Xiaojuan Chen

In vivo bioluminescence imaging of transplanted islets and early detection of graft rejection

Background. Bioluminescence imaging (BLI) modalities are being developed to monitor islet transplant mass and function in vivo. The aim of this study was to use the BLI system to determine how the change in functional islet mass correlated to metabolic abnormalities during the course of alloimmune rejection in a murine transplant model. Methods. Islets obtained from a transgenic mouse strain (FVB/NJ-luc) that constitutively expressed firefly luciferase were transplanted to various implantation sites of syngeneic wild-type FVB/NJ or allogeneic Balb/C streptozotocin-induced diabetic recipients. In vivo graft luminescent signals were repeatedly measured after transplantation using the BLI system and related to blood glucose levels and graft site histologic findings. Results. The BLI signals were detected in as few as 10 islets implanted in the renal subcapsular space, intrahepatic, intraabdominal, and subcutaneous locations. There was a linear relationship between the number of islets transplanted and luminescence intensity. In isografts, stable luminescence intensity signals occurred within 2 weeks of transplantation and remained consistent on a long-term basis (18 months) after transplantation. In allografts, after normoglycemia was achieved and stable luminescence intensity occurred, graft bioluminescent intensity progressively decreased several days before permanent recurrence of hyperglycemia as a result of histologically proven rejection ensued. Conclusions. Bioluminescence imaging is a sensitive method for tracking the fate of islets after transplantation and is a useful method to detect early loss of functional islet mass caused by host immune responses even before overt metabolic dysfunction is evident. Bioluminescence imaging holds promise for use in designing and testing interventions to prolong islet graft survival.

Sequential Kidney/Islet Transplantation Using Prednisone-Free Immunosuppression

Islet transplantation is becoming established as a treatment option for type I diabetes in select patients. Individuals with type I diabetes who have previously received a successful kidney allograft may be good candidates for islet transplantation. They have already assumed the risks of chronic immunosuppression, so the added procedural risk of a subsequent islet transplant would be minimal. Furthermore, because of the preimmunosuppressed state it is possible that islet‐after‐kidney transplantation may result in a more efficient early islet engraftment. Consequently, insulin independence might be achieved with significantly fewer islets than the approximately 8–10u2003000 islet equivalents/kg/b.w. currently required. A mass that usually demands two or more cadaveric donors. A case of successful islet‐after‐kidney transplantation is described using the steroid‐free Edmonton immunosuppression protocol. Characteristics of the final islet product are: a) islet equivalents: 265u2003888 (4100 islet equivalents/kg/b.w.); b) islet purity: 75–80%; c) viability:u2003>u200395% (trypan blue exclusion); and d) mean islet potency (static low‐high glucose challenge): 4.16u2003±u20031.91‐fold increase. Post‐transplant the patients hypoglycemic episodes abated. Exogenous insulin requirements were eliminated at week 12 post‐transplant as basal and Ensure® (Abbott Laboratories, Abbott Park, IL, USA) oral glucose stimulated C‐peptide levels peaked and stabilized. Twenty‐four‐hour continuous glucose monitoring confirmed moment‐to‐moment glycemic control, and periodic nonfasting finger stick glucose determinations over the next month confirmed glycemia was controlled. Hemoglobin A1c levels declined from a pretransplant level of 6.9% to 5.3%. Renal allograft function remained changed.

Accumulation of cadmium in insulin-producing β cells

Evidence suggests that chronic low level cadmium exposure impairs the function of insulin-producing β cells and may be associated with type-2 diabetes mellitus. Herein, we describe the cadmium content in primary human islets and define the uptake kinetics and effects of environmentally relevant cadmium concentrations in cultured β cells. The average cadmium content in islets from 10 non-diabetic human subjects was 29 ± 7 nmol/g protein (range 7 to 72 nmol/g protein). Exposure of the β-cell line MIN6 to CdCl2 concentrations between 0.1 and 1.0 µmol/L resulted in a dose- and time-dependent uptake of cadmium over 72 h. This uptake resulted in an induction of metallthionein expression, likely enhancing cellular cadmium accumulation. Furthermore, cadmium accumulation resulted in an inhibition of glucose stimulated insulin secretion in MIN6 cells and primary mouse islets. Our results indicate that this impairment in β-cell function is not due to an increase in cell death or due to an increase in oxidative stress. We conclude that mouse β cells accumulate cadmium in a dose- and time-dependent manner over a prolonged time course at environmentally relevant concentrations. This uptake leads to a functional impairment of β-cell function without significant alterations in cell viability, expression of genes important for β-cell function or increase in oxidative stress.

