Monoclonal Antibodies against Pancreatic Islet‐Cell‐Surface Antigens Selected by Flow Cytofluorometry
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Abstract:
BALB/c mice were immunized with human islets of Langerhans and spleen cells from two mice. found to develop cell‐surface antibodies against insulin‐producing rat islet tumour RIN‐5F cells, were fused with mouse myeloma cells. Antibody‐producing hybrids were cloned on the basis of their production of surface antibodies reactive with paraformaldehyde‐fixed RIN‐5F cells by indirect immunofluorescence analysis in the fluorescence‐activated cell sorter. Among 236 primary clones, eight stable cell lines producing islet‐cell‐surface antibodies were eventually cloned. Antibody 2G3 (IgM) reacted with viable normal rat islet cells and high insulin‐producing rat islet tumour RIN5‐A2 cells, while 3G3(IgM) only reacted with RIN5‐A2 cells. AntibodyβB1 (IgG1) reacted with all islet cells tested and detected an M 1 21k component in immunoblotting experiments with RIN‐5AH cell plasma membrane proteins electrophoretically transferred to nitrocellulose filters. Antibody 7F6 (IgM) reacted with all islet and non‐islet cells tested and delected bands of M 1 66k and 27k by immunoblotting. Antibodies γB3,γB6, γC2, and 6BI (all IgM) showed varying degrees of binding to different islet cells, but reacted only weakly with non‐islet human cells. It is concluded that monoclonal antibodies against pancreatic islet cells may define specific endocrine islet‐cell‐surface determinants.Keywords:
Immunofluorescence
Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (∆OCRglc) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of ∆OCRglc obtained at both institutions were compared. Islet specificity was determined by comparing ∆OCRglc in islet and nonislet tissue. The ∆OCRglc ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (∆OCRglc was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The ∆OCRglc was statistically independent of percent beta cells, demonstrating that ∆OCRglc is governed more by islet quality than by islet composition. The ∆OCRglc, but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ∆OCRglc as a measurement of islet quality for human islet transplantation. Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (∆OCRglc) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of ∆OCRglc obtained at both institutions were compared. Islet specificity was determined by comparing ∆OCRglc in islet and nonislet tissue. The ∆OCRglc ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (∆OCRglc was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The ∆OCRglc was statistically independent of percent beta cells, demonstrating that ∆OCRglc is governed more by islet quality than by islet composition. The ∆OCRglc, but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ∆OCRglc as a measurement of islet quality for human islet transplantation. As stated in 1990, in order for human islet transplantation to become a consistent and reliable treatment option for diabetics, standardized methods to assess human islet quality must be established (1Ricordi C Gray DW Hering BJ et al.Islet isolation assessment in man and large animals.Acta Diabetol Lat. 1990; 27: 185-195Crossref PubMed Scopus (521) Google Scholar). Seventeen years later, the lack of validated assessment methods still precludes using information about islet quality as release criteria for transplantation and diagnosing causes for success or failure of the procedure (2Wonnacott K Update on regulatory issues in pancreatic islet transplantation.Am J Ther. 2005; 12: 600-604Crossref PubMed Scopus (21) Google Scholar). Recently, however, efforts to identify and establish assays for human islet quality assessment have intensified (3Sweet IR Gilbert M Jensen R et al.Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality.Transplantation. 2005; 80: 1003-1011Crossref PubMed Scopus (42) Google Scholar, 4Fraker C Timmins MR Guarino RD et al.The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: Oxygen consumption as a potential measure of islet potency.Cell Transplant. 2006; 15: 745-758Crossref PubMed Scopus (48) Google Scholar, 5Papas KK Colton CK Nelson RA et al.Human islet oxygen consumption rate and DNA measurements predict diabetes reversal in nude mice.Am J Transplant. 2007; 7: 707-713Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 6Goto M Holgersson J Kumagai-Braesch M Korsgren O The ADP/ATP ratio: A novel predictive assay for quality assessment of isolated pancreatic islets.Am J Transplant. 