Supporting the Research Likarda Performs

The publications supporting the research performed at Likarda are listed below. If you are unable to access or read any of the following publications, please contact us and we will assist you with gaining access.

A simple, reliable method for high-throughput screening for diabetes drugs using 3D β-cell spheroids.

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Differences in insulin biosynthesis pathway between small and large islets do not correspond to insulin secretion.

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Arum Palaestinum with isovanillin, linolenic acid and β-sitosterol inhibits prostate cancer spheroids and reduces the growth rate of prostate tumors in mice.

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A Simple Method to Replace Islet Equivalents for Volume Quantification of Human Islets.

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Comparison of Antimicrobial and Wound Healing Agents on Oral Fibroblast Viability and In-vivo Bacterial Load

Oral rinses are designed to restore healing (often for mouth sores) or kill oral bacteria. The balance between bactericidal and restorative effects is not often achieved, leading to unwanted side effects of the products. In this study we ran comparisons of currently available cleansing rinses, measuring possible toxic effects on gingival fibroblasts invitro and their bactericidal effects in-vivo.

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Assessment of re-aggregated human pancreatic islets for secondary drug screening

Insulin secretion from isolated pancreatic islets is a pivotal assay in developing novel insulin secretagogues, given its good correlation with in-vivo efficacy. Because the supply of human islets is limited, this assay is typically run with rodent islets, which do not address species differences and are low-throughput, because of the size matching or volume normalization required. Here we have evaluated the suitability of human re-aggregated islets for this assay.

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KU-32, a Novel Drug for Diabetic Neuropathy, Is Safe for Human Islets and Improves In Vitro Insulin Secretion and Viability

KU-32 is a novel, novobiocin-based Hsp90 inhibitor that protects against neuronal glucotoxicity and reverses multiple clinical indices of diabetic peripheral neuropathy in a rodent model. However, any drug with potential for treating diabetic complications must also have no adverse effects on the function of pancreatic islets. Thus, the goal of the current study was to assess the effect of KU-32 on the in vitro viability and function of human islets.

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Engineering islets for improved performance by optimized reaggregation in a micromold.  

Ramachandran K, Williams SJ, Huang HH, Novikova L, Stehno-Bittel L.
Tissue Eng Part A. 2013 Mar;19(5-6):604-12.

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Isolated islets can provide a source of tissue for research, transplantation, and drug discovery to develop therapies for diabetes. Empirical modeling of islet diffusion barriers demonstrated that only the outermost layers of cells were exposed to glucose and sufficient oxygen levels, resulting in core cell death. Islets under a diameter of 100 μm exhibited a lower diffusion barrier, superior survival rates, and improved functional properties. Utilizing these observations, we engineered optimal islets by dispersing them into single cells and reaggregating them over several days in a micromold. These custom-designed micromolds contained conical-shaped recesses that enhanced reaggregation of cells into a defined geometry. The engineered islets, or Kanslets, were all under 100 μm in diameter, and had the same general cellular composition as native islets. Kanslets continued to produce new insulin molecules and had microvilli on the islet surface, much like native islets. The engineered islets had a statistically higher viability (percent of live cells), and increased glucose diffusion compared to native islets. In addition, they remained responsive to varying glucose levels by secreting insulin. When transplanted into diabetic rats, engineered islets performed reduced random blood glucose to normal levels within 48 h. Optimally, engineering islets may be a suitable alternative to utilizing native, isolated islet tissue for a variety of applications. Reaggregating tissue in an optimized manner using our engineered micromold approach has immense impact for three-dimensional tissue production and its subsequent use in research, drug discovery, and the clinic.

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Long-term liraglutide treatment is associated with increased insulin content and secretion in β-cells, and a loss of α-cells in ZDF rats. 

Schwasinger-Schmidt T, Robbins DC, Williams SJ, Novikova L, Stehno-Bittel L.
Pharmacol Res. 2013 Oct;76:58-66.