Transfection of pancreatic islets using polyvalent DNA-functionalized gold nanoparticles

BACKGROUNDnTransplantation of pancreatic islets is an effective treatment for select patients with type 1 diabetes. Improved cellular therapy results may be realized by altering the gene expression profile of transplanted islets. Current viral and nonviral vectors used to introduce nucleic acids for gene regulation hold promise, but safety and efficacy shortcomings motivate the development of new transfection strategies. Polyvalent gold nanoparticles (AuNPs) densely functionalized with covalently immobilized DNA oligonucleotides (AuNP-DNA) are new single entity transfection and gene regulating agents (ie, not requiring lipids, polymers, or viral vectors for cell entry) able to enter cells with high efficiency and no evidence of toxicity. We hypothesize that AuNP-DNA conjugates can efficiently transfect pancreatic islets with no impact on viability or functionality, and can function to regulate targeted gene expression.nnnMETHODSnAuNPs were surface-functionalized with control and antisense DNA oligonucleotides. Purified murine and human islets were exposed to AuNP-DNA conjugates for 24 hours. Islet AuNP-DNA uptake, cell viability, and functionality were measured. Furthermore, the ability of antisense AuNP-DNA conjugates to regulate gene expression was measured using murine islets expressing eGFP.nnnRESULTSnCollectively, fluorescent confocal microscopy, transmission electron microscopy, mass spectrometry, and flow cytometry revealed substantial penetration of the AuNP-DNA conjugates into the inner core of the islets and within islet cells. No change in cellular viability occurred and the insulin stimulation index was unchanged in treated versus untreated islets. Transplantation of AuNP-DNA treated islets cured diabetic nude mice. Functionally, antisense eGFP AuNP-DNA conjugates reduced eGFP expression in MIP-eGFP islets.nnnCONCLUSIONnPolyvalent AuNP-DNA conjugates may represent the next generation of nucleic acid-based therapeutic agents for improving pancreatic islet engraftment, survival, and long-term function.

Proinflammatory cytokines induce NF-κB-Dependent/NO-independent chemokine gene expression in MIN6 β cells1

Background. Interactions between chemokines IP-10, MCP-1, and RANTES and their receptors may mediate graft rejection following islet transplantation. The mechanisms regulating chemokine gene expression in pancreatic islet cells have not been well characterized. We examined the cytokine-induced gene expression profiles for several chemokines in a transformed pancreatic β-cell line (MIN6) cotreated with an inhibitor of nitric oxide synthase and in a mutated clone of MIN6 made to overexpress a dominant negative inhibitor of NF-κB (IκBαM). n nMethods. MIN6 and MIN6-IκBαM (Bm) cells were cultured in mixtures of IL-1β and TNF-α or IL-1β, TNF-α, and IFN-γ plus/minus the iNOS inhibitor l-NMMA. RT-PCR and RNase Protection Assay were used to measure mRNA expression for the following chemokines: IP-10, MIP-1α, MIP-1β, MCP-1, and RANTES. Enzyme linked immunosorbant assay was used to measure IP-10 and MCP-1 protein release. n nResults. Cytokine-treated MIN6 and Bm demonstrated increased expression of genes for IP-10 and MCP-1. Expression in MIN6 was first detected at 2 h of incubation and peaked at 6 h. MIN6 demonstrated a more marked increase in chemokine gene expression for both IP-10 and MCP-1 and a more marked increase in IP-10 protein release than did Bm. There was no detectable gene expression for MIP-1α, MIP-1β, or RANTES from MIN6 or Bm. l-NMMA completely blocked NO production from MIN6 and Bm but had no effect on chemokine gene expression in either MIN6 or Bm. n nConclusions. These results suggest that β cells produce a complement of rejection-relevant chemokines in response to a proinflammatory stimulus and that pathways governing cytokine-induced chemokine gene expression in MIN6 are dependent on NF-κB but independent of NO.

