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.

Comprehensive assessment of deceased donor kidneys with clinical characteristics, pre-implant biopsy histopathology and hypothermic mechanical perfusion parameters is highly predictive of delayed graft function.

Background: Due to the current high demand for transplant tissue, an increasing proportion of kidney donors are considered extended criteria donors, which results in a higher incidence of delayed graft function (DGF) in organ recipients. Therefore, it is important to fully investigate the risk factors of DGF, and establish a prediction system to assess donor kidney quality before transplantation.Methods: A total of 333 donation after cardiac death kidney transplant recipients were included in this retrospective study. Both univariate and multivariate analyses were used to analyze the risk factors of DGF occurrence. Receiver operating characteristic (ROC) curves were used to analyze the predictive value of variables on DGF posttransplant.Results: The donor clinical scores, kidney histopathologic Remuzzi scores and hypothermic mechanical perfusion (HMP) parameters (flow and resistance index) were all correlated. 46 recipients developed DGF postoperatively, with an incidence of 13.8% (46/333). Multivariate logistic regression analysis of the kidney transplants revealed that the independent risk factors of DGF occurrence post-transplantation included donor score (OR = 1.12, 95% CI 1.06-1.19, p < 0.001), Remuzzi score (OR = 1.21, 95% CI 1.02-1.43, p = 0.029) and acute tubular injury (ATI) score (OR = 4.72, 95% CI 2.32-9.60, p < 0.001). Prediction of DGF with ROC curve showed that the area under the curve was increased to 0.89 when all variables (donor score, Remuzzi score, ATI score and HMP resistance index) were considered together.Conclusions: Combination of donor clinical information, kidney pre-implant histopathology and HMP parameters provide a more accurate prediction of DGF occurrence post-transplantation than any of the measures alone.

Outcomes for primary kidney transplantation from donation after Citizens' death in China: a single center experience of 367 cases.

BACKGROUND: The cases of donation after brain death followed by circulatory death (DBCD) and donation after cardiac death (DCD) have been increased year by year in China. Further research is needed to understand in the outcomes and risk factors of delayed graft function (DGF) in order to minimize the risk of DGF and ameliorate its potential impact on long-term outcomes. This study was to explore the differences in outcomes between DBCD and DCD transplant and the main risk factors for DGF in DBCD. METHODS: Retrospective analysis of the clinical data of 367donations after citizens' death kidney transplant procedures (donors and recipients) between July 2012 and August 2015 at our center. RESULTS: During the study period, the donation success rate was 25.3%. 164 cases of DBCD and 35 cases of DCD had been implemented and 367 kidneys were transplanted. The incidence of DGF in DBCD group were significantly lower than that of DCD group (12.0% vs. 27.0%, p = 0.002). The 1-year percent freedom from acute rejection (AR) was significantly higher in DBCD group compared with it of DCD group (94% vs. 82%, p = 0.036). Multivariate logistic regression analysis of the kidney transplants revealed that the high risk factors for DGF after renal transplantation in DBCD were history of hypertension (Odds Ratio [OR] = 5.88, 95% CI: 1.90 to 18.2, p = 0.002), low blood pressure (BP < 80 mmHg) (OR = 4.86, 95% CI: 1.58 to 14.9, p = 0.006) and serum creatinine of donor (OR = 1.09, 95% CI: 1.03 to 1.16, p = 0.003) before donation. CONCLUSIONS: The outcomes of DBCD could be better than DCD in DGF and AR. The main risk factors for DGF in DBCD kidney transplants are donors with a history of hypertension, low blood pressure, and serum creatinine of donor before donation.

Influence of de novo donor-specific antibody on early renal allograft function recovery.

