Gamma irradiation of isolated rat islets pretransplantation produces indefinite allograft survival in cyclosporine-treated recipients

In this study we have examined the use of low-dose gamma-irradiation for the reduction of islet immunogenicity in the strong allogeneic combination of WAG rat islets transplanted into diabetic AUG recipients. First, we determined that gamma-irradiation reduced immunogenicity in vitro by use of a modified MLR with WAG islets as stimulators and AUG splenocytes as responders. We then determined the maximum dose of gamma-irradiation that could be used (250 rads) before islet function was affected. As 250 rads islet pretreatment alone was ineffective in prolonging allograft survival, we combined the pretreatment with a short course (days 0, 1, 2; 30 mg/kg) of cyclosporine. We found that CsA was only effective in significantly prolonging allograft survival when given subcutaneously in olive oil. The CsA treatment alone gave a significantly prolonged survival time for the islet allografts (median, 37 days vs. 6 days for controls), but when combined with the 250 rads islet pretreatment a synergistic effect was seen with 100% becoming long-term survivors (greater than 100 days). The long-term surviving AUG rats from both the CsA alone group and the CsA plus 250 rads pretreated islets group were challenged with WAG dendritic cells (DC). The islets from the 250 rads pretreated group were subsequently rejected (day 6) while the CsA alone group were not affected. The role of low dose gamma-irradiation when combined with CsA treatment of islet graft recipients in inducing specific unresponsiveness will be discussed.     

Low-temperature culture of human islets isolated by the distention method and purified with Ficoll or Percoll gradients

Islets were isolated from human pancreases by the distention method and purified by centrifugation on Ficoll or Percoll gradients. The purity of the final preparations was 60% to 90% islets, making it possible to culture the islet preparations in vitro. Perifusion of the islet preparations after 1, 3, or 7 days of culture at 24 degrees C indicated that the islets were viable and would respond to glucose stimulation. If the islets were returned to 37 degrees C culture for 1 day, then the insulin secretory response at 7 days was comparable with the response during the first day of culture at 37 degrees C. Extending the culture period at 24 degrees C to 14 days damaged the islets as indicated by the lack of response to glucose stimulation. The Ficoll technique was a much more effective method for purifying the islets, since it provided approximately twice the yield of islets and insulin compared with the Percoll procedure. The mean yield of purified islets by the Ficoll technique was 2180 +/- 325 islets/gm of pancreas with a calculated islet mass of 3.7 +/- 0.8 mm3/gm. The findings that massive numbers of purified human islets can be obtained by centrifugation on Ficoll gradients and that the purified islet preparations remain morphologically and functionally intact during 7 days' culture at 24 degrees C make it possible to assess the function of donor islets before human islet transplantation, transplant the islets via the portal vein, and use low-temperature culture as a possible approach for altering the immunogenicity of donor human islets.     

Secondary cytotoxic allograft responses in vitro. III. The immunogenicity of allogeneic membrane fragments.

The immunogenicity of murine membrane fragments was tested both in primary and in secondary in vitro cytotoxic allograft responses, and compared to that of intact allogeneic stimulator cells. Allogeneic membrane fragments induced poor proliferative and cytotoxic responses in normal splenic responder cells. However reexposure of immune responder T cells to allogeneic membrane fragments triggered the generation of highly reactive cytotoxic T lymphocytes (CTL). Moreover, the generation of secondary CTL as induced by allogeneic membrane fragments was preceded by only a marginal cell proliferation. The results obtained are compatible with the concept that serologically defined (SD) antigens alone are capable of triggering alloimmune T cells to differentiate highly reactive secondary CTL.     