2006; 6: 2483-2487Crossref PubMed Scopus (95) Google Scholar, 7Sabek OM Marshall DR Minoru O Fraga DW Gaber AO OP-142 gene expression profile of nonfunctional human pancreatic islets: Predictors of transplant failure?.Transplant Proc. 2005; 37: 3441-3443Crossref PubMed Scopus (8) Google Scholar, 8Hermann M Pirkebner D Draxl A Margreiter R Hengster P "Real-time" assessment of human islet preparations with confocal live cell imaging.Transplant Proc. 2005; 37: 3409-3411Crossref PubMed Scopus (18) Google Scholar, 9Ichii H Inverardi L Pileggi A et al.A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations.Am J Transplant. 2005; 5: 1635-1645Crossref PubMed Scopus (176) Google Scholar, 10Barnett MJ McGhee-Wilson D Shapiro AM Lakey JR Variation in human islet viability based on different membrane integrity stains.Cell Transplant. 2004; 13: 481-488Crossref PubMed Scopus (83) Google Scholar, 11Armann B Hanson MS Hatch E Steffen A Fernandez LA Quantification of basal and stimulated ROS levels as predictors of islet potency and function.Am J Transplant. 2007; 7: 38-47Crossref PubMed Scopus (68) Google Scholar, 12Street CN Lakey JR Shapiro AM et al.Islet graft assessment in the Edmonton Protocol: implications for predicting long-term clinical outcome.Diabetes. 2004; 53: 3107-3114Crossref PubMed Scopus (189) Google Scholar). In order to develop a quality assessment methodology that can be standardized for use throughout the islet transplantation community, two requirements must be met. First and foremost, the test must be predictive of transplant outcome. We and others have reported on studies demonstrating the ability of incremental changes of parameters reflecting electron transport in response to glucose (∆OCRglc and ∆cytochrome c reductionglc) but not absolute values to predict efficacy of human islets after transplantation into mice (3Sweet IR Gilbert M Jensen R et al.Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality.Transplantation. 2005; 80: 1003-1011Crossref PubMed Scopus (42) Google Scholar,4Fraker C Timmins MR Guarino RD et al.The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: Oxygen consumption as a potential measure of islet potency.Cell Transplant. 2006; 15: 745-758Crossref PubMed Scopus (48) Google Scholar). Using receiver operating characteristic (ROC) analysis, ∆OCRglc and ∆cytochrome c reductionglc correctly picked the transplanted islets with the highest secretory response between 80% and 90% of the time. Although another group has championed the predictive ability of absolute levels of OCR (5Papas KK Colton CK Nelson RA et al.Human islet oxygen consumption rate and DNA measurements predict diabetes reversal in nude mice.Am J Transplant. 2007; 7: 707-713Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), results of other studies observed that this parameter was very variable and depended on conditions that are unrelated to the islet cell's ability to survive (3Sweet IR Gilbert M Jensen R et al.Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality.Transplantation. 2005; 80: 1003-1011Crossref PubMed Scopus (42) Google Scholar,4Fraker C Timmins MR Guarino RD et al.The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: Oxygen consumption as a potential measure of islet potency.Cell Transplant. 2006; 15: 745-758Crossref PubMed Scopus (48) Google Scholar). The second requirement is that tests must be performed in a standardized protocol so that all participating laboratories obtain reproducible results. To date, there are no reports that any assays proposed for use as human islet quality control have been thus standardized. Without standardization, no universal release criteria can be established. Furthermore, this critical goal will also be essential for carrying out any test of an assay's ability to predict human islet function after transplantation into humans. Since in general less than five transplants are done by any one center per year (13Collaborative Islet Transplant Registry..Annual Report. The EMMES Corporation, Rockville, MD2006Google Scholar), it is clear that any test of potency assay will have to involve multiple centers. Therefore it is imperative that candidate assessment methodologies can be transferred to multiple laboratories and that results between the laboratories are consistent for such clinical trials to be carried out. Each laboratory must have the same or equivalent equipment, the technicians trained to adhere strictly to standardized protocols, and proper validation that each laboratory obtains accurate results. In this report, we have carried out studies aimed at determining the concordance between concomitant measurements obtained at the University of Washington (UW) and the City of Hope (COH). Although not rigorously tested, it stands to reason that the larger the contribution of nonislet tissue in the sample to the quality assessment parameter, the less sensitive the parameter will be to islet quality. Since oxygen is consumed by nearly every cell in the body, oxygen consumption rate as a test offers no islet specificity and its use would presumably