Indu Nair

TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN.

Akt kinase is activated by transforming growth factor-β1 (TGF-β) in diabetic kidneys, and has important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells. However, the mechanisms of Akt activation by TGF-β are not fully understood. Here we show that TGF-β activates Akt in glomerular mesangial cells by inducing the microRNAs (miRNAs) miR-216a and miR-217, both of which target PTEN (phosphatase and tensin homologue), an inhibitor of Akt activation. These miRNAs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-β and miR-192 through E-box-regulated mechanisms, as shown previously. Akt activation by these miRs led to glomerular mesangial cell survival and hypertrophy, which were similar to the effects of activation by TGF-β. These studies reveal a mechanism of Akt activation through PTEN downregulation by two miRs, which are regulated by upstream miR-192 and TGF-β. Due to the diversity of PTEN function, this miR-amplifying circuit may have key roles, not only in kidney disorders, but also in other diseases.

Glucose‐Stimulated Increment in Oxygen Consumption Rate as a Standardized Test of Human Islet Quality

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 (ΔOCRglc) 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 ΔOCRglc obtained at both institutions were compared. Islet specificity was determined by comparing ΔOCRglc in islet and nonislet tissue. The ΔOCRglc 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 (ΔOCRglc was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The ΔOCRglc was statistically independent of percent beta cells, demonstrating that ΔOCRglc is governed more by islet quality than by islet composition. The ΔOCRglc, but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing ΔOCRglc as a measurement of islet quality for human islet transplantation.

In vivo imaging of transplanted islets with 64Cu-DO3A-VS-Cys40-Exendin-4 by targeting GLP-1 receptor.

Glucagon-like peptide 1 receptor (GLP-1R) is highly expressed in pancreatic islets, especially on β-cells. Therefore, a properly labeled ligand that binds to GLP-1R could be used for in vivo pancreatic islet imaging. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), a more stable agonist of GLP-1 such as Exendin-4 is a preferred imaging agent. In this study, DO3A-VS-Cys(40)-Exendin-4 was prepared through the conjugation of DO3A-VS with Cys(40)-Exendin-4. The in vitro binding affinity of DO3A-VS-Cys(40)-Exendin-4 was evaluated in INS-1 cells, which overexpress GLP-1R. After (64)Cu labeling, biodistribution studies and microPET imaging of (64)Cu-DO3A-VS-Cys(40)-Exendin-4 were performed on both subcutaneous INS-1 tumors and islet transplantation models. The subcutaneous INS-1 tumor was clearly visualized with microPET imaging after the injection of (64)Cu-DO3A-VS-Cys(40)-Exendin-4. GLP-1R positive organs, such as pancreas and lung, showed high uptake. Tumor uptake was saturable, reduced dramatically by a 20-fold excess of unlabeled Exendin-4. In the intraportal islet transplantation models, (64)Cu-DO3A-VS-Cys(40)-Exendin-4 demonstrated almost two times higher uptake compared with normal mice. (64)Cu-DO3A-VS-Cys(40)-Exendin-4 demonstrated persistent and specific uptake in the mouse pancreas, the subcutaneous insulinoma mouse model, and the intraportal human islet transplantation mouse model. This novel PET probe may be suitable for in vivo pancreatic islets imaging in the human.

Generation of human islets through expansion and differentiation of non-islet pancreatic cells discarded (pancreatic discard) after islet isolation.

Objectives: Islet transplantation is hampered by the shortage of donor tissues. Our objective was to generate islet-like cell clusters (ICCs) from cultures of non-islet pancreatic cells. Methods: The starting cultured cells came from the non-islet fractions of human pancreases after enzymatic digestion and purification for the purpose of islet isolation. Initially, these cells expanded in monolayer cultures and became confluent on collagen-coated flasks. After trypsination and suspension of these cells in a defined islet differentiation medium, the cells aggregated to form ICCs. Results: The initial cell population consisted of less than 1% of insulin-positive cells, 44% amylase-positive cells, and 41% cytokeratin (CK) 7-positive, or CK19+ cells, but PDX-1+ cells were absent. Cells from later stages of the monolayer cultures showed signs of dedifferentiation/transdifferentiation. At the time of harvesting, more than 90% of the cells were positive for CK 7/19 and PDX-1, but less than 1% of the cells were insulin-positive. After aggregation, the ICCs appeared redifferentiated, and contained glucose-responsive, insulin-secreting cells with an insulin content measuring 20% of that found in freshly isolated islets isolated from the same pancreas. ICCs transplanted into athymic mice and removed after 4 months did acquire the morphology of mature islets, indicating further maturation of the ICCs in vivo after transplantation. Human C-peptide was detected in recipient animal sera. Conclusion: Using the specified culture methods, non-islet pancreas cells can generate cell clusters resembling islets. These ICCs, obtained from fractions of the pancreas that are otherwise discarded, continue to differentiate after transplantation to become mature islets.