BACKGROUND: The aim of the present study is to investigate the impact of de novo donor-specific antibodies (dnDSA) on early graft function, to provide objective reference for early clinical diagnosis and reasonable individualized treatment. METHODS: 305 cases of renal transplant patients for the first time were observed in this study. Follow-up time for all recipients was 6 months after operation. HLA antibody, DSA, renal function were monitored after transplant. RESULTS: In total of 305 cases, 66 cases (21.64%) were HLA antibody positive and 21 cases (6.89%) showed acute rejection (AR) in 6 months after transplant. The HLA antibody-positive patients included six cases of dnDSA-positive and 60 cases of dnDSA-negative. The incidence of AR was 2.09% (5/239) in HLA antibody-negative patients, 18.33% (11/60) in HLA antibody positive with DSA-negative patients, and 83.33% (5/6) in HLA antibody-positive patients with DSA-positive. There was a big difference between DSA-negative and DSA-positive patients (p < 0.01). The recovery time of AR patients with DSA-positive were longer than DSA-negative patients, and the recovery graft function of AR patient with DSA-positive were not as good as those with DSA-negative. CONCLUSIONS: The appearance of dnDSA in the early stage of kidney transplantation is a warning sign of AR occurrence. Dynamic monitoring of HLA antibody and DSA could predict the state of graft function, and play an important role in the prevention of AR, timely and effectively.

[Endothelial cells promote islet survival and function].

OBJECTIVE: To investigate islet graft survival and function after co-culture and co-transplantation with vascular endothelial cells (ECs) in diabetic rats. METHODS: We isolated ECs, and assessed the viability of isolated islets in a group of standard culture and a group of co-culture with ECs. Then we put the diabetic rats in 4 groups: an islet transplantation group, an islet graft with EC transplantation group, an EC transplantation group, and a PBS control group. Blood glucose and insulin concentrations were measured daily. Cell morphology and cell markers were investigated by immunohistochemical staining and electron microscope. RESULTS: Normal morphology was shown in more than 90% of AO/PI staining positive islets while co-cultured with ECs for 7 days. Insulin release assays showed a significantly higher simulation index co-culture except for the first day (P<0.05). There was a significant difference in concentrations of blood glucose and insulin among the 4 groups after 3 days after the transplantation (P<0.05). CONCLUSION: EC-islet co-culture can improve the function and survival of isolated islets in vitro, and EC-islet co-transplantation can effectively prolong the islet graft survival in diabetic rats.

The causal effect of triglyceride and high blood pressure on IgA nephropathy: a Mendelian randomization study.

Background: It has been reported that high blood pressure (HBP) and triglyceride (TG) are considered risk factors in immunoglobulin A nephropathy (IgAN). This study aimed to explore the causalities between HBP and TG, and IgAN on the basis of Mendelian randomization (MR) analysis. Methods: Firstly, the genome-wide association study (GWAS) summary data of IgAN (GCST90018866) and two exposure factors, TG (ukb-d-30870_raw) and HBP (ukb-a-437), were sourced from the GWAS Catalog and Integrative Epidemiology Unit (IEU) OpenGWAS databases, respectively. In this study, five methods were utilized to perform MR analysis after picking out single nucleotide polymorphisms (SNPs) as instrumental variables, including MR-Egger, weighted median, simple mode, weighted mode, and inverse variance weighted (IVW), followed by the sensitivity analysis containing the heterogeneity, horizontal pleiotropy test and leave-one-out (LOO) analysis. Finally, the enrichment analysis and interaction network construction of genes corresponding to SNPs of HBP and TG were performed. Results: The univariate MR results revealed that HBP and TG regarded as risk factors were causally related to IgAN [TG: p = 0.046, odds ratio (OR) = 1.065, 95% confidence interval (CI) = 1.001-1.133; HBP: p = 7.09 × 10-7, OR = 1.970, 95% CI = 1.507-2.575] based on random-effect IVM method, of which TG had a weaker impact. The reliability of these univariate MR results was certified by the sensitivity analysis, in which there was no horizontal pleiotropy and exaggerated influence of each SNP. Furthermore, HBP was markedly causally related to IgAN (p = 0.000512) with the help of multivariate MR analysis, rather than TG (p = 0.332). Therefore, when HBP and TG occur simultaneously, HBP is a direct influencing factor on IgAN. Ultimately, a total of 208 and 153 genes separately corresponding to SNPs of TG and HBP were included in enrichment analysis, and thereinto, genes relevant to TG were mainly enriched in lipid homeostasis and cholesterol metabolism, while genes concerned with HBP played their roles in regulation of cell growth, aldosterone synthesis and secretion and so forth. Conclusion: TG and HBP as risk factors were causally connected with IgAN, of which HBP was strongly related to the onset of IgAN, providing more reliable evidence for further exploring the relationship between TG and HBP and IgAN.

