich-Gregoir ureterovesical implantation for kidney transplants.

OBJECTIVE: To compare the outcomes of standard Lich-Gregoir technique and a modified one-stitch technique of ureteroneocystostomy in renal transplantation. PATIENTS AND METHODS: Data from 645 transplant recipients by two different ureteroneocystostomy techniques were retrospectively reviewed at the first Affiliated Hospital, Medical College of Xi'an Jiaotong University, between January 2002 and December 2007. RESULTS: There were 418 recipients in the Lich-Gregoir group and 227 in new one-stitch group. The overall ureteral complication rate for new one-stitch technique was 19.8 % (n = 45) as opposed to 15.79 % (n = 66) for the Lich-Gregoir technique. No significantly different rate of ureteral complications occurred in two groups (P > 0.05). In comparison, there was a higher proportion of hematuria at the limit of statistical significance in new one-stitch group (P < 0.05). Average operative time for the modified one-stitch and Lich-Gregoir techniques was 8.8 ± 1.4 and 21.9 ± 6.1 min, respectively (P < 0.05). Urinary tract infections, delayed graft function and rejection rates were not significantly different between the two groups (P > 0.05). CONCLUSION: Although the modified one-stitch technique may predispose patients to higher rates of hematuria, it has no significant difference in ureteral complications compared with the Lich-Gregoir group. Based on this large series and data analyses, we believe that this new technique will become one of our multiple choices in our setting.

Immune monitoring in kidney transplant recipients could predict acute rejection by a new method: flow cytometric microcarrier assay.

BACKGROUND: Peripheral blood lymphocytes (PBL) of kidney transplant recipients stimulated in vitro release tumor necrosis factor (TNF)-α and interferon (IFN)-γ into the supernate as detected by a flow cytometric microcarrier assay (FCMA) that we used to predict acute rejection episodes. METHODS: Fifty-two kidney transplant recipients were divided into 2 groups; stable function (STA; n = 30) and acute rejection (ARG; n = 22) for comparison with healthy volunteers (n = 10). PBL were stimulated for 8 hours with phorbol myphnistate acetate and ionomycin, thereafter detecting TNF-α and IFN-γ in culture supernates by FCMA. Receiver operating characteristics (ROC) procedures were used to assess the sensitivity and specificity to predict acute rejection. RESULTS: The fluorescence intensity of TNF-α and IFN-γ in culture supernates was significant higher among healthy controls than STA: 68.38 ± 28.59 vs 51.08 ± 34.05, respectively (P < .05). The intensity of TNF-α and IFN-γ in ARG (144.47 ± 81.21 and 116.61 ± 53.89, respectively) was significant higher than STA (P < .001). The sensitivity and specificity to predict acute rejection were 86.4% and 86.7%, respectively, when analyzed by ROC curves combining TNF-α and IFN-γ. The intensity in noncultured plasma from ARG or STA was significant lower than that in culture supernates from ARG and STA with sensitivity and specificity to predict acute rejection episodes of 63.6% and 73.3%, respectively, when combining TNF-α and IFN-γ. CONCLUSIONS: Monitoring the expression of TNF-α and IFN-γ in cell culture supernates after stimulation of kidney transplant recipient PBL in vitro using FCMA predicted acute rejection episodes.

Decreasing loss of cryopreserved-thawed rat islets by coculture with Sertoli cells.

OBJECTIVE: Cryopreserved-thawed rat islets were cocultured with Sertoli cells to examine whether they could decrease the loss and improve islet function. METHODS: Islets and Sertoli cells were harvested from the pancreas and the testis of Sprague-Dawley rats, respectively. Cryopreserved, stored islets were thawed and divided into groups of coculture with Sertoli cells versus single cells. We measured islets recovery rate and function. Apoptotic-related proteins and gene expressions were detected by Western blot and reverse-transcriptase polymerase chain reaction. Soluble factors secreted by Sertoli cells in to the supernate were detected by enzyme-linked immunosorbent assay. We compared islet graft survival times in diabetic mice. RESULTS: In contrast to the single culture controls, thawed islets cocultured with Sertoli cells exhibited improved morphology. Recovery rates and insulin secretion were significantly higher among coculture cells. Four soluble factors were detected in supernates from Sertoli cell cultures including transforming growth factor-ß, insulin-like growth factor-1, epidermal growth factor, and basic fibroblast growth factor. Expression of proapoptotic Bax and caspase 3, 7 were down-regulated while that of antiapoptotic Bcl-2 was up-regulated. Cotransplantation with Sertoli cells significantly prolonged islet graft survival. CONCLUSION: These results suggested that coculture with Sertoli cells significantly improved islet yields and function after thawing and depressed islet apoptosis.