Mixed Chimerism and Growth Factors Augment β Cell Regeneration and Reverse Late-Stage Type 1 Diabetes

Combination therapy that induces mixed chimerism and augments both β cell neogenesis and replication reverses late-stage type 1 diabetes in NOD mice. Two Treatments Are Better than One The mission of the immune system is to defend the body, fighting off infections and cancer. However, sometimes, immune cells mistake healthy normal cells for dangerous foreign invaders. Type 1 diabetes develops when immune cells attack and destroy the insulin-producing β cells in the pancreatic islets. In the absence of insulin, glucose levels increase in the blood and urine, which can be fatal if untreated. The only curative treatment for type 1 diabetes is islet transplantation, but this therapy is restricted by limited donor availability and lifelong immunosuppression for the recipients. Moreover, islet transplants only last about 3 years before the islets succumb to chronic rejection. Thus, other approaches are being tried. One approach, establishing mixed hematopoietic chimerism, can limit autoimmune destruction of islets but only works before the islets are destroyed. Another, administration of gastrin and epidermal growth factor (EGF), stimulates β cell neogenesis, but does not prevent further autoimmune attack. Now, Wang et al. combine these approaches to cure late-stage type 1 diabetes in a mouse model of type 1 diabetes. The authors treated nonobese diabetic mice with a combination of a radiation-free, nontoxic conditioning regimen to induce mixed chimerism and gastrin/EGF to induce β cell regeneration. This combination therapy reversed late-stage type 1 diabetes and improved insulin sensitivity. New β cells were formed following this therapy, and these cells survived and accumulated as a result of mixed chimerism-mediated inhibition of the autoimmune reaction. If this success holds true in human patients, this combination therapy may provide an improved curative therapy for type 1 diabetes. Type 1 diabetes (T1D) results from an autoimmune destruction of insulin-producing β cells. Currently, islet transplantation is the only curative therapy for late-stage T1D, but the beneficial effect is limited in its duration, even under chronic immunosuppression, because of the chronic graft rejection mediated by both auto- and alloimmunity. Clinical islet transplantation is also restricted by a severe shortage of donor islets. Induction of mixed chimerism reverses autoimmunity, eliminates insulitis, and reverses new-onset but not late-stage disease in the nonobese diabetic (NOD) mouse model of T1D. Administration of gastrin and epidermal growth factor (EGF) also reverses new-onset but not late-stage T1D in this animal model. Here, we showed that combination therapy of induced mixed chimerism under a radiation-free nontoxic anti-CD3/CD8 conditioning regimen and administration of gastrin/EGF augments both β cell neogenesis and replication, resulting in reversal of late-stage T1D in NOD mice. If successfully translated into humans, this combination therapy could replace islet transplantation as a long-term curative therapy for T1D.

64Cu labeled sarcophagine exendin-4 for microPET imaging of glucagon like peptide-1 receptor expression