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.

Blockade of the nuclear factor kappa B pathway prolonged islet allograft survival.

Nuclear factor kappa B (NF-κB) pathway is known for its important role in the upregulation of various inflammatory mediators and its "switch regulator" functionality for transcription factor gene networks which control cytokine-induced ß-cell dysfunction and death. In this study, the islets were divided into the control group, Ad-green fluorescent protein, and the adenovirus transfected with inhibitor kappa B group. The proliferation index of peripheral blood mononuclear cells and the islets apoptosis index were examined after mixed lymphocyte-islet reaction with inverted fluorescence microscopy. Moreover, mRNA expression of inflammatory cytokines was measured by reverse transcription polymerase chain reaction. The islet graft survival time in diabetic rats, insulin in grafts, and cytokine concentrations in the supernatant were determined by immunohistochemistry and enzyme-linked immunosorbent assay. We found that blocking of NF-κB activation in ß-cells significantly downregulated inflammatory chemokine production by islets cells in vitro and in vivo, inhibited T-cell recruitment into the pancreatic islets, inhibited ß-cell dysfunction, and effectively prolonged the survival time of islet grafts. The results presented in this work highlight a novel mechanism of blocking NF-κB activation in ß-cells for the treatment of islet cell transplantation.

Assessment of different biomarkers provides valuable diagnostic standards in the evaluation of the risk of acute rejection.

Acute rejection (AR) is a strong risk factor for chronic rejection in renal transplant recipients. Accurate and timely diagnosis of AR episodes is very important for disease control and prognosis. Therefore, objectively evaluated the immune status of patients is essential in the field of post-transplantation treatment. This longitudinal study investigated the usefulness of five biomarkers, human leukocyte antigen (HLA)-G5 and sCD30 level in sera, intracellular adenosine triphosphate (iATP) release level of CD4(+) T cells, and granzyme B/perforin expression in peripheral blood mononuclear cells (PBMCs) and biopsies, to detect AR and the resolution of biomarkers in a total of 84 cases of renal transplantation. The data demonstrated that recipients with clinical or biopsy proven rejection significantly increased iATP release level of CD4(+) T cells, and elevated sCD30 but lowered HLA-G5 level in sera compared with individuals with stable graft function. Expression levels of granzyme B and perforin were also elevated in PBMCs and graft biopsies of AR patients. Taken together, we identified that upregulation of sCD30, iATP, granzyme B, perforin, and downregulation of HLA-G5 could provide valuable diagnostic standards to identify those recipients in the risk of AR. And iATP may be a better biomarker than others for predicting the graft rejection episode.

Combination therapy with diltiazem plus CsA/MMF/Pred or CsA/Aza/Pred triple immunosuppressive regimens for use in clinical kidney transplantation in Northwestern China.