Effects of astragalosides on cultured islets after cryopreservation in rats.

OBJECTIVE: To explore the effects of AST (astragalosides) on cultured rat islet yield, purity, and function after cryopreservation in rats. METHODS: Pancreatic islets were isolated from 30 Sprague-Dawley rats using the standard technique of collagenase P digestion and discontinuous Ficoll gradient purification. After thaw, the islets were randomly divided into AST group and control group (n=15). Next, the islet cells were cultured in AST-containing medium or standard medium for 7, 14, and 21 days after cryopreservation and thaw. The quantity, purity, and survival rate were calculated in the two groups before and after culture. Then the in vitro and in vivo function was observed in diabetic rats after islet transplantation. RESULTS: The quantity and purity of islets had no difference between the two groups before culture (P>.05) while the difference after culture was significantly (P<.05). The survival rate of islets was 48% in AST group and 32% in the control group 21 days after thaw (P<.05). After 3 days, there was significantly a higher simulation index in the AST group than in the control group (P<.05). There was a significant difference in blood glucose and insulin concentrations between the groups after 3 days (P<.05). CONCLUSION: AST can be added to the culture medium to reduce the loss of islet cryopreservation and be intravenously injected to improve culture islet function in vitro and prolong islet graft survival in diabetic rats.

Prolongation of islet graft survival using concomitant transplantation of islets and vascular endothelial cells in diabetic rats.

BACKGROUND: The transplantation of isolated islets of Langerhans is nearing acceptance as treatment of type 1 diabetes mellitus. Because the arterial and venous connections of the pancreas are disrupted during islet isolation, islets must be revascularized after transplantation. OBJECTIVE: To observe whether increased numbers of vascular endothelial cells in islets can affect the angiogenesis and function of the grafts. MATERIALS AND METHODS: Rats with streptozocin-induced diabetes were divided into 3 groups. The rats in group 1 received islet grafts under the capsule of the left kidney; rats in group 2 received combined vascular endothelial cell and islet transplants; and rats in group 3 served as controls. After the transplantation procedure, blood glucose and insulin concentrations were evaluated daily. Hematoxylin-eosin and immunohistochemical staining was used to detect expression of vascular endothelial growth factor antibodies in the diabetic rat kidneys. The mean microvascular density was also calculated. RESULTS: At 3 days posttransplantation, blood glucose and insulin concentrations returned to normal in group 2, however, they declined only slightly in group 1, and moderate hyperglycemia was present. There was a significant difference in blood glucose and insulin concentrations between the 2 groups after 3 days (P < .05). The mean (SD) microvascular density in group 2 was markedly higher than that in group 1 (12.58 [1.81] vs 10.38 [0.97] P = .04). CONCLUSION: This study suggests that concomitant transplantation of isolated islets with endothelial cells can prolong islet graft survival in diabetic rats.

Improved islet survival and funtion with rat endothelial cells in vitro co-culture.

OBJECTIVE: Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To preserve its function, transplanted islets must be revascularized because arterial and venous connections are disrupted during islet isolation. The current paradigm is that islet revascularization originates from the transplant recipient. This study was designed to test whether the function of isolated islets can be retained by co-culture with thoracic aorta endothelial cells in vitro. METHODS: Sprague-Dawley rats were used in this study. The endothelial cells (ECs) were isolated from the thoracic aorta. The viability of the isolated islets was assessed by acridine orange/propidium iodide (AO/PI) double staining. The islets were either placed in standard cultures (group A) or in co-cultures with ECs (group B). Islet viablity was assessed by an insulin release assay. RESULTS: The islets in group B exhibited normal morphology with >90% staining positive as detected by AO/PI with 7 days. Insulin release assays showed a significantly higher simulation index (SI) in group B compared with group A (P < .05) except on the first day. CONCLUSION: This study suggested that co-cultrue of freshly isolated rat islets with ECs improves postculture survival and islet function in vitro.