Conditional and specific inhibition of NF-κB in mouse pancreatic β cells prevents cytokine-induced deleterious effects and improves islet survival posttransplant

BACKGROUNDnIslets are susceptible to damage by proinflammatory cytokines via activation of transcription factor NF-κB. We hypothesized that inhibition of NF-κB activity will decrease cytokine-mediated β-cell injury and improve islet transplant functional outcome.nnnMETHODSnWe created a transgenic mouse expressing a degradation resistant N-terminally deleted IκBα (ΔNIκBα) under the control of a commercially available tetracycline-controlled transcriptional activation system using a rat insulin promoter. Isolated islets from transgenic and control mouse strains were exposed to cytokines in vitro and assayed or transplanted.nnnRESULTSnWestern blot analysis showed that ΔNIκBα was significantly increased with doxycycline treatment. Cytokine-induced NF-κB activation was significantly decreased in transgenic (0.065 ± 0.013 absorbance value/μg protein) vs control islets (0.128 ± 0.006; P < .05). Suppression of cytokine-mediated NF-κB activity decreased expression of inducible nitric oxide synthase, monocyte chemoattractant protein-1, and interferon-γ inducible protein-10 RNA transcripts, and significantly decreased nitric oxide production in transgenic islets (0.084 ± 0.043 μM/μg protein) vs. controls (0.594 ± 0.174; P < .01). The insulin stimulation index in islets exposed to cytokines was higher in transgenic vs controls (1.500 ± 0.106 vs 0.800 ± 0.098; P < .01). Syngeneic transplants of a marginal mass of intraportally infused transgenic islets resulted in a reversion to euglycemia in 69.2% of diabetic recipients at a mean of 7.8 ± 1.1 days vs. 35.7% of control islet recipients reverting at a mean of 15.8 ± 2.9 days (P < .05).nnnCONCLUSIONnConditional and specific suppression of NF-κB activity in β cells protected islets from cytokine-induced dysfunction in vitro and in vivo. These results provide a proof of principle that inhibition of NF-κB activity in donor islets enhances function and improves the outcome of islet transplantation.

Prolonging islet allograft survival using in vivo bioluminescence imaging to guide timing of antilymphocyte serum treatment of rejection.

Background. Bioluminescence imaging (BLI) is a sensitive and noninvasive method for tracking the fate of transplanted islets. The aim of this study was to investigate whether early detection of rejection by BLI can aid in the timing of antilymphocyte serum (ALS) treatment for prolonging islet graft survival. Methods. Transgenic islets (200 per recipient) expressing the firefly luciferase from FVB/NJ strain (H-2q) mice were transplanted under the kidney capsule of streptozotocin-induced diabetic allogeneic Balb/c strain (H-2d) mice. BLI signals and serum glucose levels were measured daily after transplant. Four groups of mice were transplanted: group 1 recipients were untreated controls (n=12), group 2 (n=8) received ALS before transplant, group 3 (n=10) received ALS at a time after transplant when normoglycemic but prompted by a reduction (∼30%) in BLI signal intensity for 2 consecutive days, and group 4 (n=5) received ALS after transplant when prompted by blood glucose levels increasing ∼20% from the normoglycemic baseline (BLI reduction ∼70%). Results. The incidence of graft loss from rejection in groups 1, 2, 3, and 4 was 92.3%, 88%, 40%, and 100%, respectively. The mean (±SE) time to graft loss in groups 1, 2, 3 and 4 was 22.5±4.8, 29.2±9.9, 53.5±17.9, and 22.1±2.4 days, respectively. Conclusions. Noninvasive imaging modalities of functional islet mass, such as BLI (but not blood glucose levels), can prompt the appropriate timing of ALS treatment of islet allograft rejection and significantly prolong graft survival or protect the grafts from permanent loss.