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The ultimate treatment goal of diabetes is to preserve and restore islet cell function. Treatment of certain diabetic animal models with incretins has been reported to preserve and possibly enhance islet function and promote islet cell growth. The studies reported here detail islet cell anatomy in animals chronically treated with the incretin analog, liraglutide. Our aim was to quantitatively and qualitatively analyze islet cells from diabetic animals treated with vehicle (control) or liraglutide to determine whether normal islet cell anatomy is maintained or enhanced with pharmaceutical treatment. We harvested pancreata from liraglutide and vehicle-treated Zucker Diabetic Fatty (ZDF) rats to examine islet structure and function and obtain isolated islets. Twelve-week-old male rats were assigned to 3 groups: (1) liraglutide-treated diabetic, (2) vehicle-treated diabetic, and (3) lean non-diabetic. Liraglutide was given SC twice daily for 9 weeks. As expected, liraglutide treatment reduced body weight by 15% compared to the vehicle-treated animals, eventually to levels that were not different from lean controls. At the termination of the study, blood glucose was significantly less in the liraglutide-treated rats compared to vehicle treated controls (485.8±22.5 and 547.2±33.1mg/dl, respectively). Insulin content/islet (measured by immunohistochemistry) was 34.2±0.7 pixel units in vehicle-treated rats, and 54.9±0.6 in the liraglutide-treated animals. Glucose-stimulated insulin secretion from isolated islets (measured as the stimulation index) was maintained in the liraglutide-treated rats, but not in the vehicle-treated. However, liraglutide did not preserve normal islet architecture. There was a decrease in the glucagon-positive area/islet and in the α-cell numbers/area with liraglutide treatment (6.5 cells/field), compared to vehicle (17.9 cells/field). There was an increase in β-cell numbers, the β- to α-cell ratio that was statistically higher in the liraglutide-treated rats (24.3±4.4) compared to vehicle (9.1±2.8). Disrupted mitochondria were more commonly observed in the α-cells (51.9±10.3% of cells) than in the β-cells (27.2±4.4%) in the liraglutide-treated group. While liraglutide enhanced or maintained growth and function of certain islet cells, the overall ratio of α- to β-cells was decreased and there was an absolute reduction in islet α-cell content. There was selective disruption of intracellular α-cell organelles, representing an uncoupling of the bihormonal islet signaling that is required for normal metabolic regulation. The relevance of the findings to long-term liraglutide treatment in people with diabetes is unknown and should be investigated in appropriately designed clinical studies.

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Small human islets comprised of more β-cells with higher insulin content than large islets. 

Farhat B, Almelkar A, Ramachandran K, Williams SJ, Huang HH, Zamierowksi D, Novikova L, Stehno-Bittel L.
Islets. 2013 Mar-Apr;5(2):87-94.

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For the past 30 years, data have suggested that unique islet populations exist, based on morphology and glucose sensitivity. Yet little has been done to determine the mechanism of these functional differences. The purpose of this study was to determine whether human islets were comprised functionally unique populations, and to elucidate a possible mechanism. Islets or pancreatic sections from 29 human donors were analyzed. Islets were isolated and measured for insulin secretion, cell composition and organization, insulin and glucagon granule density and insulin content. Insulin secretion was significantly greater in small compared with large islets. In sectioned human pancreata, β-cells comprised a higher proportion of the total endocrine cells in small islets (63%) than large islets (39%). A higher percentage of β-cells in small islets contacted blood vessels (44%) compared with large islets (31%). Total insulin content of isolated human islets was significantly greater in the small (1323 ± 512 μIU/IE) compared with large islets (126 ± 48 μIU/IE). There was less immunostaining for insulin in the large islets from human pancreatic sections, especially in the core of the islet, compared with small islets. The results suggest that differences in insulin secretion between large and small islets may be due to a higher percentage of β-cells in small islets with more β-cells in contact with blood vessels and a higher concentration of insulin/β-cell in small islets.