Clinical application of Cordyceps sinensis on immunosuppressive therapy in renal transplantation.

OBJECTIVE: We sought to explore the adjunctive effects of Cordyceps sinensis (CS) in clinical renal transplantation. MATERIALS AND METHODS: Patients (n = 202) were divided randomly by lottery into a treatment (n = 93) and a control group (n = 109). Patients in the treatment group were treated with CS 1.0 g 3 times a day in addition to the immunosuppressive regimen given to the control group. We compared patient and graft survivals, incidence, time and severity of acute rejection episodes, chronic allograft nephropathy (CAN), hepatotoxicity and nephrotoxicity, biochemistry parameters including indicators of liver and kidney functions, fats, proteinuria, dosages, and whole blood concentrations of cyclosporine (CsA). RESULTS: Patient and graft survival rates, serum creatinine (SCr), and blood urea nitrogen (BUN) were not significantly different between the 2 groups (P > .05). Serum uric acid (UA) and 24-hour urinary total protein (24-hour UTP) were significantly lower in the treatment group than in the control group (P < .05). The incidences (11.83% vs 15.60%) and times to acute renal allograft rejection (23.48 +/- 7.22 vs 22.27 +/- 8.03 days posttransplantation) were not significantly different between the treated and control groups (P > .05). Patients receiving thymoglobulin antirejection therapy (3 cases) were fewer in the heated versus control group (13 cases; P = .014). The incidences of hepatotoxicity and nephrotoxicity in the treated group were 12.90% and 19.35%, significantly lower than 24.77% and 33.94% in the control group, respectively (P < .05). At 2 to 6 months posttransplantation, the CsA dosages in the treated group were significantly lower than those in the control group (P < .05). The whole blood trough CsA concentrations in the treated group were significantly lower than those in the control group at 3 to 6 months posttransplantation (P < .05). The decreasing trends of the 2 aforementioned parameters in the treatment group were approximately linear among treated subjects compared with approximately quadratic in the control group (P < .05). The incidence of CAN in the treated group was 7.53%, which was significantly lower than 18.35% in the control group (P = .024). The 24-hour UTP level in CAN patients within the treated group was significantly lower than the control group after transplantation (P = .045). The differences in total bilirubin, SCr, serum UA, and total cholesterol levels among otherwise normal patients in the treated group were significantly lower than those among the control group (P < .05). CONCLUSIONS: The use of CS may allow decreased dosages and concentrations of CsA causing fewer side effects without an increased risk of acute rejection. In addition, CS with reduced dose CsA may decrease proteinuria and retard CAN progression.

Mycobacterium tuberculosis infection in solid organ transplant recipients: experience from a single center in China.

OBJECTIVE: We sought to explore the prevalence, clinical manifestations, diagnostic procedures, and treatment of tuberculosis (TB) after solid organ transplantation. PATIENTS AND METHODS: In this study, we retrospectively analyzed data of 1947 renal transplant recipients and 85 liver transplant recipients. RESULTS: TB developed in 28 organ transplant recipients with a prevalence of 1.38% (28/2032). The median interval between transplantation and development of TB was 32 months (range, 1-142 months). Mycobacterium tuberculosis isolation, histologic signs of caseating granulomas, and TB-DNA detection directly supported the diagnosis in 10 (35.71%), 7 (25.00%), and 5 (17.86%) patients, respectively. In addition, 6 patients (21.43%) highly suspected of TB infection received tentative antituberculosis treatment with favorable responses. Most renal transplant recipients (22/25; 78.57%) received isoniazid, rifampicin (or rifabutin), and ethambutal (or pyrazinamide) for a mean duration of 10 months (range, 6-14 months). Three liver transplant recipients received a different protocol: isoniazid, rifabutin, ethambutal, and ofloxacin for 3 months; then isoniazid and rifabutin for 6 months. Upon follow-up, 8 subjects (28.57%) died; 5 of the deaths were related to TB. During the antituberculosis therapy, toxic hepatitis was seen in 12 patients (42.86%); cyclosporine levels decreased in 15 patients (53.57%); and allograft rejection developed in 6 of them. CONCLUSIONS: The peak incidences of TB in liver and kidney transplantations are in the first year and after the first year posttransplantation, respectively. Response to antituberculosis treatment should be considered to make a diagnosis among patients highly suspected of TB infections. Except in special circumstances, antituberculosis treatment protocols including isoniazid and rifampicin for about 10 months seem significantly effective and tolerable for non-liver transplant patients. Fluoroquinolones should be emphasized in posttransplantation TB treatment.