OBJECTIVE: The effects of diltiazem on 1692 kidney transplant recipients under the immunosuppressive regimen of cyclosporine A (CsA) in combination with either mycophenolate mofetil or azothioprine were assessed. The two treatment groups were compared for blood concentrations of CsA, the extent of acceptable dosage reduction for the maintenance of immunotherapy, potential effects of kidney protection, and promotion of graft function. METHOD: We monitored changes of blood concentrations of CsA in the two different patient treatment groups for post-transplant graft function, episodes of acute rejection, and hepatic and renal toxicity in 1640 renal transplant recipients after treatment with diltiazem. RESULTS: In patients treated with the triple immunosuppressive regimen consisting of CsA, azothioprine, and prednisolone (Pred), the sub-group of patients receiving the diltiazem treatment saw a significantly reduced CsA dosage in comparison to the non-diltiazem group (control group 1) (P < 0.05), but the blood concentrations of CsA of the diltiazem group were higher than those of control group 1 (P < 0.01). Of the patients treated with CsA, mycophenolate mofetil, and Pred, the sub-group of patients also treated with diltiazem showed similar effects: CsA dosage was reduced (P < 0.01) and the blood concentrations of CsA significantly increased (P < 0.01) in comparison with those of control group 2. In addition, recovery time of graft function decreased to 4.7 ± 1.8 days and 3.9 ± 1.4 days in the two diltiazem treatment groups, respectively (P < 0.05), and the rate of acute rejection decreased to 21 (p < 0.05) and 7.9% (P < 0.01), respectively. CONCLUSION: In our cohort of renal transplantation patients, co-administration of CsA and diltiazem increased CsA blood concentration, thereby resulting in a reduction in its required dosage treatment, which lightened the patients' economic burden while improving primary and long-term kidney function by promoting the recovery of graft function and decreasing hepatic and renal toxicity. The co-administration of diltiazem may also reduce the rate of acute rejection, especially in patients who also receive the triple immunosuppressive regimen consisting of CsA, mycophenolate mofetil, and Pred.

Improved survival and function of rat cryopreserved islets by coculture with sertoli cells.

In order to investigate how to improve the function and survival of cryopreserved islets, we cocultured cryopreserved thawed rat islets with rat Sertoli cells. After thawing, the islets were divided into the Sertoli cell coculture group and the control group. Using light and transmission electron microscopes, we examined the morphology of islets and measured their apoptosis index (AI) and insulin release stimulation index (SI). Moreover, we measured apoptosis protein and mRNA by western-blot and reverse transcription polymerase chain reaction and cytokine concentrations in supernatant by ELISA. We examined islet graft survival time in diabetic mice and detected insulin in grafts by immunohistochemistry. We found that the morphology, AI, and SI of the coculture group were all significantly improved. The relative expression levels of cleaved caspase-3 P20, P11, and caspase-7 in the coculture group were lower than those in the control group. Compared with the control group, the expression level of Bax was decreased, but that of Bcl-2 was increased. After transplantation, islet survival in the coculture group was similar to that of fresh islets but longer than that in the control group. These results suggest that coculture with rat Sertoli cells significantly improves the yield and function of rat cryopreserved thawed islets by effectively reducing islet apoptosis.

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.

Long-term follow-up of co-administration of diltiazem and cyclosporine in Chinese kidney transplant recipients.

BACKGROUND: Co-administration of diltiazem and cyclosporine A (CsA) in kidney transplant recipients shows improvement of renal transplantation outcomes. METHODS: We respectively analyzed 1531 kidney transplant recipients treated by different immunosuppressive therapy schemes from 1986 to 2003. They were divided into three groups depending on their immunosuppressive therapy schemes: control group with a standard triple therapy without use of diltiazem; study group I with the combination of diltiazem and the standard triple therapy but slightly low CsA; study group II with combination of diltiazem and a modified standard triple therapy but lower CsA. The CsA blood concentrations, posttransplant complications, and long-term survival in the three groups were compared. RESULTS: The results showed that the patient and allograft survival in the study group II was 69.9 and 65.1%, respectively, significantly higher than that in the control group (50.7 and 47.6%). Occurrence of hepatotoxicity and nephrotoxicity episodes was higher in the control group than those in the study group I and the study group II. The incidence of acute rejection in the control group was 30.3% (23/76), similar to 28.0% (184/657) in study group I, but statistically significantly higher than 7.6% (61/798) in the study group II. CONCLUSION: Combination of diltiazem and CsA in the kidney allograft recipients tends to reduce the CsA oral dosage, improve patient survival, and decrease the occurrence of hepatotoxicity and nephrotoxicity.

