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Background--A major problem in the islet field is the high selectivity exercised in accepting cadaveric pancreas for islet isolation. This practice is based on experience that indicates that islet yield and posttransplant function are related to donor demographics and injury mechanisms. Objective--To examine factors influencing islets recovery and in vivo function with emphasis on donor-related factors. Methods--Islets were isolated from 99 human donor pancreata, and islet yield was reported as islet equivalent per gram pancreatic tissue. Donor, procurement, and isolation factors were collected for each isolation and correlation statistics were performed between these variables and islet yield. Results--Results indicated a differential effect of enzyme mixes on yield with Collagenase P digestion most suitable for increased ischemic time (R[2]=0.1; P<.08), Liberase with small donor pancreas size and elevated preprocurement glucose (R[2]=0.15; P<.02), and Serva with female donors (R[2]=0.17; P<.06). Islets from 29 isolations were further tested by transplantation under the kidney capsule of immune-deficient NOD-SCID mice. Although all 29 preparations had acceptable in vitro perfusion parameters indicating viability, only 19 functioned in vivo with serum levels of insulin >5 U/mL and C peptide >1.5 ng/mL. No significant differences in donor, procurement, and isolation factors were evident between the islet preparations that functioned in vivo and those that were nonfunctional. Conclusions--These data demonstrate that although yield is affected by a variety of donor factors and enzyme mixes, these factors do not affect islet in vivo function. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cell-derived β-cells (SC-β-cells) represent an alternative cell source for transplantation in diabetic patients. Although mitogens could in theory be used to expand β-cells, adult β-cells very rarely replicate. In contrast, newly formed β-cells, including SC-β-cells, display higher proliferative capacity and distinct transcriptional and functional profiles. Through bidirectional expression modulation and single-cell RNA-seq, we identified SPOCK2, an ECM protein, as an inhibitor of immature β-cell proliferation. Human β-cells lacking SPOCK2 presented elevated MMP2 expression and activity, leading to β-integrin-FAK-c-JUN pathway activation. Treatment with the MMP2 protein resulted in pronounced short- and long-term SC-β-cell expansion, significantly increasing glucose-stimulated insulin secretion in vitro and in vivo. These findings suggest that SPOCK2 mediates fetal β-cell proliferation and maturation. In summary, we identified a molecular mechanism that specifically regulates SC-β-cell proliferation and function, highlighting a unique signaling milieu of SC-β-cells with promise for the robust derivation of fully functional cells for transplantation., Competing Interests: Competing interests: KB, APJ, and MB—filed patents on the role of SPOCK2 (P.448318) and MMP2 (P.448319) in hPSC-derived β-cells., (© 2025. The Author(s).)

This study was to determine whether extracellular vesicles (EVs) derived from insulin-producing cells (IPCs) can modulate naïve mesenchymal stromal cells (MSCs) to become insulin-secreting. MSCs were isolated from human adipose tissue. The cells were then differentiated to generate IPCs by achemical-based induction protocol. EVs were retrieved from the conditioned media of undifferentiated (naïve) MSCs (uneducated EVs) and from that of MSC-derived IPCs (educated EVs) by sequential ultracentrifugation. The obtained EVs were co-cultured with naïve MSCs.The cocultured cells were evaluated by immunofluorescence, flow cytometry, C-peptide nanogold silver-enhanced immunostaining, relative gene expression and their response to a glucose challenge.Immunostaining for naïve MSCs cocultured with educated EVs was positive for insulin, C-peptide, and GAD65. By flow cytometry, the median percentages of insulin-andC-peptide-positive cells were 16.1% and 14.2% respectively. C-peptide nanogoldimmunostaining providedevidence for the intrinsic synthesis of C-peptide. These cells released increasing amounts of insulin and C-peptide in response to increasing glucose concentrations. Gene expression of relevant pancreatic endocrine genes, except for insulin, was modest. In contrast, the results of naïve MSCs co-cultured with uneducated exosomes were negative for insulin, C-peptide, and GAD65. These findings suggest that this approach may overcome the limitations of cell therapy., (© 2024. The Author(s).)