have to be limited to samples that are very pure. In contrast, beta cells, due to the presence of the high-Km hexokinase IV [i.e. glucokinase (14Matschinsky FM Ellerman JE Metabolism of glucose in the islets of Langerhans.J Biol Chem. 1968; 243: 2730-2736Abstract Full Text PDF PubMed Google Scholar)], are uniquely sensitive to glucose in the physiologic range. Even when handpicking islets, there will be contamination from nonislet tissue in the sample. Therefore, in this study, the contribution of nonislet cells to ∆OCRglc was determined in order to evaluate the error due to the presence of impurities. All assessments of islet quality need to be normalized in some way for results to be meaningful. In some assays such as ATP/ADP, cytochrome c reduction and cellular viability staining, ratios are generated implicitly by the analysis. However, the determination of oxygen consumption must always be represented as a function of the mass of tissue being analyzed. Some groups have used DNA and purity as a measure of islet mass (4Fraker C Timmins MR Guarino RD et al.The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: Oxygen consumption as a potential measure of islet potency.Cell Transplant. 2006; 15: 745-758Crossref PubMed Scopus (48) Google Scholar,5Papas KK Colton CK Nelson RA et al.Human islet oxygen consumption rate and DNA measurements predict diabetes reversal in nude mice.Am J Transplant. 2007; 7: 707-713Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In our previous studies, we chose to normalize all of our data to the number of handpicked islets (3Sweet IR Gilbert M Jensen R et al.Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality.Transplantation. 2005; 80: 1003-1011Crossref PubMed Scopus (42) Google Scholar). It is possible that the size of each islet picked could be selected to fall within a small size distribution (between 150 and 200 μ). However, since the islet composition may differ (15Brissova M Fowler MJ Nicholson WE et al.Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.J Histochem Cytochem. 2005; 53: 1087-1097Crossref PubMed Scopus (563) Google Scholar), ∆OCRglc may be a function of both the beta cell fraction and the bioenergetic competency (i.e. the quality) of the islets. If ∆OCRglc is independent of the percentage of beta cells, then ∆OCRglc can be used as a standalone measurement, and normalizing the data to a measure of beta cell mass would not be necessary. Overall, the data presented in this report and our previous study (3Sweet IR Gilbert M Jensen R et al.Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality.Transplantation. 2005; 80: 1003-1011Crossref PubMed Scopus (42) Google Scholar) indicate that measuring ∆OCRglc using a flow culture system meets a number of requisites for initiating a multi-center test of the methodology for human islet quality assessment. Kreb's Ringer Bicarbonate buffer was used for perifusions and was made up as described previously (3Sweet IR Gilbert M Jensen R et al.Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality.Transplantation. 2005; 80: 1003-1011Crossref PubMed Scopus (42) Google Scholar). Carbachol (CCh) and glucose were purchased from Sigma-Aldrich (St. Louis, MO). Human islets were provided for research use by the Southern California Islet Cell Resources Center, at the COH (Duarte, CA) with the approval of the COH Institutional Review Board and with the written informed consent from each organ donor for research use. Pancreata were digested by a modified Ricordi method (6Goto M Holgersson J Kumagai-Braesch M Korsgren O The ADP/ATP ratio: A novel predictive assay for quality assessment of isolated pancreatic islets.Am J Transplant. 2006; 6: 2483-2487Crossref PubMed Scopus (95) Google Scholar) using Liberase-HI collagenase (Roche Molecular Biochemicals, Indianapolis, IN) and then purified on a continuous Biocoll (Biochrom, Berlin, Germany) gradient in a cooled COBE 2991 Cell Processor (Gambro BCT, Lakewood, CO). Fractions were collected from the COBE, and fractions that had a purity of greater than 70% were pooled and cultured in Miami Media #1 (Mediatech Inc., Herndon, VA) for 2–6 days prior to analysis. For experiments on nonislet tissue, the remaining tissue in the bag was washed extensively in media. Two days prior to performing the perifusions, the number and size distribution of islets in a culture flask were determined, where each islet was normalized to the equivalent volume of an islet that is 150 μ in diameter (termed an islet equivalent [IEQ]) (1Ricordi C Gray DW Hering BJ et al.Islet isolation assessment in man and large animals.Acta Diabetol Lat. 1990; 27: 185-195Crossref PubMed Scopus (521) Google Scholar). An aliquot of islets (5000 IEQ) was shipped overnight to Seattle in a 50 mL conical tube filled completely with shipping medium consisting