[Living-related donor kidney transplantation in 158 patients].

OBJECTIVE: To introduce clinical experience for living-related donor kidney transplantation (LDKT) by reviewing LDKT clinical data. METHODS: A total of 158 patients underwent LDKT. Expect for 7 patients donated by their spouses, the others had blood relationship donors. Donor-recipient HLA matching showed 2 patients had 5-loci mismatch, 5 with 4-loci mismatch, 88 with 3-loci mismatch, 50 with 2-loci mismatch, 12 with 1-loci mismatch, the other 1 with 0-loci mismatch. All of the 158 donors underwent open nephrectomy, 35 of whom donated the right kidneys and the other 123 donated the left kidneys. Triple immunosuppressive regimen consisted of calcineurin inhibitors or FK506, MMF or AZa, and steroid. RESULTS: All donors were healthy after the operation. All donors were followed up for 6 to 12 months and blood exams showed that inosine levels were normal. The longest kidney transplant functional survival time was 10 years to up June 2008. The one year patient/graft survival rate was 95.5%. Delayed graft function (DGF) occurred in 5 patients, 4 of whom recovered in 2-5 weeks. Five patients died, 4 of whom died of post-operational pulmonary infection within 3-5 months, with no transplantational complications. The other one died of pulmonary bleeding during dialysis while treating for DGF. One patient received a second deceased kidney transplant because of hyperacute rejection during the surgery. Five developed acute rejection 1 month after the operation (incidence rate 3.16%), 4 of whom were cured by administration of methylprednisolone, and the other one returned to dialysis because of renal toxicity of cyclosporine. Three patients had positive chronic rejection, 2 of whom lost graft function in 1.5-3.5 years. Eight patients developed pulmonary infection and 4 of them were cured. CONCLUSION: Sufficient LDKT pre-operational assessment, satisfactory tissue matching and reduced ischemia time may result in lower incidence of DGF, acute rejection and higher patient/graft survival rate. In LDKT, importance should also be attached to the prevention of DGF and graft rejection. Rational dosage of immunosuppressants is advocated to prevent secondary infective complications. Donor specifications and all around evaluation of the living-related donors should also be emphasized to minimize the harm to the donors. Long term follow-up is also essential to ensure donors' post-operational healthy life.

Application of kushenin on patients with chronic hepatitis C after renal transplantation.

OBJECTIVE: To evaluate the efficacy of kushenin in treating patients with chronic hepatitis C after renal transplantation. METHODS: Fifty-five patients were randomly assigned by lottery to the treatment group (29 cases) and control group (26 cases). The same immunosuppression therapy was given to all patients in both groups. Patients in the treatment group were treated with kushenin 0.6 g once a day, while those in the control group were treated with conventional liver protective agents such as vitamins. The treatment duration of both groups was 3 months. The incidences of serious hepatitis and acute rejection reaction, serum biochemistry parameters including indicators of liver and kidney functions, hepatic fibrosis index, and serum HCV-RNA were compared between the two groups. RESULTS: (1) The incidence of serious hepatitis in the treatment group and the control group was 3.45% (1/29 cases) and 11.54% (3/26 cases), respectively, which was insignificantly different between the two groups (P=0.335). (2) The incidence of acute rejection in the treatment group was 6.90% (2/29 cases) and that in the control group was 7.69% (2/26 cases), showing insignificant difference (P=0.335). (3) The differences in serum alanine aminotransferase (ALT), direct bilirubin (DBIL), hyaluronic acid (HA), propeptide collagen type III (PC III), laminin (LN), collagen type IV (Col IV) levels between the two groups were insignificant before transplantation (P>0.05), while the above-mentioned parameters in the treatment group were significantly lower than those in the control group after transplantation (P<0.05). The difference in serum creatinine (SCr) and endogenous creatinine clearance rate (CCr) between the two groups was insignificant before and after transplantation (P>0.05). (4) The negative conversion rate of HCV-RNA in the treatment group was 31.03% (9/29 cases), significantly higher than the value of 11.54% (3/26 cases) in the control group after transplantation (P<0.05). (5) The levels of serum ALT and DBIL in patients with HCV-RNA converted to negative were significantly lower than those with still-positive HCV-RNA (P<0.05). CONCLUSIONS: Kushenin has a certain effect on inhibiting the proliferation of HCV, protecting liver cells, and anti-liver fibrosis. On the other hand, it has no obvious influence on renal allograft function. Thus, the drug is clinically safe and effective for use in treating patients with chronic hepatitis C after renal transplantation.