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Elimination of T cell reactivity to pancreatic β cells and partial preservation of β cell activity by peptide blockade of LFA-1:ICAM-1 interaction in the NOD mouse model.

Dotson AL, Novikova L, Stehno-Bittel L, Benedict SH.
Clin Immunol. 2013 Aug;148(2):149-61.

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In insulin dependent diabetes mellitus (T1D), self-reactive T cells infiltrate pancreatic islets and induce beta cell destruction and dysregulation of blood glucose. A goal is to control only the self-reactive T cells, leaving the remainder of the T cell population free to protect the host. One approach is blockade of the second signal for T cell activation while allowing the first (antigen-specific) signal to occur. This work proposes that small peptides that block interaction of second signals delivered through the counter receptors LFA-1:ICAM-1 will induce attacking T cells (receiving the antigen signal) to become anergic or undergo apoptosis. In NOD mice, the peptides eliminated T cell reactivity against pancreatic antigens and reduced cellular infiltration into islets, which retained stronger density of insulin staining at five weeks after cessation of therapy. In in vitro studies the peptides induced nonresponsiveness during activation of T cells from mice and from human peripheral blood.

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A novel three-dimensional stromal-based model for in vitro chemotherapy sensitivity testing of leukemia cells.

Ramachandran K, Stehno-Bittel L, Van Veldhuizen P, Lin TL, Kambhampati S, Garimella R., Aljitawi OS, Li D, Xiao Y, Zhang D
Leuk Lymphoma. 2013 May 15

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The disparate response of leukemia cells to chemotherapy in vivo, compared to in vitro, is partly related to the interaction of leukemic cells and the three-dimensional bone marrow stromal microenvironment. We investigated the effects of chemotherapy agents on leukemic cell lines co-cultured with human bone marrow mesenchymal stem cells (hu-BM-MSCs) in a three-dimensional model (3D). Comparison was made to leukemic cells treated in suspension, or grown on a hu-BM-MSC monolayer (2D conditions). We demonstrated that leukemic cells cultured in 3D were more resistant to drug-induced apoptosis compared to cells cultured in 2D or in suspension. We also demonstrated significant differences in leukemic cell response to chemotherapy using different leukemic cell lines cultured in 3D. We suggest that the differential responses to chemotherapy in 3D may be related to the expression of N-cadherin in the co-culture system. This unique model provides an opportunity to study leukemic cell responses to chemotherapy in 3D.

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Expression and regulation of nampt in human islets.

Kover K, Tong PY, Watkins D, Clements M, Stehno-Bittel L, Novikova L, Bittel D, Kibiryeva N, Stuhlsatz J, Yan Y, Ye SQ, Moore WV.
PLoS One. 2013;8(3):e58767.

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Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the mammalian NAD+ biosynthesis of a salvage pathway and exists in 2 known forms, intracellular Nampt (iNampt) and a secreted form, extracellular Nampt (eNampt). eNampt can generate an intermediate product, nicotinamide mononucleotide (NMN), which has been reported to support insulin secretion in pancreatic islets. Nampt has been reported to be expressed in the pancreas but islet specific expression has not been adequately defined. The aim of this study was to characterize Nampt expression, secretion and regulation by glucose in human islets. Gene and protein expression of Nampt was assessed in human pancreatic tissue and isolated islets by qRT-PCR and immunofluorescence/confocal imaging respectively. Variable amounts of Nampt mRNA were detected in pancreatic tissue and isolated islets. Immunofluorescence staining for Nampt was found in the exocrine and endocrine tissue of fetal pancreas. However, in adulthood, Nampt expression was localized predominantly in beta cells. Isolated human islets secreted increasing amounts of eNampt in response to high glucose (20 mM) in a static glucose-stimulated insulin secretion assay (GSIS). In addition to an increase in eNampt secretion, exposure to 20 mM glucose also increased Nampt mRNA levels but not protein content. The secretion of eNampt was attenuated by the addition of membrane depolarization inhibitors, diazoxide and nifedipine. Islet-secreted eNampt showed enzymatic activity in a reaction with increasing production of NAD+/NADH over time. In summary, we show that Nampt is expressed in both exocrine and endocrine tissue early in life but in adulthood expression is localized to endocrine tissue. Enzymatically active eNampt is secreted by human islets, is regulated by glucose and requires membrane depolarization.