Metabolic syndrome (MetS) is an increasing global health threat and strong risk factor for type 2 diabetes (T2D). MetS causes both hyperinsulinemia and islet size overexpansion, and pancreatic β-cell failure impacts insulin and proinsulin secretion, mitochondrial density, and cellular identity loss. The low-density lipoprotein receptor knockout (LDLr-/-) model combined with high-fat diet (HFD) has been used to study alterations in multiple organs, but little is known about the changes to β-cell identity resulting from MetS. Osteocalcin (OC), an insulin-sensitizing protein secreted by bone, shows promising impact on β-cell identity and function. LDLr-/- mice at 12 months were fed chow or HFD for 3 months ± 4.5 ng/h OC. Islets were examined by immunofluorescence for alterations in nuclear Nkx6.1 and PDX1 presence, insulin-glucagon colocalization, islet size and %β-cell and islet area by insulin and synaptophysin, and mitochondria fluorescence intensity by Tomm20. Bone mineral density (BMD) and %fat changes were examined by Piximus Dexa scanning. HFD-fed mice showed fasting hyperglycemia by 15 months, increased weight gain, %fat, and fasting serum insulin and proinsulin; concurrent OC treatment mitigated weight increase and showed lower proinsulin-to-insulin ratio, and higher BMD. HFD increased %β and %islet area, while simultaneous OC-treatment with HFD was comparable to chow-fed mice. Significant reductions in nuclear PDX1 and Nkx6.1 expression, increased insulin-glucagon colocalization, and reduction in β-cell mitochondria fluorescence intensity were noted with HFD, but largely prevented with OC administration. OC supplementation here suggests a benefit to β-cell identity in LDLr-/- mice and offers intriguing clinical implications for countering metabolic syndrome.

Purpose: In the aftermath of a nuclear disaster or accident, survivors will suffer from radiation-induced normal tissue damage. Recovery after radiation exposure is dictated by several factors, one of which is degree of shielding at time of exposure. This study aims to characterize the short and late term changes in kinetics and magnitude of pancytopenia and blood chemistry in a model of heterogeneous radiation exposure, or partial body irradiation (PBI), compared to whole body irradiation (WBI)., Materials and Methods: Male C57BL/6 mice, 8-10 weeks of age, were WBI at 6 different doses (6, 6.1. 6.15, 6.2, 6.5, and 7.5 Gy) to establish the LD 50 . To determine the effect of shielding on blood cell counts and chemistry, animals were either WBI at 6 Gy (LD 22 30) or 6 Gy PBI with one leg shielding (LD 0 30). Complete blood counts and chemistry were measured at 1, 5-, 10-, 20-, 30- and 120-days post-irradiation., Results and Conclusions: Irradiated animals had significant depletion of white blood cells, red blood cells and platelets up to 10 days post-irradiation. Separation between PBI and WBI were observed at 10- and 20-days post-irradiation at which point PBI animals showed sign of recovery while overall cell count remains depleted in WBI animals up to 30 days post-irradiation. In addition, significant changes were found in parameters indicative of hematopoietic injury including hemoglobin count, hematocrit count and white blood cell population. Significant changes were observed in kidney function with changes to blood urea nitrogen and calcium concentration at 5-days post-irradiation. At 10-days post-irradiation. liver function changes differentiated WBI from PBI animals. Long-term, irradiated animal's chemistry values and many blood counts were not significantly different from Sham. In conclusion, partial shielding ensured complete survival and demonstrated a different recovery kinetics of blood and chemistry parameters after irradiation compared to survivors of whole body irradiation and no single hemopoietic parameter was able to consistently differentiate irradiated from Sham animals. This seems to indicate that there is no single robust hemopoietic parameter to differentiate those exposed from those who were not due to the inherent variability in individual responses. Furthermore, there were no significant long-term effects on these blood parameters between survivors of WBI and PBI except that shielding accelerated recovery.

Islet and β-cell function is intrinsic to glucose homeostasis. Pancreatectomy and islet autotransplantation (PIAT) for chronic pancreatitis (CP) treatment is a useful model for assessing islet function in the absence of immune-suppression and to perform extensive presurgical metabolic evaluations not possible from deceased donors. We recently showed that in CP-PIAT patients, preoperative islet identity loss presented with postoperative glycemic loss. Here, we examine presurgical islet function using Homeostatic Model Assessment-Beta Cell Function (%) (HOMA-β) and glycemic variables and compared them with postsurgical insulin independence and their predicted alignment with Secretory Unit of Islet Transplant Objects (SUITO) and beta cell score after transplantation (BETA-2) scores., Methods: Seven CP-PIAT patients were assessed for β-cell function metrics, including pretransplant and 6-mo posttransplant HOMA-β using insulin and C-peptide and evaluations of proposed insulin independence by SUITO and BETA-2 graft function equations. These were compared with oral glucose tolerance tests and pancreas histological samples taken at the time of transplant, examined for β-cell maturity markers., Results: Pre-PIAT, HOMA-β (60%-100%) associated with post-PIAT insulin independence. This association was only moderately supported by post-PIAT SUITO threshold scores (≥26) but robustly by BETA-2 scores (≥16.2). Appropriate posttransplant oral glucose tolerance test curves were found in those patients with normal pretransplant HOMA-β values. Preoperative low serological β-cell function was displayed by concurrent evidence of β-cell identity alterations including colocalization of insulin and glucagon, loss of urocortin-3, and increased intra-islet vimentin in patients who were insulin-dependent post-PIAT., Conclusions: These data encourage HOMA-β assessment before PIAT for estimating posttransplant insulin independence., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