of CMRL medium 1066 without NaHCO3 (Mediatech) supplemented with 0.5% Human Serum Albumin (ZLB Behring, Kankakee, IL), 10 μM Trolox® (Sigma-Aldrich) and 10 mM nicotinamide and maintained at approximately 16°C. Upon arrival, the islets were subsequently cultured in Miami Media #1 at 37 °C for 18 h prior to perifusion analysis. The islet flow culture system has been described in detail previously (16Sweet IR Cook DL Wiseman RW et al.Dynamic perifusion to maintain and assess isolated pancreatic islets.Diabetes Technol Ther. 2002; 4: 67-76Crossref PubMed Scopus (45) Google Scholar). A total of 750 islets were hand picked using a P200 pipet into a sterile 1.7 mL microfuge tube based on size and intactness of islet capsule and were mixed with 0.12 mg Cytodex beads (Amersham Biosciences Corp., Piscataway, NJ), sucked into PE50 tubing with a 1 mL syringe, and after turning off the pump, gently loaded into the chamber onto the bottom layer of 0.4 mg of Cytopore beads (Amersham) that had been layered on top of a polyethylene frit. Cytopore beads were layered on to the islets, and then the top frit was lowered until it was almost touching the Cytopore beads. Absolute levels of OCR were calculated as the flow rate times the difference between inflow and outflow oxygen tension measured by detecting the phosphorescence lifetime of an oxygen-sensitive dye that was painted inside the perifusion chamber (17Sweet IR Khalil G Wallen AR et al.Continuous measurement of oxygen consumption by pancreatic islets.Diabetes Technol Ther. 2002; 4: 661-672Crossref PubMed Scopus (84) Google Scholar). Inflow oxygen tension remains constant during the course of the experiment (17Sweet IR Khalil G Wallen AR et al.Continuous measurement of oxygen consumption by pancreatic islets.Diabetes Technol Ther. 2002; 4: 661-672Crossref PubMed Scopus (84) Google Scholar), and was determined at the conclusion of each experiment after inhibiting cellular respiration by the addition of antimycin A (18Sweet IR Cook DL DeJulio E et al.Regulation of ATP/ADP in pancreatic islets.Diabetes. 2004; 53: 401-409Crossref PubMed Scopus (80) Google Scholar). Phosphorescent lifetimes were measured equivalently but with two different machines at the two institutes. At the COH, a PMOD 5000 Frequency Domain Phosphorometer (Oxygen Enterprises Ltd, Philadelphia, PA) was used as previously described (3Sweet IR Gilbert M Jensen R et al.Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality.Transplantation. 2005; 80: 1003-1011Crossref PubMed Scopus (42) Google Scholar) with the end of the excitation light guide (1/8", Edmund Industrial Optics, Barrington, NJ) illuminated by a 405-nm LED just touching the outside of the glass opposite where the dye was painted and the detecting light guide was situated at a 90 degree angle. At the UW, lifetime was detected using an MFPF-100 multi-frequency phase fluorometer lifetime measurement system made by TauTheta Instruments (Boulder, CO) (19Sweet IR Gilbert M Contribution of calcium influx in mediating glucose-stimulated oxygen consumption in pancreatic islets.Diabetes. 2006; 55: 3509-3519Crossref PubMed Scopus (39) Google Scholar). The changes in OCR in response to glucose (∆OCRglc) and CCh (∆OCRCCh) were calculated as the difference in OCR averaged from 30 to 45 min following the change in media composition, and the 15 min prior to the change. Insulin was measured by collecting outflow fractions and measuring its concentration by ELISA per manufacturer instructions (Mercodia Inc, Winston Salem, NC). ISR was calculated as the flow rate times the insulin concentration. The change in ISR in response to each agent was calculated as the ratio of ISR averaged from 30 to 45 min following the change in media composition, over the average ISR during the 15 min prior to the change (termed the stimulation index [SI]). The fraction of beta cells to total cells in each preparation was determined by immunofluorescent staining of sections of intact, fixed islet clusters for insulin and the other major islet/pancreatic cell types and quantitation by laser scanning cytometry (20Kamentsky LA Kamentsky LD Microscope-based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data.Cytometry. 1991; 12: 381-387Crossref PubMed Scopus (228) Google Scholar,21Darzynkiewicz Z Bedner E Li X Gorczyca W Melamed MR Laser-scanning cytometry: A new instrumentation with many applications.Exp Cell Res. 1999; 249: 1-12Crossref PubMed Scopus (265) Google Scholar). Briefly, an aliquot of each islet preparation was fixed in 10% formalin, imbedded in paraffin, sectioned (5 μm), and stained with a combination of antibodies from the following: guinea pig anti-human insulin serum (LINCO, St. Charles, MO), mouse anti-human glucagon (Sigma-Aldrich), rabbit anti-human somatostatin (DAKO, Carpinteria, CA), rabbit anti-human pancreatic polypeptide (LINCO), rabbit anti-human amylase (Sigma-Aldrich), mouse anti-human cytokeratin 19 (DAKO) and counterstained with 4',6-diamidino-2-phenylindole (DAPI) (22Todorov I Omori K Pascual M et al.Generation of human islets through expansion and differentiation of non-islet pancreatic cells discarded (pancreatic discard) after islet isolation.Pancreas. 