Pain Control, Glucose Control, and Quality of Life in Patients With Chronic Pancreatitis After Total Pancreatectomy With Islet Autotransplantation: A Preliminary Report.

BACKGROUND: Total pancreatectomy (TP) is offered as a last treatment option for pain relief in patients with chronic pancreatitis. Concurrent islets autotransplantation (TP-IAT) may improve glucose control. METHODS: We analyzed results in 20 recent patients who underwent TP-IAT at The University of Chicago. The median observation period was 28 months (2-38). Data were collected prospectively then analyzed retrospectively. RESULTS: The number of patients requiring opioids daily for pain control decreased from 16 (80%) prior to surgery to 2 (13%) 1 year after, with only 1 (6.5%) patient experiencing persistent phantom pancreatic pain. Opioid requirements decreased from a median 56.3 (0-240) morphine equivalent dose to 5 (0-130) on day 75 and to 0 (0-30) at 1-year follow up. Five patients (25%) completely stopped insulin support prior to day 75 while maintaining hemoglobin A1c of 5.9% (5-6.3). Eight (53%) patients were insulin free at 1 year with A1c of 6% (5.5-6.8) and a similar rate persisted in next 2 years. For the remaining patients, the more islet function that was preserved, the less insulin they required and A1c was closer to optimal. Quality of Life (QoL) measured by SF36 Physical (PCS) and Mental (MCS) Component Score improved on day 75 (P < .001) and maintained improvement later on. Both PCS and MCS improved regardless of whether patient requires insulin support or not. CONCLUSIONS: Improvements of QoL with pain resolution and good glucose control can be achieved after TP-IAT in properly selected patients with CP and intractable pain, regardless of patient insulin support status.

External Validation of the Newly Developed BETA-2 Scoring System for Pancreatic Islet Graft Function Assessment.

BACKGROUND: BETA-2 score using a single fasting blood sample was developed to estimate beta-cell function after islet transplantation (ITx) and was validated internally by a high ITx volume center (Edmonton). The goal was to validate BETA-2 externally, in our center. METHODS: Areas under receiver operating characteristic curves (AUROCs) were obtained to see if beta score or BETA-2 would better detect insulin independence and glucose intolerance. RESULTS: We analyzed values from 48 mixed meal tolerance tests (MMTTs) in 4 ITx recipients with a long-term follow-up to 140 months (LT group) and from 54 MMTTs in 13 short-term group patients (ST group). AUROC for no need for insulin support was 0.776 (95% confidence interval [CI] 0.539-1, P = .02) and 0.922 (95% CI 0.848-0.996, P < .001) for beta score and 0.79 (95% CI 0.596-0.983, P = .003) and 0.941 (95% CI 0.86-1, P < .001) for BETA-2, in LT and ST groups, respectively, and did not differ significantly. In LT group BETA-2 score ≥ 13.03 predicted no need for insulin supplementation with sensitivity of 98%, specificity of 50%, positive predictive value (PPV) of 93%, and negative predictive value (NPV) of 75%. In ST group the optimal cutoff was ≥13.63 with sensitivity of 92% and specificity, PPV, and NPV 82% to 95%. For the detection of glucose intolerance BETA-2 cutoffs were <19.43 in LT group and <17.23 in ST group with sensitivity > 76% and specificity, PPV, and NPV > 80% in both groups. CONCLUSION: BETA-2 score was successfully validated externally and is a practical tool allowing for frequent and reliable assessments of islet graft function based on a single fasting blood sample.

Improved coating of pancreatic islets with regulatory T cells to create local immunosuppression by using the biotin-polyethylene glycol-succinimidyl valeric acid ester molecule.

