Prolongation of islet xenograft survival by cryopreservation

Our attempt to reduce islet immunogenicity by slow cooling to -40 degrees C, storage at -196 degrees C, and rapid thawing is based on the differential susceptibility of various cell types to a freeze-thaw process. Five hundred rat islets (greater than or equal to 100 micron) were immediately implanted or cryopreserved and then implanted beneath the renal capsule of streptozocin-induced diabetic mice with or without an injection of anti-lymphocyte serum at the time of transplantation. Thirteen days after transplantation, all fresh xenografts had rejected, whereas 37.5% of cryopreserved grafts were still functioning. In immunosuppressed mice, 6.2% of fresh xenografts and 54.5% of cryopreserved grafts were functioning 19 days after transplantation. These results show that cryopreservation can extend xenograft survival.     

Interferon-mediated induction of Ia antigen expression on isolated murine whole islets and dispersed islet cells

Islets from male B10.BR mice (H-2k) were isolated by the collagenase technique, handpicked with a Pasteur pipette, and incubated (either intact or after dispersion with Dispase) for 0, 3, 5, 7, 10, or 14 days in tissue culture medium supplemented with either lymphokine supernatants or recombinant murine interferon-gamma. Islets and single cells were examined for IAk molecules by use of indirect immunofluorescence. Ia-positive islet cells were identified on the surface of islets incubated with 5-10% lymphokine for greater than 4 days or with 10, 100, or 1000 ng/ml interferon for greater than 6 days. Islets incubated in unsupplemented medium were Ia negative. Incubation with 5% lymphokine induced Ia expression on 10-40% dispersed islet cells cultured for greater than 9 days. Dual immunofluorescent staining for Ia and insulin revealed that Ia-positive cells included both beta- and non-beta-cells.     

FTY720在异种胰岛移植排斥反应中的作用

目的　研究FTY72 0单独用药和联合应用FTY72 0及环孢素A (CsA)在抑制猪 大鼠异种胰岛移植排斥反应中的作用。方法　大鼠肾被膜下移植成年猪胰岛 (APIs) 2 μl,单独应用FTY72 0 ( 1.0mg/kg·d和 3 .0mg/kg·d)和联合应用FTY 72 0及CsA(FTY 72 0 1.0mg/kg·d +CsA15mg/d和FTY72 0 3 .0mg/kg·d +CsA15mg/kg·d)。移植后 12d应用免疫组织化学染色方法来观察移植物内细胞浸润情况。结果　单独应用FTY72 0不能抑制API异种移植排斥反应的发生 ;在FTY72 0 +CsA组 ,排斥反应过程被明显抑制。在移植后第 12天 ,可以见到大量完整的API ,仅偶尔有细胞浸润。内分泌组织完整。结论　在猪 大鼠异种胰岛细胞模型中 ,联合应用FTY72 0及CsA可有效抑制排斥反应直到移植后第 12天 ,且未出现任何明显毒性作用。     

Neonatal pig islets induce a lower T-cell response than adult pig islets in IDDM patients

BACKGROUND: Pancreatic pig islets may provide a substitute in the future for difficult to obtain human islets for transplantation in insulin-dependent diabetes millitus (IDDM) patients. However, the immune response to xenografts may significantly hamper this approach. Because neonatal tissue is believed to be less immunogenic, we examined whether the T-cell response to neonatal pig islets differs from the response to adult islets. METHODS: The T-cell proliferative response to different concentrations of sonicated neonatal and adult pig islets, as well as to insulin and mitogens, was tested in 21 recent onset IDDM patients and 21 healthy controls. We determined the presence of various circulating islet autoantibodies and their association with the T-cell response in IDDM patients. RESULTS: In the IDDM patients, sonicated adult pig islets (at 1 microg protein/ml) induced a significantly higher frequency (12 of 21 vs. 1 of 21, p<0.001) and magnitude (2.58+/-0.44 vs. 1.38+/-0.13, p<0.02) of positive T-cell responses than neonatal islets at the same concentration. Similar results were obtained with a 10-fold higher concentration of islet sonicate. There was no significant association between the individual T-cell responses and the presence of circulating autoantibodies in IDDM patients. CONCLUSION: These results indicate that neonatal pig islets induce a lower T-cell reactivity than adult islets, suggesting that the neonatal tissue may be immunologically more suitable for future islet xenotransplantation.     

High-dosed nicotinamide decreases early graft failure after pig to nude rat intraportal islet transplantation.