Polyglycolic Acid Fibrous Scaffold Improving Endothelial Cell Coating and Vascularization of Islet.

BACKGROUND: Improving islet graft revascularization has become a crucial task for prolonging islet graft survival. Endothelial cells (ECs) are the basis of new microvessels in an isolated islet, and EC coating has been demonstrated to improve the vascularization and survival of an islet. However, the traditional method of EC coating of islets has low efficiency in vitro. This study was conducted to evaluate the effect of a polyglycolic acid (PGA) scaffold on the efficiency of islet coating by ECs and the angiogenesis in the coated islet graft. METHODS: A PGA fibrous scaffold was used for EC coating of islet culture and was evaluated for its efficiency of EC coating on islets and islet graft angiogenesis. RESULTS: In in vitro experiments, we found that apoptosis index of ECs-coating islet in PGA group (27% ± 8%) was significantly lower than that in control group (83% ± 20%, P < 0.05) after 7 days culture. Stimulation index was significantly greater in the PGA group than in the control group at day 7 after ECs-coating (2.07 ± 0.31 vs. 1.80 ± 0.23, P < 0.05). vascular endothelial growth factor (VEGF) level in the PGA group was significantly higher than the coating in the control group after 7 days culture (52.10 ± 13.50 ng/ml vs. 16.30 ± 8.10 ng/ml, P < 0.05). Because of a tight, circumvallated, adhesive and three-dimensional growth microenvironment, islet cultured in a PGA scaffold had higher coating efficiency showing stronger staining intensity of enzyme than those in the control group after 14 days of culture following ECs-coating. For in vivo study, PGA scaffold significantly prolonged the average survival time of EC-coated islet graft after transplantation compared with control group (15.30 ± 5.60 days vs. 8.30 ± 2.45 days, P < 0.05). The angiogenesis and area of survived grafts were more in the PGA group compared with the control group by measuring the mean microvessel density (8.60 ± 1.21/mm2 vs. 5.20 ± 0.87/mm2, P < 0.05). In addition, expression of VEGF and tyrosin-protein kinase receptor (Tie-2) gene increased in PGA scaffold group than that in control group by real-time reverse transcription-polymerase chain reaction analysis. CONCLUSIONS: These results demonstrate that the efficiency of EC coating of islets was successfully increased by culturing ECs on a PGA scaffold. This method enhances the function, survival, and vascularization of isolated islets in vitro and in vivo.

Inactivation of p27kip1 Promoted Nonspecific Inflammation by Enhancing Macrophage Proliferation in Islet Transplantation.