Lessons from in vitro perifusion of pancreatic islets isolated from 80 human pancreases

We report the average insulin response to acute glucose measured by in vitro perifusion of pancreatic islets isolated from 80 consecutive human organs. Different perifusion parameters were considered [basal release, stimulation index (SI), time to peak, incremental area under the curve delta-AUC alpha)], and the correlation among them was determined. SI positively correlated with delta-AUC alpha (p < 0.001, r = 0.80) while negatively with time to peak (p < 0.05, r = -0.23). We also evaluated several variables of the isolation procedure that might affect responsiveness to glucose by human islets. Sex and age of pancreas donors, cold ischemia time, duration of the digestion, collagenase concentration, and lot characteristics (collagenase, trypsin, clostripain, and proteases activity), and final islet yield were considered. Multivariate regression analysis showed only an independent association between SI and the concentration of collagenase (p = 0.01).     

微囊化猪胰岛的免疫原性研究

目的 经过体外实验,明确微囊化新生猪胰岛样细胞团（ＩＣＣ）的免疫原性。方法 通过胰岛－淋巴细胞混合培养（ＭＬＩＣ）后的淋巴细胞转化试验。以游离的新生猪ＩＣＣ和空微囊为对照,比较微囊ＩＣＣ组与对照组的淋巴细胞转化值。结果 微囊ＩＣＣ组淋巴细胞转化值显著低于游离ＩＣＣ组（Ｐ〈０．０５）,但明显高于空囊组（Ｐ〈０．０５）。结论 微囊包裹后的新生猪ＩＣＣ的免疫原性比游离的新生猪ＩＣＣ的免疫原性显著降低。提     

Effects of high glucose on insulin secretion by isolated rat islets and purified beta-cells and possible role of glycosylation

We investigated the effect of 24 h of exposure to various glucose concentrations on insulin secretion by isolated rat pancreatic islets and purified rat beta-cells. Compared with islets cultured with standard medium (5.5 mM glucose), islets cultured with 16.7 mM glucose showed a higher basal insulin release (means +/- SE, 3.0 +/- 0.5 vs. 0.7 +/- 0.2%, n = 8, P less than .005) and reduced glucose-stimulated insulin secretion (2.4 +/- 0.3 vs. 5.8 +/- 0.4%, n = 8, P less than .005). Similar results were also obtained with purified beta-cells. The effect of high glucose was time dependent (present after 12 h, maximal after 24 h) and reversible: when islets cultured with high glucose were transferred to standard medium, normal responsiveness to glucose was restored within 8 h and normal basal release within 24 h. Mannitol, 3-O-methylglucose, and 2-deoxyglucose were not able to mimic the effects of glucose. Islets or purified beta-cells cultured in the presence of high glucose had a normal response when stimulated with glyburide, dibutyryl cyclic AMP, and isobutylmethylxanthine. Tunicamycin, an inhibitor of N-terminal glycosylation, prevented glucose-induced desensitization when added during 24 h of islet culture with 16.7 mM glucose. Swainsonine, another agent that influences glycosylation, had a similar effect. Our study indicates 1) that 24 h of exposure to high glucose induces a specific and reversible impairment of insulin secretion in response to glucose, 2) that this is a direct effect of glucose on beta-cells, and 3) that islet glucose metabolism and glycosylation processes may play a critical role in determining glucose desensitization.     

Oxygen tension in isolated transplanted rat islets and in islets of rat whole-pancreas transplants

Despite similar immunosuppressive protocols having been applied, the success rate of pancreatic islet transplantation has been much lower than that of whole-pancreas transplantation. We compared the oxygen tension in syngeneically transplanted isolated rat islets and islets of syngeneic rat whole-pancreatic grafts, with native islets, using Clark-type microelectrodes. An oxygen tension of approximately 40 mmHg was recorded in both native islets and in islets of the whole-pancreas graft. Isolated islets transplanted under the renal capsule had a markedly lower oxygen tension ( approximately 6 mmHg). The exocrine parenchyma of the native and of the transplanted pancreatic gland had an oxygen tension of approximately 30 mmHg. The lower oxygen tension in transplanted isolated islets may be one explanation for the more frequent failures of transplanted islets compared with the outcome when the whole pancreatic gland is transplanted.     

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.