BACKGROUND: Clinical and experimental data indicate that early failure of intraportally grafted islets is mediated by inflammation. Although nicotinamide (NA) has the potential to protect rodent islets it is unknown whether large mammalian islets can be protected in an inflammatory environment. Therefore, we investigated NA-mediated protection of pig islets intraportally transplanted into diabetic nude rats characterized by involvement of NO in intrahepatic graft failure. METHODS: Nonfasting serum glucose levels were evaluated after intraportal transplantation (TX) of 4000 pig islets or intraportal sham-TX in diabetic nude rats infused for 7 days with saline (0 mg), 500 mg, or 1000 mg NA/kg/day. The nitrate/nitrite serum levels were colorimetrically quantified 0, 24, and 48 hr posttransplant. RESULTS: Graft survival after 21 days was not improved (2/13) by 500 mg NA compared to 0 mg NA (1/22) and 500 mg sham-TX (0/7). A total of 1000 mg NA promoted sustained normoglycemia in transplanted rats (10/18, P<0.001 vs. 0 mg NA, P<0.05 vs. 500 mg NA) but deteriorated hyperglycemia in 1000 mg NA sham-TX (P<0.01 vs. 0 mg sham-TX). Regeneration of endogenous islets determined as pancreatic insulin content was only measured in islet recipients receiving 1000 mg NA (P<0.001). Posttransplant NO levels were not affected by NA and increased in all recipients two-fold (P<0.05 vs. day 0). CONCLUSIONS: Compared with efficient administration in syngeneic rodent models NA has to be applied in significant higher doses for protection of xenografted pig islets implying that protection of islets from large mammalians after transplantation into a proinflammatory organ seems feasible. In contrast to other observations graft survival was not mediated by interference of NA with hepatic NO generation.     

Encapsulation of rat islets of Langerhans prolongs xenograft survival in diabetic mice

Rat islets encapsulated in alginate-polylysine membranes were implanted intraperitoneally into nonimmunosuppressed streptozocin-induced diabetic mice. Diabetes was reversed within 3 days, and the animals remained normoglycemic for up to 144 days, with a mean xenograft survival of 80 days. This was significantly greater than nonencapsulated islets, which functioned for less than 14 days. The graft survival rate at 50 days was greater than 80%. Xenografts of rat islets encapsulated in alginate-polyornithine membranes also had a prolonged survival rate. This study demonstrates that encapsulation of pancreatic islets in semipermeable membranes can prolong xenograft survival in the absence of immunosuppression.     

Reduced NO production improves early canine islet xenograft function: a role for nitric oxide in islet xenograft primary nonfunction

Isolated canine islets transplanted to hyperglycemic rats fail to restore euglycemia in almost all cases, although the grafted islet tissue appears to be morphologically intact for up to 48 h following transplantation. Cytokines typically produced in the xenograft environment (e.g., IL-1 and TNF) inhibit insulin biosynthesis and secretion from isolated pancreatic islets, and are associated with the production of nitric oxide (NO). To further define the relationship between NO production and islet xenotransplantation, the inhibition of NO in a splenocyte/islet coculture system, and the in vivo effect of this inhibition on canine islet xenotransplantation, was investigated. Splenocytes (SPLC) from Lewis rats were cocultured with canine islets (freshly isolated or cultured 7 days), supernatant removed, and NO concentration (NO2) determined by optical density (Griess reaction, 550 nm, expressed as nmol nitrite/10(6) cells/18 h). Lipopolysaccharide (LPS) was used as a positive control of SPLC production of NO. Stimulation by LPS resulted in maximal NO production (2.20 +/- 0.16 nmol/10(6) cells/18 h, p < 0.001 compared to baseline values of 0.73 +/- 0.04 nmol/10(6) cells/18 h). In the presence of NO inhibitors (NMA, polymyxin B, hydrocortisone, aminoguanidine, DMSO), nitrite levels did not significantly rise above unstimulated values. Freshly isolated canine islets did stimulate NO production (1.26 +/- 0.12 nmol/10(6) cells/18 h, p < 0.001). In contrast, cultured canine islets did not stimulate NO production (0.84 +/- 0.09 nmol/10(6) cells/18 h). Transplantation of freshly isolated canine islets to STZ-diabetic recipient Lewis rats resulted in amelioration of hyperglycemia in only 50% (n = 6) of recipients 12 h posttransplant, with a return to hyperglycemia at all subsequent time points. Transplantation of 7-day cultured canine islets resulted in amelioration of hyperglycemia in 88% of recipients 12 h posttransplant and 63% of recipients 24 h posttransplant [p = 0.028, mean survival time (MST) = 1.0 days, n = 8]. Transplantation of canine islet xenografts with aminoguanidine therapy (BID, n = 11) resulted in amelioration of hyperglycemia in 100% of recipients at 12 h posttransplant, decreasing to 82% by 24 h following transplantation (p = 0.002, MST = 0.9 days). These results demonstrate that freshly isolated canine islets are potent stimulators of NO production by rat SPLC in vitro, and that culture of canine islets, or addition of NO inhibitors, abrogates stimulated NO production. These results also demonstrate a statistically significant improvement (p < 0.001) in early function of canine islet xenografts following 7 days of islet culture prior to transplant, and following recipient treatment with aminoguanidine. These studies suggest that the production of NO in the microenvironment of the graft site may adversely affect engraftment and function of canine islets, and suggest that the abrogation of islet-stimulated NO production may improve engraftment following islet xenotransplantation.     