BACKGROUND: We showed that T regulatory (Treg) cells can be attached to the surface of pancreatic islets providing local immunoprotection. Further optimization of the method can improve coating efficiency, which may prolong graft survival. In this study, we compared the effectiveness of two different molecules used for binding of the Tregs to the surface of pancreatic islets. Our aim was to increase the number of Treg cells attached to islets without compromising islets viability and function. METHODS: The cell surface of human Treg cells and pancreatic islets was modified using biotin-polyethylene glycol-N-hydroxylsuccinimide (biotin-PEG-NHS) or biotin-PEG-succinimidyl valeric acid ester (biotin-PEG-SVA). Then, islets were incubated with streptavidin as islet/Treg cells binding molecule. Treg cells were stained with CellTracker CM-DiL dye and visualized using a Laser Scanning Confocal Microscope. The number of Treg cells attached per islets surface area was analyzed by Imaris software. The effect of coating on islet functionality was determined using the glucose-stimulated insulin response (GSIR) assay. RESULTS: The coating procedure with biotin-PEG-SVA allowed for attaching 40% more Treg cells per 1 µm(2) of islet surface. Although viability was comparable, function of the islets after coating using the biotin-PEG-SVA molecule was better preserved than with NHS molecule. GSIR was 62% higher for islets coated with biotin-PEG-SVA compared to biotin-PEG-NHS. CONCLUSION: Coating of islets with Treg cells using biotin-PEG-SVA improves effectiveness with better preservation of the islet function. Improvement of the method of coating pancreatic islets with Treg cells could further facilitate the effectiveness of this novel immunoprotective approach and translation into clinical settings.

Donor height in combination with islet donor score improves pancreas donor selection for pancreatic islet isolation and transplantation.

To maximize the islet isolation yield for successful islet transplantation, the key task has been to identify an ideal pancreas donor. Since implementation of the islet donor score in donor selection, we have consistently obtained higher islet yields and transplantation rates. In this study, we tested whether assessing donor height as an independent variable in combination with the donor score could improve the pancreas donor selection. Donor and islet isolation information (n = 22) were collected and studied between 2011 and 2012. Pearson correlation analysis was used in statistical analysis. Donor height as an independent variable was significantly correlated to the weight of the pancreas, pre-Islet Equivalents (pre-IEQ), post-IEQ, and IDS (P < .05). When donor with height of 179 cm ± 3 was selected in combination with IDS > 80, the clinical islet transplantation rate reached 80%.

Analysis of 2-chloro-2'-deoxyadenosine incorporation into cellular DNA by quantitative polymerase chain reaction.

Thermus aquaticus (Taq) DNA polymerase elongation is blocked by several DNA adducts. This property has been exploited in polymerase chain reaction (PCR) methods to analyze cellular DNA damage and repair after exposure to damaging agents. Such methods have not been applied previously to detect nucleoside analog incorporation into cellular DNA. 2-Chloro-2'-deoxyadenosine (CldAdo), a deoxyadenosine analog, is clinically effective for hairy cell leukemia. CldAdo is taken up by cells, converted to the triphosphate, and incorporated into cellular DNA. Here, we measured by primer extension the ability of CldAMP residues in 98-base single-stranded DNA to block Taq elongation. In contrast to control DNA, no full-length 98-mers were produced on CldAMP-containing templates, and Taq polymerase was halted at the first CldAMP site. We then examined the possibility of using quantitative PCR to measure CldATP incorporation into the N-ras gene after incubation of cultured human leukemia cells with CldAdo or with cisplatin as a positive control for DNA damage. Treatment with either drug resulted in reduced amounts of amplified DNA product compared to untreated cells. CldAMP residues within cellular DNA inhibited PCR amplification in a dose-dependent manner; 100 nM CldAdo produced approximately 0.4 CldAMP sites within a 523-bp region of the N-ras sequence. Thus, PCR analysis with Taq polymerase provides a sensitive assessment of nucleoside analog incorporation after cellular exposure to antileukemic drugs.

2-Chloro-2'-deoxyadenosine, an antileukemic drug, has an early effect on cellular mitochondrial function.