2006; 32: 130-138Crossref PubMed Scopus (39) Google Scholar). The fraction of each cell type was determined using an iCys Imaging Cytometer (CompuCyte, Cambridge, MA). All mice were housed in specific pathogen free (SPF) conditions at the Animal Resources Center (ARC) of the Beckman Institute at the COH. The NOD-SCID mice were obtained from the ARC Breeding colony at COH, which were derived from breeder animals received from Jackson Laboratories (Bar Harbor, ME). The use of animals and the animal procedures were approved by the City of Hope Research Animal Care Committee. Diabetes was induced by intraperitoneal injection once/day for 3 days of 50 mg/kg streptozotocin (Sigma-Aldrich) that was freshly dissolved in citrate buffer. Blood samples were taken from the tail and measured using the One-Touch Ultra Blood Glucose Monitoring System (Lifescan Inc., Milpitas, CA). Animals were considered diabetic following two consecutive blood glucose measurements >400 mg/dL. The transplantation of 1000–2000 IEQ under the renal capsule was performed as described previously (23Rush BT Fraga DW Kotb MY et al.Preservation of human pancreatic islet in vivo function after 6-month culture in serum-free media.Transplantation. 2004; 77: 1147-1154Crossref PubMed Scopus (22) Google Scholar,24Gaber AO Fraga D Kotb M Lo A Sabek O Latif K Human islet graft function in NOD-SCID mice predicts clinical response in islet transplant recipients.Transplant Proc. 2004; 36: 1108-1110Crossref PubMed Scopus (28) Google Scholar). Posttransplant blood glucose measurements were taken two to three times per week. Islets were considered efficacious if the average blood glucose 3–4 weeks after transplantation remained below 200 mg/dL. Nephrectomies were performed to ensure graft dependence of glycemic reduction. Kinetic profiles were obtained in response to glucose and CCh. For each islet assessment at each institute, two perifusions were done in parallel, and the responses were averaged to yield a single value of either ∆OCRglc or ∆OCRCCh. The coefficient of variation (CV) was calculated as 100 × SD/average. Using simple linear regression, p values were calculated in order to test the association between the following pairs of variables: ∆OCRglc and the percentage of beta cells in the sample, ∆OCRCCh and ∆OCRglc, and finally between ∆OCRCCh and the percentage of nonislet cells in the sample. To determine the agreement between the measurements done at the two sites, intraclass correlation coefficients (between-preparation variance/[between-preparation variance + between-labs variance for each preparation]) were calculated as described (25Koepsell TD Weiss NS Epidemiologic Methods: Studying the Occurrence of Illness.. Oxford University Press, New York2003: 222Google Scholar). For the evaluation of the ability of OCR and ∆OCRglc to predict transplant efficacy, ROC curves, plots of the sensitivity (true positives/[true positive + false negatives]) versus 1 − specificity (true negatives/[true negatives + false positives]), were generated as previously described (3Sweet IR Gilbert M Jensen R et al.Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality.Transplantation. 2005; 80: 1003-1011Crossref PubMed Scopus (42) Google Scholar). After purification of pancreatic homogenate, the islet and nonislet fractions were cultured for 2 days under identical conditions. In parallel, 750 islets or nonislet cell clusters of similar size were handpicked and loaded into perifusion chambers. The protocol for obtaining ∆OCRglc and ∆OCRCCh involved a 90-min equilibration period where the level of glucose in the media was 3 mM, followed by a 45-min period with 20 mM glucose and 45 min with 20 mM glucose plus 10 μM CCh (Figure 1). In contrast to the islets, where the average ∆OCRglc was 0.123 ± 0.024 nmol/min/100 islets (n = 4), the nonislet tissue response was insignificant (0.007 ± 0.003 nmol/min/100 clusters). The unstimulated OCR was similar in the two tissue types (0.197 ± 0.025 vs. 0.151 ± 0.043 for nonislet and islet tissue), and the response by nonislet tissue to CCh was larger than that for islets (∆OCRCCh was 0.053 ± 0.012 vs. 0.035 ± 0.002), indicating that the nonislet tissue was viable and responsive to stimuli. For 20 different islet isolations, islets were assessed at the COH for both the percent beta cells in the preparation and ∆OCRglc (Figure 2). The mean ± SD, and CV for the percent beta cells and for ∆OCRglc were 40.0 ± 