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A replacement for islet equivalents with improved reliability and validity. 

Huang HH, Ramachandran K, Stehno-Bittel L.
Acta Diabetol. 2012 Feb 3.

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slet equivalent (IE), the standard estimate of isolated islet volume, is an essential measure to determine the amount of transplanted islet tissue in the clinic and is used in research laboratories to normalize results, yet it is based on the false assumption that all islets are spherical. Here, we developed and tested a new easy-to-use method to quantify islet volume with greater accuracy. Isolated rat islets were dissociated into single cells, and the total cell number per islet was determined by using computer-assisted cytometry. Based on the cell number per islet, we created a regression model to convert islet diameter to cell number with a high R (2) value (0.8) and good validity and reliability with the same model applicable to young and old rats and males or females. Conventional IE measurements overestimated the tissue volume of islets. To compare results obtained using IE or our new method, we compared Glut2 protein levels determined by Western Blot and proinsulin content via ELISA between small (diameter ≤ 100 μm) and large (diameter ≥ 200 μm) islets. When normalized by IE, large islets showed significantly lower Glut2 level and proinsulin content. However, when normalized by cell number, large and small islets had no difference in Glut2 levels, but large islets contained more proinsulin. In conclusion, normalizing islet volume by IE overestimated the tissue volume, which may lead to erroneous results. Normalizing by cell number is a more accurate method to quantify tissue amounts used in islet transplantation and research.

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KU-32, a novel drug for diabetic neuropathy, is safe for human islets and improves in vitro insulin secretion and viability. 

Farmer K, Williams SJ, Novikova L, Ramachandran K, Rawal S, Blagg BS, Dobrowsky R, Stehno-Bittel L.
Exp Diabetes Res. 2012;2012:671673.

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KU-32 is a novel, novobiocin-based Hsp90 inhibitor that protects against neuronal glucotoxicity and reverses multiple clinical indices of diabetic peripheral neuropathy in a rodent model. However, any drug with potential for treating diabetic complications must also have no adverse effects on the function of pancreatic islets. Thus, the goal of the current study was to assess the effect of KU-32 on the in vitro viability and function of human islets. Treating human islets with KU-32 for 24 hours showed no toxicity as assessed using the alamarBlue assay. Confocal microscopy confirmed that with a minimum of 2-day exposure, KU-32 improved cellular viability by blocking apoptosis. Functionally, isolated human islets released more glucose-stimulated insulin when preincubated in KU-32. However, diabetic BKS-db/db mice, a model for type 2 diabetes, administered KU-32 for 10 weeks did not show any significant changes in blood glucose and insulin levels, despite having greater insulin staining/beta cell in the pancreas compared to untreated BKS db/db mice. In summary, KU-32 did not harm isolated human islets and may even be protective. However, the effect does not appear significant enough to alter the in vivo metabolic parameters of diabetic mice.

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Organ-based response to exercise in type 1 diabetes.ISRN Endocrinol. 

Stehno-Bittel L.
2012;2012:318194. doi: 10.5402/2012/318194.

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While significant research has clearly identified sedentary behavior as a risk factor for type 2 diabetes and its subsequent complications, the concept that inactivity could be linked to the complications associated with type 1 diabetes (T1D) remains underappreciated. This paper summarizes the known effects of exercise on T1D at the tissue level and focuses on the pancreas, bone, the cardiovascular system, the kidneys, skeletal muscle, and nerves. When possible, the molecular mechanisms underlying the benefits of exercise for T1D are elucidated. The general benefits of increased activity on health and the barriers to increased exercise specific to people with T1D are discussed.

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Low insulin content of large islet population is present in situ and in isolated islets. 