2-Chloro-2'-deoxyadenosine [CldAdo (cladribine)], a novel effective antileukemic agent, was examined for its effects on cellular mitochondrial function and DNA content after long term (< or = 7-day) incubation of cultured CCRF-CEM human leukemia cells. Dideoxycytidine (ddC), which is known to have a delayed effect on mitochondrial DNA content, was used as a positive control to monitor mitochondrial dysfunction. CldAdo at 6-16 nM was toxic to cells within 24 hr, which is in contrast to 300 nM ddC, which had no effect on cell growth for the first 4 days of treatment. Cellular lactic acid production was used to monitor concomitant perturbations in oxidative phosphorylation during drug treatment. Unlike the delayed increase in lactate observed with ddC exposure, CldAdo-treated cells exhibited a 2-2.4-fold increase in lactate levels after 2 days of exposure to 16 nM CldAdo. By days 4 and 7, however, lactate production returned to control levels. Shorter incubations with CldAdo revealed that lactate levels began to increase within 12 hr of drug exposure, paralleling cytotoxicity. We also examined mitochondrial DNA content during drug treatment by competitive polymerase chain reaction. ddC (300 nM) reduced mitochondrial DNA levels from approximately 1000 copies/untreated cell to approximately 130 copies/cell after 7 days of exposure. In contrast, cytotoxic doses of CldAdo had little or no effect on mitochondrial DNA content during the 1-week incubation. Thus, the early CldAdo-induced perturbation of mitochondrial function was not associated with a loss of mitochondrial DNA per cell. In addition, no evidence of DNA laddering, indicative of cellular apoptosis, was detected at these dosage levels and treatment times.

In vitro transcription of DNA containing 2-chloro-2'-deoxyadenosine monophosphate.

2-Chloro-2'-deoxyadenosine (cladribine [CldAdo]) represents one of the most promising therapeutic agents for the treatment of pediatric leukemias and adult hairy cell leukemia. We examined whether CldAdo incorporation into DNA inhibited subsequent transcription in vitro using purified phage RNA polymerases. Control (Ade-containing) and 2-chloroadenine (ClAde)-substituted DNA strands that contained a RNA polymerase promoter sequence were synthesized by a modified asymmetric polymerase chain reaction. Complementary (+) and (-) strands were annealed, incubated with phage RNA polymerase, and analyzed with denaturing PAGE. When ClAde was present in both strands, the yield of full-length transcripts (approximately equal to 100 bases) was reduced by approximately equal to 90% relative to control DNA. Transcription was also reduced to a slightly lesser degree when substitutions occurred in only one of two strands. The observed low transcript levels on ClAde-containing DNA were due in part to the presence of the analogue within the promoter region. With gel shift binding assays, we demonstrated that RNA polymerase did not bind as well to ClAde-containing promoters. Polymerase/DNA complex formation was decreased by approximately equal to 80% compared with that on control unsubstituted promoters. In addition, on binding to the substituted promoter, RNA polymerase had an altered conformation that led to enhanced proteolytic clipping by endoproteinase Glu-C. Transcript sequence analysis indicated that SP6 RNA polymerase read through template ClAde residues with no apparent misincorporation into RNA. Our results provide insight into a novel effect of this nucleoside analogue that may explain its cytotoxicity in nondividing cells.

A human factor that recognizes DNA substituted with 2-chloroadenine, an antileukemic purine analog.

2-Chloro-2'-deoxyadenosine (cladribine), an analog of deoxyadenosine, is an important new drug for the treatment of hairy cell leukemia and other forms of adult and pediatric leukemia. By a gel-shift binding assay, we identified an activity in HeLa nuclear extracts that recognizes and binds to oligonucleotides substituted with 2-chloroadenine (ClAde). The activity was specific for ClAde residues because control oligomers did not readily compete out the complex. The binding factor was a monomeric protein that was resistant to inactivation by heating at 45 degrees C but sensitive to heating at 65 degrees C, proteinase K treatment, and 5 mM ZnCl2. This protein, designated ClAde recognition protein (CARP), appeared to be related to a protein that recognized other forms of DNA damage. Gel-shift binding reactions with ultraviolet (UV)-irradiated oligomers revealed a UV-specific protein/DNA complex that had an electrophoretic mobility similar to that of the CARP/DNA complex, and CARP binding to ClAde-containing oligomers was readily competed out by UV-irradiated DNA. Moreover, CARP activity was present in extracts prepared from UV-sensitive xeroderma pigmentosum group A cells but not in a subset of cells from group E, suggesting that CARP was similar to a previously described repair associated factor, xeroderma pigmentosum-E binding factor. Our findings support a possible repair process for ClAde residues incorporated into cellular DNA.