9.7%, 24.4% and 0.133 ± 0.075 nmol/min/100 islets, 56.6% respectively. The range of ∆OCRglc spanned a change of 9.3-fold, whereas the range of percent beta cells only spanned 2.3-fold. With a slope of 0 (95% CI: −0.004 to 0.004; p = 0.96), results from the linear regression showed no association between ∆OCRglc and the percentage of beta cells in the sample. This suggests that ∆OCRglc is not dependent on the percentage of beta cells in handpicked samples but more strongly controlled by the variation in the energetic state. This may alleviate the necessity of normalizing ∆OCRglc results thereby simplifying the process of determining the adequacy of a sample for transplant. Simultaneously at each institute, islets were handpicked and assessed for ∆OCRglc and ∆OCRCCh using the protocol shown in Figure 1. At each site, two perifusions were run in parallel and the calculated steady state changes from the two kinetic curves were averaged. The measurements of ∆OCRglc and ∆OCRCCh at the two sites are tabulated (Table 1, Panel A), and each assessment was normalized to the average of all assessments obtained at each site, and then plotted against the parallel measurement carried out at the other institute (Figure 3.) The average ∆OCRglc and ∆OCRCCh for each site differed by only 9.4% and 2.7%, respectively. To evaluate the agreement of results obtained at the two sites, intraclass correlation coefficients were calculated for ∆OCRglc and ∆OCRCCh (0.92 and 0.89, respectively). The intraclass correlation coefficient approaches 1 if the two labs measure identical ∆OCR values, and the high values obtained indicated that the difference between the values at the two centers was not very different than the measurement error of the method. In contrast to the ∆OCR data, there was much less agreement between the two centers with respect to the SIs of ISR (Table 1, Panel B): the intraclass correlation coefficients for SI-ISRglc and SI-ISRCCh were 0.45 and 0.64, respectively and SI-ISRglc at the COH was almost double that obtained at the UW, despite similar average basal ISR measured at the two institutes (0.37 vs. 0.32 ng/min/100 islets at the COH vs. UW, respectively).Table 1Summary of glucose- and carbachol-stimulated oxygen consumption rate (∆OCRglc and ∆OCRCCh)(A) or stimulation index of insulin secretion rate by glucose and carbachol (SI-ISRglc and SI-ISRCCh) (B) determined at the UW or COHPanel APerifusion∆OCRgic∆OCRCChUWCOHUWCOH10.1500.1660.2500.19220.0820.0830.0330.03230.1000.0760.1600.15740.1750.1290.0590.04750.1260.1190.1180.08060.1540.0850.1270.13770.1190.0900.0300.02080.0230.008ND0.02890.1760.1560.0980.189100.1930.1600.0560.073110.0970.1110.0480.054120.1540.188NDND130.0720.0790.0460.119140.0430.0120.0710.026Average0.119 ± 0.0140.104 ± 0.0140.091 ± 0.0190.089 ± 0.017Panel BPerifusionSI-ISRglcSI-ISRCChUWCOHUWCOH10.72.41.51.520.20.62.11.531.41.31.20.744.33.01.91.151.31.11.31.261.02.11.51.071.13.11.21.28ND4.3ND1.095.63.81.50.1101.79.41.91.611321.51.31.0121.61.0ND0.5130.87.51.21.5140.88.00.81.0Average1.8 ± 0.43.5 ± 0.81.4 ± 0.11.1 ± 0.1OCR and ISR were measured using the protocol in Figure 1, and then calculated as the change in steady state values as described in Methods. Each value (nmol/min/100 islets) represents the average of two determinations obtained in parallel. Open table in a new tab OCR and ISR were measured using the protocol in Figure 1, and then calculated as the change in steady state values as described in Methods. Each value (nmol/min/100 islets) represents the average of two determinations obtained in parallel. Originally it was hypothesized that ∆OCRCCh may provide supplemental information by stimulating muscarinic receptors, a neural pathway distinct from fuel sensing. The correlation between ∆OCRglc and ∆OCRCCh (Figure 4A) was weak and there was no apparent association between the two in linear regression (slope = 0.64; 95% CI: −0.07–1.36; p = 0.07). This lack of correlation may indicate that CCh and glucose are measuring different aspects of the islet preparations, which could be a function of the islets and/or the presence of variable contributions from nonislet tissue due to the fact that CCh response is not specific for islets. We therefore compared ∆OCRCCh to the percent nonislet cells as reflected by cells not staining for the islet hormones (Figure 4B). In contrast to ∆OCRglc, which was independent of the percentage of beta cells (Figure 2), ∆OCRCCh is strongly dependent on the percentage of nonislet cells, with the ∆OCRCCh increasing 0.002 nmol/min/100 islets (95% CI: 0.001–0.004; p = 0.003) for each percent increase in nonislet cells. The clear relation between these two parameters makes it unlikely that CCh could be used as a supplemental test agent, and demonstrated the need for islet specific effectors in probing islet quality. In order to test the utility of ∆OCRglc, OCR (at high glucose) and SI-ISRglc to provide an objective measure of islet quality, islets were tested in parallel for their ability to lower blood sugar in diabetic mice 3–4 weeks following their transplantation under the kidney capsule. The islet isolations, perifusions and transplantation studies were all carried out at the COH. Outcomes of ∆OCRglc isolations fell into three regions (Figure 5A): above 0.165 nmol/min