Comparative Study of Regenerative Potential of β Cells From Young and Aged Donor Mice Using a Novel Islet Transplantation Model

Background. The effect of ageing on &bgr;-cell regeneration under hyperglycemia has not been defined and may best be addressed using a unique islet-transplantation model. Methods. Streptozotocin-induced diabetic FVB/NJ mice were rendered normoglycemic with a therapeutic mass of syngeneic islets implanted in the epididymal fat pad, followed by a subrenal capsular implantation of a subtherapeutic mass of 25 islets from young (3 months) or old (24 months) mice. Three weeks after the second transplant, the islet containing fat pad was removed to reintroduce hyperglycemia. Bromodeoxyuridine (BrdU) was provided to mice continuously in drinking water. Islet grafts under the kidney capsule were harvested at different time points and examined for markers of &bgr;-cell regeneration by immunohistochemistry. Results. After a 7-day labeling, BrdU was detected in 54.2% or 53.0% &bgr; cells of the young or old islet grafts, respectively, under hyperglycemia when compared with 3.3% in grafts under normoglycemia. Ki67-positive &bgr; cells were enhanced from a baseline level of 0.5% to 5.2% (young islets) or 4.0% (old islets) on day 7 of hyperglycemia, then decreased to 2.4% on day 21, at which time point an accumulative 75.3% or 66.8% BrdU-positive &bgr; cells was detected in the young or old grafts, respectively. No statistic difference in the percent BrdU- or Ki67-positive &bgr; cells was detected between the young and aged grafts at any time point studied. Conclusions. These data reveal that islet &bgr; cells from aged mice can replicate in response to hyperglycemia after transplantation at a capacity and frequencies not significantly different than that of the young adult ones.

In Vivo Detection of Extrapancreatic Insulin Gene Expression in Diabetic Mice by Bioluminescence Imaging

Background Extrapancreatic tissues such as liver may serve as potential sources of tissue for generating insulin-producing cells. The dynamics of insulin gene promoter activity in extrapancreatic tissues may be monitored in vivo by bioluminescence-imaging (BLI) of transgenic mice Tg(RIP-luc) expressing the firefly luciferase (luc) under a rat-insulin gene promoter (RIP). Methods The Tg(RIP-luc) mice were made diabetic by a single injection of the pancreatic β-cell toxin streptozotocin. Control mice were treated with saline. Mice were subject to serum glucose measurement and bioluminescence imaging daily. On day eight of the treatment, mice were sacrificed and tissues harvested for quantitative luciferase activity measurement, luciferase protein cellular localization, and insulin gene expression analysis. Results Streptozotocin-induced diabetic Tg(RIP-luc) mice demonstrated a dramatic decline in the BLI signal intensity in the pancreas and a concomitant progressive increase in the signal intensity in the liver. An average of 5.7 fold increase in the liver signal intensity was detected in the mice that were exposed to hyperglycemia for 8 days. Ex vivo quantitative assays demonstrated a 34-fold induction of the enzyme activity in the liver of streptozotocin-treated mice compared to that of the buffer-treated controls. Luciferase-positive cells with oval-cell-like morphology were detected by immunohistochemistry in the liver samples of diabetic mice, but not in that of non-treated control transgenic mice. Gene expression analyses of liver RNA confirmed an elevated expression of insulin genes in the liver tissue exposed to hyperglycemia. Conclusions BLI is a sensitive method for monitoring insulin gene expression in extrapancreatic tissues in vivo. The BLI system may be used for in vivo screening of biological events or pharmacologic activators that have the potential of stimulating the generation of extrapancreatic insulin-producing cells.

Bioluminescent Imaging of Transplanted Islets

Bioluminescence imaging (BLI) modalities have been developed, refined, and used broadly in the study of small animal models of human biology and disease, including monitoring the fate of transplanted islets in vivo in real time. In order to advance our understanding of the pathophysiology and immunobiology of islet transplantation as they occur in living animals, islet grafts tagged with light-emitting luciferase can be implanted in a mouse islet transplantation model and assessed using in vivo BLI. We have utilized transgenic islets expressing the firefly luciferase as donor islets in syngeneic and allogeneic islet transplant mouse models for monitoring islets in vivo by BLI after they have been transplanted at different sites of the mice, including the intrahepatic site via portal vein injection. The sensitive and non-invasive BLI system allows better understanding of the dynamic fate of transplanted islets and the relationships among the islet mass that ultimately engrafts, the quality of graft function, and overall glucose homeostasis. It permits detection of early changes in islet graft function or mass due to rejection to prompt timely therapeutic intervention and change the fate of the graft. This chapter details some of the procedures for islet isolation, transplantation, and imaging as well as considerations of using the BLI system in the field of islet transplantation research.