Prolongation of rat islet xenograft survival in the liver of IFN-gamma-deficient mice

The cellular mechanisms involved in islet xenograft rejection remain undetermined. In the present study, the role of interferon-gamma (IFN-gamma) in rat islet xenograft rejection was examined with the use of IFN-gamma-deficient mice as recipients and the results were compared with allografts. There was no significant difference in the survival of intrahepatic islet allografts in IFN-gamma-deficient mice compared with that in wild-type mice. In contrast, a marked prolongation of rat islet xenograft survival was obtained in IFN-gamma-deficient mice without immunosuppression when compared with the survival in wild-type mice. In order to dissect the difference, infiltrating cells in the liver in association with rejection were examined with flow cytometry. An expansion of CD8 T cells was seen in the liver of wild-type mice rejecting xenografts compared with isografts. There was no significant change in other cell populations. In IFN-gamma-deficient mice, the expansion of CD8 T cells was seen in the liver rejecting xenografts; however, the time of development was markedly delayed by the time of rejection. These findings suggest that the acute rejection of rat islet xenografts in mice is IFN-gamma-dependent although the exact mechanisms remain unknown.     

Long-term survival, morphology and in vitro function of isolated pig islets under different culture conditions.

BACKGROUND: Islet culture aims to optimize islet survival and to reduce islet immunogenicity. To achieve these objectives, culture periods at 37 degrees C and 22-24 degrees C are mainly used. METHODS: This study compares the influence of donor age (juvenile vs. adult), temperature (22 degrees C vs. 37 degrees C), and serum supplementation (10% newborn calf serum [NCS] with 10% pig serum) on morphological integrity and in vitro function of porcine islets during long-term culture (LTC). RESULTS: After 21 days at 22 degrees C, the survival rate of cultured islets isolated from juvenile donors was lower than of adult islets (23+/-0.9% vs. 88+/-2.8%, P<0.001). Compared with 37 degrees C, LTC at 22 degrees C increased survival of adult islets and DNA recovery (92+/-2.5% vs. 45+/-4.8%, P<0.001; 72+/-4.1% vs. 30+/-5.1%, P<0.001) and reduced viability (62+/-8% vs. 89+/-5%, P<0.05). LTC at 22 degrees C was associated with a reduction of insulin content (85+/-9 vs. 152+/-10 microU/islet equivalents [IEQ], P<0.01), 24 hr-insulin secretion (82+/-7 vs. 552+/-91 microU/ day/IEQ, P<0.001), and integrated dynamic insulin response to glucose (1093+/-124 vs. 3074+/-708 microU/60 min/100 IEQ, P<0.05), compared with 37 degrees C LTC. Histologic analysis revealed disintegration of islet periphery after 22 degrees C, whereas smoothly shaped islets were present after 37 degrees C LTC. Integrity after 14 days at 37 degrees C was significantly better preserved when medium CMRL 1066 was supplemented with 10% porcine serum, compared with 10% NCS (40+/-2.3% vs. 21+/-6.7%, P<0.05), contrasting with 22 degrees C (52+/-4.0% vs. 59+/-3.7%, not significant). CONCLUSIONS: This study demonstrates that survival of cultured porcine islets is increased at 22 degrees C, whereas in vitro function and viability are better preserved at 37 degrees C. Survival at 37 degrees C can be improved by adding homologous serum to the medium.     

A CD200FC immunoadhesin prolongs rat islet xenograft survival in mice

BACKGROUND: A solubilized form of the CD200 molecule, CD200Fc, has been shown to suppress allograft rejection and development of collagen-induced arthritis in mice. We investigated whether the same molecule could prolong survival of rat islet xenografts. METHODS: Streptozocin-treated mice, receiving injections with anti-asialo-GM1 antibody, received rat islets ( approximately 400/mouse) under the kidney capsule or injected into the portal vein, along with rapamycin treatment. Thereafter mice received injections of CD200Fc (10 microg/mouse/injection) or control mouse IgG2. Blood glucose was monitored daily. Some mice received additional injections of anti-CD200/-CD200R monoclonal antibodies. RESULTS: Portal vein delivery of islets led to more extended resolution of diabetes than did transplantation under the kidney capsule. CD200Fc further prolonged survival in either case, an effect abolished by anti-CD200 or F(ab')2 anti-CD200R mAbs, but not by whole anti-CD200R (anti-CD200R Ig). Spleen cells taken from CD200Fc-treated mice showed polarization to type-2 cytokine production (interleukin-4, interleukin-10) on restimulation with rat splenocytes in culture, in comparison to cells from control mice (type-1 cytokines, interlulin-2, interferon-gamma). CONCLUSION: CD200:CD200R interactions are important in regulating rat islet xenograft survival.     