Islet transplantation suffers from low efficiency caused by nonspecific inflammation-induced graft loss after transplantation. This study reports increased islet loss and enhanced inflammatory response in p27-deficient mice (p27-/-) and proposes a possible mechanism. Compared with wild type, p27-/- mice showed more severe functional injury of islet, with increased serum levels of inflammatory cytokines IL-1 and TNF-α, inducing macrophage proliferation. Furthermore, the increased number, proapoptotic proteins, and nuclear factor-kappa b (NF-κB) phosphorylation status of the infiltrating macrophages were accompanied by increased TNF-α mRNA level of islet graft site in p27-/- mice. Moreover, in vitro, we found that macrophages were still activated and cocultured with islet and promoted islet loss even blocking the direct effect of TNF-α on islets. Malondialdehyde (MDA, an end product of lipid peroxidation) in islet and media were increased after cocultured with macrophages. p27 deficiency also increased macrophage proliferation and islet injury. Therefore, p27 inactivation promotes injury islet graft loss via the elevation of proliferation and inflammatory cytokines secretion in infiltrating macrophages which induced nonspecific inflammation independent of TNF-α/nuclear factor-kappa b pathway. This potentially represents a promising therapeutic target in improving islet graft survival.

Small intestinal submucosa improves islet survival and function during in vitro culture.

AIM: To evaluate the recovery and function of isolated rat pancreatic islets during in vitro culture with small intestinal submucosa (SIS). METHODS: Pancreatic islets were isolated from Wistar rats by standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and Euroficoll purification. Purified islets were cultured in plates coated with multilayer SIS (SIS-treated group) or without multilayer SIS (standard cultured group) for 7 and 14 d in standard islet culture media of RPMI 1640. After isolation and culture, islets from both experimental groups were stained with dithizone and counted. Recovery of islets was determined by the ratio of counts after the culture to the yield of islets immediately following islet isolation. Viability of islets after the culture was assessed by the glucose challenge test with low (2.7 mmol/L) and high glucose (16.7 mmol/L) solution supplemented with 50 mmol/L 3-isobutyl-1-methylxanthine (IBMX) solution. Apoptosis of islet cells after the culture was measured by relative quantification of histone-complexed DNA fragments using ELISA. RESULTS: After 7 or 14 d of in vitro tissue culture, the recovery of islets in SIS-treated group was significantly higher than that cultured in plates without SIS coating. The recovery of islets in SIS-treated group was about twice more than that of in the control group. In SIS-treated group, there was no significant difference in the recovery of islets between short- and long-term periods of culture (95.8+/-1.0% vs 90.8+/-1.5%, P>0.05). When incubated with high glucose (16.7 mmol/L) solution, insulin secretion in SIS-treated group showed a higher increase than that in control group after 14 d of culture (20.7+/-1.1 mU/L vs 11.8+/-1.1 mU/L, P<0.05). When islets were placed in high glucose solution containing IBMX, stimulated insulin secretion was higher in SIS-treated group than in control group. Calculated stimulation index of SIS-treated group was about 23 times of control group. In addition, the stimulation index of SIS-treated group remained constant regardless of short- and long-term periods of culture (9.5+/-0.2 vs 10.2+/-1.2, P>0.05). Much less apoptosis of islet cells occurred in SIS-treated group than in control group after the culture. CONCLUSION: Co-culture of isolated rat islets with native sheet-like SIS might build an extracellular matrix for islets and provide possible biotrophic and growth factors that promote the recovery and subsequent function of islets.

Isolation and culture of adult Sertoli cells and their effects on the function of co-cultured allogeneic islets in vitro.