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Many factors influence the outcome of islet transplantation. As islets in the early posttransplant setting are supplied with oxygen by diffusion only and are in a hypoxic state in the portal system, we tested whether small human islets are superior to large islets both in vitro and in vivo. We assessed insulin secretion of large and small islets and quantified cell death during hypoxic conditions simulating the intraportal transplant environment. In the clinical setting, we analyzed the influence of transplanted islet size on insulin production in patients with type 1 diabetes. Our results provide evidence that small islets are superior to large islets with regard to in vitro insulin secretion and show a higher survival rate during both normoxic and hypoxic culture. Islet volume after 48 h of hypoxic culture decreased to 25% compared with normoxic culture at 24 h due to a preferential loss of large islets. In human islet transplantation, the isolation index (islet volume as expressed in islet equivalents/islet number), or more simply the islet number, proved to be more reliable to predict stimulated C-peptide response compared with islet volume. Thus, islet size seems to be a key factor determining human islet transplantation outcome.
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It has been proposed that islet transplants comprised primarily of small rather than large islets may provide better graft function, due to their lower susceptibility to hypoxic damage. Our aim was to determine whether islet size correlated with in vivo graft function in islet transplant recipients with C peptide-negative type 1 diabetes when islets have undergone pretransplant islet culture.Human pancreatic islets were isolated, cultured for 24 hours and infused by standardized protocols. Ninety-minute stimulated C-peptide concentrations were determined during a standard meal tolerance test 3 months posttransplant. The islet isolation index (IEq/islet number) was determined immediately after isolation and again before transplantation (after tissue culture). This was correlated with patient insulin requirement or stimulated C-peptide.Changes in insulin requirement did not significantly correlate with islet isolation index. Stimulated C-peptide correlated weakly with IEq at isolation (P = 0.40) and significantly with IEq at transplantation (P = 0.018). Stimulated C-peptide correlated with islet number at isolation (P = 0.013) and more strongly with the islet number at transplantation (P = 0.001). In contrast, the correlation of stimulated C-peptide and islet isolation index was weaker (P = 0.018), and this was poorer at transplantation (P = 0.034). Using linear regression, the strongest association with graft function was islet number (r = 0.722, P = 0.001). Islet size was not related to graft function after adjusting for islet volume or number.These data show no clear correlation between islet isolation index and graft function; both small and large islets are suitable for transplantation, provided the islets have survived a short culture period postisolation.
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After islet isolation, diffusion has become the main mechanism to transport oxygen and nutrients into the core of islets. However, diffusion has limitations, by which nutrients cannot effectively reach the core of large islets and can eventually cause core cell death and islet loss. This problem can be resolved by dispersing islets into single islet cells, but single islet cells do not exhibit insulin release function in in vitro culture. In this study, we intended to establish a new islet engineering approach by forming islet cell clusters to improve islet survival and function. Therefore, alginate gels were used to encapsulate islet cells to form artificial islets after dispersion of islets into single cells. The shape of the islet cell clusters was similar to native islets, and the size of the islet cell clusters was limited to a maximum diameter of 100 μm. By limiting the diameter of this engineered islet cell cluster, cell viability was nearly 100%, a significant improvement over natural islets. Importantly, islet cell clusters express the genes of islets, including Isl-1, Gcg, and insulin-1, and insulin secretion ability was maintained in vitro.