In vitro derivation of pancreatic β-cells from human pluripotent stem cells holds promise as diabetes treatment. Despite recent progress, efforts to generate physiologically competent β-cells are still hindered by incomplete understanding of the microenvironment's role in β-cell development and maturation. Here, we analyze the human mesenchymal and endothelial primary cells from weeks 9-20 fetal pancreas and identify a time point-specific microenvironment that permits β-cell differentiation. Further, we uncover unique factors that guide in vitro development of endocrine progenitors, with WNT5A markedly improving human β-cell differentiation. WNT5A initially acts through the non-canonical (JNK/c-JUN) WNT signaling and cooperates with Gremlin1 to inhibit the BMP pathway during β-cell maturation. Interestingly, we also identify the endothelial-derived Endocan as a SST+ cell promoting factor. Overall, our study shows that the pancreatic microenvironment-derived factors can mimic in vivo conditions in an in vitro system to generate bona fide β-cells for translational applications., (© 2022. The Author(s).)

Purpose: Compared with photon cranial radiation therapy (X-CRT), proton cranial radiation therapy (P-CRT) offers potential advantages in limiting radiation-induced sequalae in the treatment of pediatric brain tumors. This study aims to identify cognitive, functional magnetic resonance and positron emission tomography imaging markers and molecular differences between the radiation modalities., Methods and Materials: Juvenile rats received a single faction of 10 Gy (relative biological effectiveness-weighted dose) delivered with 6 MV X-CRT or at the midspread out Bragg peak of a 100 MeV P-CRT beam. At 3, 6, and 12 months post-CRT, executive function was measured using 5-choice serial reaction time task. At ∼12 months post-CRT, animals were imaged with 18 F-Flurodeoxy-glucose positron emission tomography imaging followed by functional ex vivo magnetic resonance imaging and stained for markers of neuroinflammation., Results: Irradiated animals had cognitive impairment with a higher number of omissions and lower incorrect and premature responses compared with sham (P ≤ .05). The accuracy of the animals' X-CRT was less than that of sham (P ≤ .001). No significant difference in rates of cognitive change were found between the radiation modalities. At 12 months post-CRT, glucose metabolism was significantly higher than sham in X-CRT (P = .04) but not P-CRT. Using diffusion tensor imaging, P-CRT brains were found to have higher white matter volume and fiber lengths compared with sham (P < .03). Only X-CRT animals had higher apparent diffusion coefficient values compared with sham (P = .04). P-CRT animals had more connectomic changes compared with X-CRT. Correlative analysis identified several imaging features with cognitive performance. Furthermore, microgliosis (P < .05), astrogliosis (P < .01), and myelin thinning (P <.05) were observed in both radiation modalities, with X-CRT showing slightly more inflammation., Conclusions: Both P-CRT and X-CRT lead to neurocognitive changes compared with sham. Although no significant difference was observed in neuroinflammation between the irradiated groups, differences were found in late-term glucose metabolism and brain connectome. Our results indicate that despite relative biological effectiveness weighting of the proton dose there are still differential effects which warrants further investigation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem cell (MSC)-based therapy for type 1 diabetes mellitus (T1DM) has been the subject matter of many studies over the past few decades. The wide availability, negligible teratogenic risks and differentiation potential of MSCs promise a therapeutic alternative to traditional exogenous insulin injections or pancreatic transplantation. However, conflicting arguments have been reported regarding the immunological profile of MSCs. While some studies support their immune-privileged, immunomodulatory status and successful use in the treatment of several immune-mediated diseases, others maintain that allogeneic MSCs trigger immune responses, especially following differentiation or in vivo transplantation. In this review, the intricate mechanisms by which MSCs exert their immunomodulatory functions and the influencing variables are critically addressed. Furthermore, proposed avenues to enhance these effects, including cytokine pretreatment, coadministration of mTOR inhibitors, the use of Tregs and gene manipulation, are presented. As an alternative, the selection of high-benefit, low-risk donors based on HLA matching, PD-L 1 expression and the absence of donor-specific antibodies (DSAs) are also discussed. Finally, the necessity for the transplantation of human MSC (hMSC)-derived insulin-producing cells (IPCs) into humanized mice is highlighted since this strategy may provide further insights into future clinical applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Refaie, Elbassiouny, Kloc, Sabek, Khater, Ismail, Mohamed and Ghoneim.)