Prolongation of islet allograft survival by coexpression of CTLA4Ig and CD40LIg in mice.

BACKGROUND: B7/CD28 and CD40/CD40L have been well established as important costimulatory pathways. Cytotoxic T lymphocyte-associated antigen-4 (CTLA4) delivers negative signals to antigen-presenting cells to down-regulate proinflammatory responses and competitively inhibits the binding of B7 and CD28. Signals from the CD40/CD40L costimulatory pathway also play an important role in acute rejection of organ grafts. METHODS: Recombinant adenoviruses Ad-sCD40LIg-IRES2-CTLA4Ig, Ad-CTLA4Ig, and Ad-sCD40LIg were constructed to express sCD40LIg and CTLA4Ig simultaneously or separately as described previously. Streptozocin-induced diabetic BALB/c mice were injected with recombinant adenovirus, receiving approximately 500 donor islets isolated from C57BL/6 mice under the left kidney capsule. Five groups were assigned according to the treatment: nontreated group, Ad-Shuttle-CMV-treated group, Ad-CTLA4Ig-treated group, Ad-sCD40LIg-treated group, and Ad-sCD40LIg-IRES2-CTLA4Ig-treated group. The islet graft mean survival time (MST) was evaluated in the present study. RESULTS: Compared to the islet graft MST of the nontreated group (7.3+/-0.82 days) or Ad-Shuttle-CMV-treated group (7.2+/-1.47 days), the Ad-CTLA4Ig-treated and Ad-CD40LIg-treated islet graft survivals in recipients were 56.3+/-13.71 days (P<.01) and 47.3+/-15.64 days (P<.05), respectively. The islet graft MST was dramatically prolonged to 116.3+/-20.32 days in the Ad-sCD40LIg-IRES2-CTLA4Ig-treated group (P<.01). CONCLUSION: Simultaneous blockade of the CD40/CD40L and B7/CD28 costimulatory pathways via coexpression of sCD40LIg and CTLA4Ig mediated by replication-defective adenovirus may be an acceptable method to induce immune tolerance.

Prolongation of renal allograft survival in rats by replication-defective recombinant adenovirus-mediated coexpression of CD40L and CTLA4Ig.

BACKGROUND: Blockade of costimulatory signals has been shown to prolong allograft survival. The aim of the present study was to investigate the effect of simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways by replication-defective adenovirus-mediated expression of secretable extracellular domain of human CD40L (shCD40L) and CTLA4Ig to prolong rats renal allograft survival. METHODS: We constructed Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig. Coexpression of shCD40L and CTLA4Ig was evaluated by confocal laser scanning microscopy. The function of these two molecules was examined in human mixed lymphocyte reactions (MLRs) in vitro and in experimental BN-to-LEWIS rat renal transplantation in vivo. RESULTS: Successful construction of Adv-shCD40L-IRES2-CTLA4Ig was confirmed by polymerase chain reaction. Coexpression of shCD40L and CTLA4Ig on human kidney cell line HK-2 cells after transfection was detected by direct immunofluorescence staining. Human MLR was inhibited to 52.2%+/-0.6% and 42.1%+/-0.2% of the vehicle control by Adv-shCD40L and Adv-CTLA4Ig, respectively. Adv-shCD40L-IRES2-CTLA4Ig resulted in further inhibition of MLR to 22.0%+/-0.2% of vehicle control. Transfection with Adv-shCD40L or Adv-CTLA4Ig alone prolonged renal graft survival to 24.8+/-2.5 days and 27.3+/-3.6 days, respectively, as compared to vehicle-treated controls (7.8+/-0.3 days). Cotransfection of both genes extended graft survival to 41.8+/-3.7 days. CONCLUSIONS: Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig, achieved simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways, Adv-shCD40L-IRES2-CTLA4 by Ig synergistically inhibited human T-cell proliferation in MLR, and prolonged rats renal allograft survival.