Porcine Islet Cell Xenotransplantation

This article reviews the rationale, sources and preparation of pig islets for xenotransplantation. Pancreatic islet cell transplantation is an attractive alternative and an effective treatment option for type 1 diabetes, however, donor pancreas shortages prevent islet transplantation from being a widespread solution as the supply cannot possibly equal the demand. Porcine islet xenotransplantation has the potential to address these shortages, and recent preclinical and clinical trials show promising scientific support. Pig islets provide a readily available source for islet transplantation, with the recent trials in non-human primates (NHPs) demonstrating their potential to reverse diabetes. The risk of zoonosis can be reduced by designated pathogen-free breeding of the donor pigs, but porcine endogenous retroviruses (PERVs) which are integrated into the genome of all pigs, are especially difficult to eliminate. However, clinical trials have demonstrated an absence of PERV transmission with a significant reduction in the number of severe hypoglycemic episodes and up to 30% reduction in exogenous insulin doses. A number of methods are currently being tested to overcome the xenograft immune rejection. Some of these methods include the production of various transgenic pigs to better xenotransplantation efficiency and the encapsulation of islets to isolate them from the host immune system. Furthermore, ongoing research is also shedding light on factors such as the age and breed of the donor pig to determine the optimal islet quantity and function

Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation

Background: Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity. Methods: Membrane integrity staining (FDA/PI), OCR normalized to DNA (OCR/DNA), islet equivalent (IE) and OCR (viable IE) normalized to recipient body weight (IE dose and OCR dose), and OCR/DNA normalized to islet size index (ISI) were used to characterize autoislet preparations (n = 35). Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis. Results: Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001). These islet characterization methods were highly correlated with II at 6–12 months post-IAT (area-under-the-curve (AUC) = 0.94 for IE dose and 0.96 for OCR dose). FDA/PI (AUC = 0.49) and OCR/DNA (AUC = 0.58) did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72). Conclusions: Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations

Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.

Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 μM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes

Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.

Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 μM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes

Chronic exposure of mouse primary PAC to nicotine reduces the levels of TPKase proteins (A) and mRNA (B).

<p>Western blot analysis was carried out using pancreatic acinar whole cell proteins (60 μg) isolated from chronic nicotine exposed mice and their controls. Real-time PCR was performed using mice gene-specific primers. Data are mean ± SE from separate sets of samples from multiple mice and were normalized relative to ARPO and calculated by the relative relationship method. *<i>P</i> < 0.01, **<i>P</i> < 0.05.</p

Chronic exposure of human PAC to nicotine reduces the levels of THTR-1 and THTR-2 proteins (A and B) and mRNA (C and D).

<p>Levels of THTR-1 and THTR-2 protein expression were determined by Western blotting. The mRNA expression was determined by quantitative PCR. Each data represents the mean ± SE of at least three independent experiments. *<i>P</i> < 0.01, **<i>P</i> < 0.05.</p

Chronic exposure of human PAC to nicotine decreases carrier-mediated [³H] thiamin uptake.

<p>Human PAC were exposed to nicotine (0.5 μM, 48 h) and carrier-mediated uptake of ³H-thiamin was determined as described under “Materials and Methods”. Data represents the mean ± SE of at least three separate uptake determinations.*<i>P</i> < 0.01.</p

Chronic exposure of mouse primary PAC to nicotine decreases the carrier-mediated [³H]thiamin uptake.

<p>Primary PAC were isolated from mice exposed to nicotine and their controls. Carrier -mediated [³H] thiamin uptake was determined as described in “Materials and Methods”. Data are means ± SE of at least three separate uptake determinations from multiple sets of mice. *<i>P</i> < 0.01.</p

Primers used for amplifying coding region of the respective genes by quantitative PCR.

<p>Primers used for amplifying coding region of the respective genes by quantitative PCR.</p