BACKGROUND: Globally, 180 million people suffer from diabetes mellitus. Islet transplantation is believed to be an almost ideal therapy for insulin-dependent patients. How to maintain the viability and the function of isolated human islets is a challenge in clinical practice. Sertoli cells are considered 'nurse cells' in the seminiferous tubules and have been used in cell graft protocols for neurodegenerative diseases and diabetes in many studies. Many researchers have used immature murine testes as the primarily source of Sertoli cells in islet transplantation because they are easily purified. Mature human Sertoli cells have been seldom investigated. In the present study, we developed a method for the isolation and culture of Sertoli cells derived from adult human testes, and investigated their effects on the function of allogeneic islets when they were cultured together in vitro. METHODS: Adult Sertoli cells were prepared successfully by two-step enzyme digestion with trypsin, collagenase and hyaluronidase. They were identified by morphological characteristics and their activity was determined by MTT colorimetry over a 28-day culture time in vitro. A glucose-stimulated insulin secretion test was performed to detect the effects of Sertoli cells on allogeneic islets' function when they were co-cultured for 21 days in vitro. RESULTS: In cultured cells, mature human Sertoli cells accounted for more than 90% of total cells. The activity of Sertoli cells reached 95% and they remained highly cytoactive for a long time in vitro (P > 0.05). Compared with the islets cultured alone, the co-cultured islets with allogeneic Sertoli cells maintained higher sensitivity to glucose stimulation for the duration of the experiment (P < 0.01). CONCLUSIONS: A method of isolation and culture of Sertoli cells from adult testes has been established. Sertoli cells could enhance allogeneic islets' function when they were co-cultured in vitro. They could be a helper cell in islet transplantation.

Effect of small intestinal submucosa on islet recovery and function in vitro culture.

BACKGROUND: The ability to maintain isolated human islet preparation in tissue culture has recently been adopted by most islet transplant centers to improve the safety and practicality of islet transplantation. However, maintaining islet viability and recovery remains a challenge in clinical setting. Extracellular matrix (ECM) is one of the most important components of islet microenvironment. The reconstruction of the cell-matrix relationship seems to be effective in improving the loss of differentiated islet structure and function. Small intestinal submucosa (SIS), a naturally occurring ECM, has been investigated to be able to promote wound healing, tissue remodeling, and cell growth. The purpose of this study was to evaluate the recovery and function of isolated rat pancreatic islets after in vitro culture with SIS. METHODS: Pancreatic islets were isolated from Wistar rats by using standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation, and EuroFicoll purification. Groups of purified islets were cultured in plates which were coated with multilayer SIS (SIS-treated group) or without (standard cultured group) for 7 days and 14 days in standard islet culture conditions of RPMI 1640 tissue culture media in humidified atmosphere containing 95% air and 5% CO2 at 37 degree centigrade. The mean recovery of islets after the culture period was determined by sizing duplicate counts of a known volume and their viability was assessed by static incubation with low glucose (2.7 mmol), high glucose (16.7 mmol) and high glucose solution supplemented with 50 mum 3-isobutyl-1-methylxanthine (IBMX) solution. RESULTS: After 7 days and 14 days of in vitro tissue culture, the SIS-treated group showed a significantly higher recovery compared with those cultured under standard conditions. The recovery in the SIS-treated group was about two times of the control group cultured in standard conditions after 14 days culture. In the SIS-treated group, there was no statistically difference between the short and long periods of culture(95.8+/-1.0% vs. 90.8+/-1.5%, P>0.05). During incubation in high glucose (16.7 mmol) solution, there was a 2-3 fold increase in insulin secretion from both groups,but the SIS-treated group showed a higher increase than the standard cultured group after 14-day culture (20.7+/-1.1 mU/L vs. 11.8+/-1.1 mU/L, P<0.05). When islets were placed in the high glucose solution supplemented with IBMX, the stimulated insulin response in the SIS-treated group was higher than that in the standard cultured group in spite of the duration of the culture. The stimulation index of the SIS-treated group was about 2-3 times of the standard cultured group. In addition, after a long period of culture, the stimulation index of the SIS-treated group was statistically equivalent with that of the short period of culture (9.5+/-0.2 vs. 10.2+/-1.2, P>0.05). CONCLUSIONS: The co-culture of isolated rat islets with native sheet-like SIS can provide an excellent extracellular matrix, possible biotrophic and growth factors that promote the recovery and subsequent function of islets after in vitro tissue culture. In view of results of this study and rapid degradation of SIS in vitro, future studies will investigate the extended duration of culture and the effect of SIS on islets in vitro.