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Clinical islet allotransplantation is dependent on the ability to achieve a high yield and purity of islets isolated from human cadaver pancreas donors. The aim of this study was to determine the factors influencing the pancreas prior to islet isolation that may alter yield and purity. The results of 50 consecutive islet isolations from cadaver donor human pancreati at the University of Chicago Medical Center from December 1991 to April 1993 were analyzed. All pancreati were first offered for whole pancreas transplantation before being considered for islet isolation. Human pancreatic islet isolation was accomplished by a modified automated method. Some islet isolations resulted in a high islet yield but low islet purity. Other resulted in well-purified islets, but a low yield. Arbitrarily, successful islet isolation is defined as that yielding over 250,000 islet equivalents (EQN) with a purity of at least 80%. The success rate of human pancreatic islet isolation was 70%. The mean final islet yield obtained from these 50 pancreati was 300,000±131,000 islet EQN. The mean purity of the final preparation was 73%±25%. By univariate analysis, five factors were found to affect significantly the yield, purity, or overall success rate of islet isolation: organ cold ischemic time, donor age, donor plasma glucose levels, donor body weight, and cause of donor death. Even when islet isolation was successful, the function of islets from hyperglycemic and older donors appear to be impaired both in vitro and in vivo. These results suggest that islet yield and purity are affected by multiple donor-related factors. Even when adequate yield and purity are obtained, islet function is also dependent on donor variables.
Allotransplantation
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We previously reported the in vitro analysis of stage differentiation of Toxoplasma gondii in murine bone marrow-derived macrophages. The purpose of this study was to generate monoclonal rat antibodies that might be suitable for investigating tachyzoite-bradyzoite interconversion in vivo with the murine model. Immunization of Fischer rats with cysts of T. gondii NTE resulted in the generation of seven monoclonal antibodies of the immunoglobulin G2a, G2b, or M isotype, which were further characterized by the immunoblot technique, immunofluorescence assay, immunohistology, and immunoelectron microscopy. Immunoblots demonstrated specific reactivity of five monoclonal antibodies with proteins with molecular masses of 40, 52, 55, 60, 64, 65, and 115 kDa. One antibody (CC2) appeared to recognize a differently expressed antigen depending on the parasite stage, reacting with a 40-kDa molecule in tachyzoites and a 115-kDa antigen in bradyzoites and oocysts. Several other monoclonal antibodies were shown to be stage specific and to react in immunofluorescence assays or in immunoblots with either tachyzoites or bradyzoites. Kinetics of stage conversion in vitro could be monitored by immunofluorescence with two of these monoclonal antibodies. Preliminary immunohistological investigations of tissue sections from infected mice demonstrated the possible usefulness of these monoclonal antibodies for future in vivo studies on stage differentiation of T. gondii in the murine system.
Immunofluorescence
Immunoelectron microscopy
Isotype
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This report confirms reproducible methods to isolate and assess viability and function of pig and human islets. We processed 10 pig and five human pancreata. The pancreata were digested by a modification of Ricordi's automated method for islet isolation. The number of islet equivalents (150 μm diameter islets) was 335,190 ± 79,345 islets per pig pancreas (5,146 ± 1,274 islets/g pig pancreas) and 323,630 ± 147,810 per human pancreas (6,252 ± 2,572 islets/g human pancreas). The majority of islets were in the range of 50–200 μm diameter, and 20% of the islet population had a size distribution of 200 pm diameter in both porcine and human models. The purity of the final preparations exceeded 90%. The secretory response of perifused islets showed a biphasic insulin release pattern in both species. Perifused fresh pig islets released 2.5 pmol/L islet−1 min−1 at 2.0 mM glucose and 6.2 pmol/L islet−1 min−1 at 16.7 mM glucose. After 7 days culture at 37°C, human islets released 1.32 pmol/L islet−1 min−1 at 2.0 mM and 12.24 pmol/L islet−1 min−1 at 16.7 mM. These results indicate that this procedure is useful to obtain pure, large, and functional islets from pig and human pancreata.
Isolation
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