The Glucagon-like peptide 1 receptor (GLP-1R) has become an important target for imaging due to its elevated expression profile in pancreatic islets, insulinoma, and the cardiovascular system. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), several studies have conjugated different chelators to a more stable analog of GLP-1 (such as exendin-4) as PET or SPECT imaging agents with various advantages and disadvantages. Based on the recently developed Sarcophagin chelator, here, we describe the construction of GLP-1R targeted PET probes containing monomeric and dimeric exendin-4 subunit. The in vitro binding affinity of BarMalSar-exendin-4 and Mal2Sar-(exendin-4)2 was evaluated in INS-1 cells, which over-express GLP-1R. Mal2Sar-(exendin-4)2 demonstrated around 3 times higher binding affinity compared with BaMalSar-exendin-4. After 64Cu labeling, microPET imaging of 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 were performed on subcutaneous INS-1 tumors, which were clearly visualized with both probes. The tumor uptake of 64Cu-Mal2Sar-(exendin-4)2 was significantly higher than that of 64Cu-BaMaSarl-exendin-4, which could be caused by polyvalency effect. The receptor specificity of these probes was confirmed by effective blocking of the uptake in both tumor and normal positive organs with 20-fold excess of unlabeled exendin-4. In conclusion, sarcophagine cage conjugated exendin-4 demonstrated persistent and specific uptake in INS-1 insulinoma model. Dimerization of exendin-4 could successfully lead to increased tumor uptake in vivo. Both 64Cu-BaMalSar-exendin-4 and 64Cu-Mal2Sar-(exendin-4)2 hold a great potential for GLP-1R targeted imaging.

Quantitative assessment of β-cell apoptosis and cell composition of isolated, undisrupted human islets by laser scanning cytometry.

Background. Assays for assessing human islet cell quality, which provide results before transplantation, would be beneficial to improve the outcomes for islet transplantation therapy. Parameters such as percent &bgr;-cell apoptosis and cell composition are found to vary markedly between different islet preparations and may serve as markers of islet quality. We have developed fluorescence-based assays using laser scanning cytometry for assessing &bgr;-cell apoptosis and islet cell composition on serial sections of intact isolated islets. Methods. Isolated human islets were fixed in formalin and embedded in paraffin. Serial sections were immunostained for the pancreatic hormones and acinar and ductal cell markers. DNA fragmentation was used to label apoptotic cells. Stained cells were quantified using an iCys laser scanning cytometer. Results. Islet preparations from 102 human pancreatic islet isolations were analyzed. For the whole set of islet preparations, we found a mean islet cell composition of 54.5%±1.2% insulin-positive, 33.9%±1.2% glucagon, 12.1%±0.7% somatostatin, and 1.5%±0.2% pancreatic polypeptide-positive cells. The apoptotic &bgr; cells were 2.85%±0.4% with a range of 0.27% to 18.3%. The percentage of apoptotic &bgr; cells correlated well (P<0.0001, n=59) with results obtained in vivo by transplantation of the corresponding islets in diabetic NODscid mice. Conclusions. The analysis of whole, nondissociated islets for cell composition and &bgr;-cell apoptosis using laser scanning cytometry gives reliable and reproducible results and could be performed both before islet transplantation and on preserved cell blocks at any time in future. Thus, they can be a powerful tool for islet quality assessment.

Induction of Chimerism Permits Low-Dose Islet Grafts in the Liver or Pancreas to Reverse Refractory Autoimmune Diabetes

OBJECTIVE To test whether induction of chimerism lowers the amount of donor islets required for reversal of diabetes and renders the pancreas a suitable site for islet grafts in autoimmune diabetic mice. RESEARCH DESIGN AND METHODS The required donor islet dose for reversal of diabetes in late-stage diabetic NOD mice after transplantation into the liver or pancreas was compared under immunosuppression or after induction of chimerism. Recipient mice were monitored for blood glucose levels and measured for insulin-secretion capacity. Islet grafts were evaluated for β-cell proliferation, β-cell functional gene expression, and revascularization. RESULTS With immunosuppression, transplantation of 1,000, but not 600, donor islets was able to reverse diabetes when transplanted into the liver, but transplantation of 1,000 islets was not able to reverse diabetes when transplanted into the pancreas. In contrast, after induction of chimerism, transplantation of as few as 100 donor islets was able to reverse diabetes when transplanted into either the liver or pancreas. Interestingly, when lower doses (50 or 25) of islets were transplanted, donor islets in the pancreas were much more effective in reversal of diabetes than in the liver, which was associated with higher β-cell replication rate, better β-cell functional gene expression, and higher vascular density of graft islets in the pancreas. CONCLUSIONS Induction of chimerism not only provides immune tolerance to donor islets, but also markedly reduces the required amount of donor islets for reversal of diabetes. In addition, this process renders the pancreas a more superior site than the liver for donor islets in autoimmune mice.