体内诱导胰岛血红素加氧酶-1过表达对异种胰岛移植物的保护作用

胞浸润数量明显少于其他两组(P<0.05).结论 CoPP诱导HO-1过表达可延长异种胰岛移植物存活,其机制可能与IL-10免疫调节有关.     

尿热休克蛋白-70在体外循环术后急性肾损伤早期诊断中的价值

目的评估尿热休克蛋白(HSP)-70在心脏体外循环心肺转流术(CPB)后急性肾损伤(AKI)早期诊断中的价值。方法选取2018年5月至2018年7月在河南省人民医院接受CPB治疗的患者为研究对象。收集入选者术前及术后0、2、4、6、8、12、24、48 h尿液标本和临床资料。按照肾脏病改善全球预后组织(KDIGO)AKI诊断标准分为AKI组和非AKI组。酶联免疫吸附法测定尿HSP-70、金属蛋白酶组织抑制因子2(TIMP-2)和胰岛素样生长因子结合蛋白7(IGFBP7)水平;免疫比浊法测定尿中性粒细胞明胶酶相关脂质运载蛋白(NGAL)水平。绘制受试者工作特征曲线(ROC),计算尿HSP-70、[TIMP-2]×[IGFBP7]、NGAL诊断CPB术后发生AKI的临界值、敏感度及特异度。结果共纳入45例患者,其中AKI组24例,非AKI组21例。AKI组术后各时间点尿HSP-70、[TIMP-2]×[IGFBP7]和NGAL水平显著高于非AKI组,组间比较差异有统计学意义(均P<0.05)。AKI组尿HSP-70在CPB术后2 h达到峰值,明显早于尿[TIMP-2]×[IGFBP7]、尿NGAL达峰值时间(分别为术后12 h和术后4 h)。术后2 h尿HSP-70≥2.1μg/L预测CPB术后AKI的曲线下面积(AUC)=1.00,灵敏度为100.0%,特异度100.0%;术后12 h尿[TIMP-2]×[IGFBP7]>19.1μg2/L2预测CPB术后AKI的AUC=0.94,灵敏度87.5%,特异度100.0%;术后4 h尿NGAL>27.4μg/L预测CPB术后AKI的AUC=0.95,灵敏度95.8%,特异度85.7%。术后2 h尿HSP-70≥2.1μg/L预测CPB术后AKI的阳性预测值为100.0%,阴性预测值100.0%。结论CPB术后AKI患者尿HSP-70水平升高早于尿[TIMP-2]×[IGFBP7]、NGAL,尿HSP-70水平监测有助于AKI的早期发现。     

Adenoviral-mediated overexpression of either membrane-bound human FasL or human decoy Fas can prolong pig islet xenograft survival in a rat transplant model

The success of pancreatic islet transplantation is limited because of the severe shortage of allogeneic pancreas donors. Accordingly, pig islets are considered to be an attractive, promising alternative. However, cell-mediated immunity, especially CD8+ cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, remains a formidable barrier to prevent long-term islet survival in xenograft recipients. Therefore, it is particularly important to explore methods to specifically prevent cell-mediated immunity against pig islets. Our group previously demonstrated that the overexpression of either membrane-bound human FasL or human decoy Fas antigen in pig endothelial cells prevented CTL xenocytotoxicity. In this study, we assessed the cytoprotective effects of adenoviral-mediated overexpression of either membrane-bound human FasL or human decoy Fas antigen in pig islets to inhibit CTL xenocytotoxicity. The CTL-mediated killing of pig islets infected with an adenoviral vector carrying either membrane-bound human FasL or human decoy Fas was significantly reduced compares with that of control pig islets transfected with adenoviral vector encoding enhanced green fluorescent protein (EGFP). Moreover, we transfected pig islets with these molecules to confirm their cytoprotective effects in in vivo studies. The significant long-term survival of pig islets expressing these molecules was elicited through days 3 to 5 posttransplantation. Thus, these results demonstrated that the remodeling of either death receptor or death ligand on pig islets by adenoviral gene transfer prevented innate cellular immunity against xeno-islet grafts facilitating long-term xenograft survival.