Huang HH, Novikova L, Williams SJ, Smirnova IV, Stehno-Bittel L.
Islets. 2011 Jan-Feb;3(1):6-13.

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While significant research has clearly identified sedentary behavior as a risk factor for type 2 diabetes and its subsequent complications, the concept that inactivity could be linked to the complications associated with type 1 diabetes (T1D) remains underappreciated. This paper summarizes the known effects of exercise on T1D at the tissue level and focuses on the pancreas, bone, the cardiovascular system, the kidneys, skeletal muscle, and nerves. When possible, the molecular mechanisms underlying the benefits of exercise for T1D are elucidated. The general benefits of increased activity on health and the barriers to increased exercise specific to people with T1D are discussed.

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Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome. 

Williams SJ, Huang HH, Kover K, Moore W, Berkland C, Singh M, Smirnova IV, MacGregor R, Stehno-Bittel L.
Organogenesis. 2010 Apr-Jun;6(2):115-24.

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For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter >150 µm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter <100 µm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 µm/min in small islets and 2.8 µm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150 µm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.

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Adhesion of pancreatic beta cells to biopolymer films.

Williams SJ, Wang Q, Macgregor RR, Siahaan TJ, Stehno-Bittel L, Berkland C., biopolymers.
2009 Aug;91(8):676-85.

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Dramatic reversal of Type 1 diabetes in patients receiving pancreatic islet transplants continues to prompt vigorous research concerning the basic mechanisms underlying patient turnaround. At the most fundamental level, transplanted islets must maintain viability and function in vitro and in vivo and should be protected from host immune rejection. Our previous reports showed enhancement of islet viability and insulin secretion per tissue mass for small islets (<125 mum) as compared with large islets (>125 mum), thus, demonstrating the effect of enhancing the mass transport of islets (i.e. increasing tissue surface area to volume ratio). Here, we report the facile dispersion of rat islets into individual cells that are layered onto the surface of a biopolymer film towards the ultimate goal of improving mass transport in islet tissue. The tightly packed structure of intact islets was disrupted by incubating in calcium-free media resulting in fragmented islets, which were further dispersed into individual or small groups of cells by using a low concentration of papain. The dispersed cells were screened for adhesion to a range of biopolymers and the nature of cell adhesion was characterized for selected groups by quantifying adherent cells, measuring the surface area coverage of the cells, and immunolabeling cells for adhesion proteins interacting with selected biopolymers. Finally, beta cells in suspension were centrifuged to form controlled numbers of cell layers on films for future work determining the mass transport limitations in the adhered tissue constructs.

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Small rat islets are superior to large islets in in vitro function and in transplantation outcomes.  

MacGregor RR, Williams SJ, Tong PY, Kover K, Moore WV, Stehno-Bittel L.
Am J Physiol Endocrinol Metab. 2006 May;290(5):E771-9.

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Barriers to the use of islet transplantation as a practical treatment for diabetes include the limited number of available donor pancreata. This project was designed to determine whether the size of the islet could influence the success rate of islet transplantations in rats. Islets from adult rats were divided into two groups containing small (diameter <125 microm) or large (diameter >150 microm) islets. An average pancreas yielded three times more small islets than large. Smaller islets were approximately 20% more viable, with large islets containing a scattered pattern of necrotic and apoptotic cells or central core cell death. Small islets in culture consumed twice as much oxygen as large islets when normalized for the same islet equivalents. In static incubation, small islets released three times more insulin under basal conditions than did large islets. During exposure to high glucose conditions, the small islets released four times more insulin than the same islet equivalencies of large islets, and five times more insulin was released by the small islets in response to glucose and depolarization with K+. Most importantly, the small islets were far superior to large islets when transplanted into diabetic animals. When marginal islet equivalencies were used for renal subcapsular transplantation, large islets failed to produce euglycemia in any recipient rats, whereas small islets were successful 80% of the time. The results indicate that small islets are superior to large islets in in vitro testing and for transplantation into the kidney capsule of diabetic rats.

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