Human Pancreatic Islets Isolated From Donors With Elevated HbA1c Levels: Islet Yield and Graft Efficacy.

The aim of this study was to investigate the effects of elevated donor HbA1c levels (type 2 diabetes, T2D) on the islet yield and functionality postisolation. In this retrospective analysis, donors for islet isolations were classified into two groups: T2D group (HbA1c ≥ 6.5%, n = 18) and normal group (HbA1c < 6.5%, n = 308). Optimum pancreas digestion time (switch time) was significantly higher in the T2D group compared to the normal group (13.7 ± 1.2 vs. 11.7 ± 0.1 min, respectively, p = 0.005). Islet yields were significantly lower in the T2D group compared to the control (T2D vs. control): islet equivalent (IEQ)/g (prepurification 2,318 ± 195 vs. 3,713 ± 114, p = 0.003; postpurification 1,735 ± 175 vs. 2,663 ± 89, p = 0.013) and islet particle number (IPN)/g (prepurification, 2,519 ± 336 vs. 4,433 ± 143, p = 0.001; postpurification, 1,760 ± 229 vs. 2,715 ± 85, p = 0.007). Islets from T2D pancreata had significantly lower viability (T2D vs. control: 91.9 ± 1.6 vs. 94.4 ± 0.3%, p = 0.004) and decreased oxygen consumption rate (DOCR) (T2D vs. control: 0.09 ± 0.01 and 0.21 ± 0.03 nmol O2 100 islets−1 min−1, p = 0.049). The islets isolated from T2D donor pancreata reversed diabetes in NOD-SCID mice in 9% (2/22) compared to islets from control donor pancreata, which reversed diabetes in 67% (175/260, p < 0.001). In conclusion, this study demonstrates that elevated HbA1c (≥6.5%) is associated with impairment of islet function and lower islet yield; however, these islets could not be suitable for clinical applications.

Mesenchymal stem cells facilitate mixed hematopoietic chimerism induction and prevent onset of diabetes in nonobese diabetic mice.

Objectives: Allogeneic mesenchymal stem cells (MSCs) and bone marrow cells (BMCs) were cotransplanted in nonobese diabetic mice after none myeloablative preconditioning and the development of chimerism, insulitis, diabetes, and graft-versus-host disease (GVHD) were monitored. Methods: Eight-week-old female nonobese diabetic mice were injected intravenously with 2 × 107 BMCs and 5 × 105 MSCs from C57BL/6 mice after treatment with 2 intraperitoneal injections of anti-CD3 antibody (days −7 and −4) and 3-Gy total body irradiation (day −1). Thereafter, blood glucose and chimerism were monitored on peripheral blood samples. Results: Stable mixed chimerism (3−>90% of donor phenotype) was induced in 63.2% of BMCs-MSCs recipients (n = 19) and 45.0% of BMCs-alone recipients (n = 20, P = 0.256). Insulitis was prevented, and euglycemia persisted for more than 18 weeks in 89.5% of BMCs-MSCs recipients including those with less than 3% chimerism and 55% of BM-alone recipients (P < 0.05). In controls, 9.1% of mice receiving preconditioning treatment alone (n = 11) and 16.7% of preconditioned mice receiving only MSCs (n = 12) were nondiabetic. Graft-versus-host disease was not detected in all mice. Conclusions: Coinjection of MSCs and BMCs increased the success rate in inducing chimerism and preventing insulitis and overt diabetes with no incidence of GVHD. Results also indicated that even microchimerism with less than 3% donor cells is sufficient for blocking autoimmunity.Abbreviations: Ab - antibody, BM - bone marrow, BMCs - bone marrow cells, FACS - fluorescence-activated cell sorting, GVHD - graft-versus-host disease, IL - interleukin, IV - intravenous(ly), L - ligand, mAb - monoclonal antibody, MLR - mixed lymphocyte reaction, MSCs - mesenchymal stem cells, NOD - nonobese diabetic, P - passage, PBLs - peripheral blood lymphocytes, PBS - phosphate-buffered saline, TBI - total body irradiation