Purpose: Cranial radiation therapy (CRT) is a common treatment for pediatric brain tumor patients. However, side effects include significant neurobehavioral dysfunction in survivors. This dysfunction may in part be caused by inflammation, including increased production of tumor necrosis factor alpha (TNFα) and its receptor TNFR1, which can activate the nuclear factor kappa light-chain enhancer of activated B cells (NF-κB). The TNFα blockade abrogates this inflammatory response, although it presents immunologic risks. Thus, modulation of pathway subsets may be preferable. Here, we test whether inhibition of NF-κB activation using an NF-κB essential modulator binding domain (NBD) peptide mitigates CRT-induced neuroinflammation and improves behavioral outcomes., Methods and Materials: Male C57BL/6J 28-day old mice were randomized to saline (sham), 5 Gy whole-brain CRT, or CRT + NBD-peptide. Brain tissue was collected after 4 hours or 3 months for Western blot or immunohistochemistry. The cortex, corpus callosum (CC), and dentate gyrus were variably imaged for NF-κB-p65, IκBα, proliferation, apoptosis, necroptosis, TNFα, TNFR1, IBA-1, doublecortin, CD11c, and GFAP. Neurobehavioral changes were assessed by open field and elevated plus maze tests 3 months post-CRT., Results: NF-κB expression increased in whole and nuclear fractions 4 hours after CRT and was abrogated by NBD treatment. Cell death increased and proliferation decreased after CRT, including within neuronal progenitors, with some loss mitigated by NBD. Increased levels of TNFα, IBA-1, and GFAP were found in the CC and cortex months after CRT and were limited by NBD. The anti-NF-κB peptide also improved neurobehavioral assessments, yielding improvements in anxiety and exploration., Conclusions: Results suggest a role for NF-κB modulation by NBD peptide in the reduction of neuroinflammation and mitigation of behavioral complications after pediatric radiation therapy., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In the aftermath of a nuclear incident, survivors will suffer the deleterious effects from acute radiation exposure. The majority of those affected would have received heterogeneous radiation exposure, reflected in hematological metrics and blood chemistry. Here, we investigated the acute and long-term changes in kinetics and magnitude of pancytopenia and blood chemistry in rats irradiated using varying degrees of body shielding. We hypothesized that, although a single blood count may not be able to differentiate the degree of radiation exposure, a combination of measurements from complete blood cell counts (CBCs) and blood chemistry tests is able to do so. Male Sprague Dawley rats, 8-10 weeks of age, received single-dose 7.5 Gy (160 kVp, 25 mA, 1.16 Gy/min) whole-body irradiation (WBI, LD 100/30 ) or partial-body irradiation (PBI), as follows: one leg shielded (1LS, LD 0/30 ), two legs shielded (2LS, LD 0/30 ) or the upper half of the body shielded (UHS, LD 0/30 ). Animal morbidity and weights were measured. Blood was drawn at 1, 5, 10, 20 and 30 days postirradiation (n = 4-11). For kidney and liver function measurements, CBC and blood chemistry analyses were performed. WBI animals on average survived 9 ± 0.4 days postirradiation. In contrast, all PBI animals survived the 30-day study period. CBC analysis revealed that both white blood cell (WBC) and platelet counts were most affected after irradiation. While WBC counts were significantly lower in all irradiated groups on days 1, 5 and 10, platelets were only significantly lower on days 5 and 10 postirradiation. In addition, on day 5 postirradiation both WBC and platelet counts were able to differentiate WBI (non-survivors) from PBI 2LS and UHS animals (survivors). Using four blood parameters (platelets, percentage lymphocytes, percentage neutrophils and percentage monocytes) on day 5 after 7.5 Gy irradiation and a linear discrimination analysis (LDA), we were able to predict the degree of body exposure in animals with a 95.8% accuracy. Alkaline phosphatase (ALP) was significantly lower in all groups on days 5 and 10 postirradiation compared to baseline. Furthermore, ALP was significantly higher in the UHS than WBI animals. The AST:ALT ratio was significantly higher than baseline in all irradiated groups on day 1 postirradiation. In conclusion, four CBC parameters, on day 5 after receiving a 7.5 Gy dose of radiation, can be employed in a LDA to differentiate various degrees of exposure (shielding). The characterization presented in this work paves the way for further studies in differences caused by heterogeneous body exposure to radiation and a new metric for biodosimetry.

Background: We explored the association of C-peptide (marker of secreted insulin), proinsulin and proinsulin ⁄C-peptide ratio (PI/C) (markers of beta-cell endoplasmic reticulum [ER] stress) with undercarboxylated (uOC) and carboxylated osteocalcin (cOC) and their ratio (uOC/cOC) in children with recently diagnosed type 1 (T1D) or type 2 diabetes (T2D), and the correlation of these variables with partial remission (PR) in children with T1D., Methods: Demographic and clinical data of children with new-onset diabetes (n = 68; median age = 12.2 years; 33.8% non-Hispanic White, 45.6% Hispanic/Latino, 16.2% African American and 4.4% other) were collected at diagnosis and during the first (V1), second (V2) and third clinical visits at 9.0, 32.0 and 175.7 weeks, respectively. Serum proinsulin, C-peptide, uOC and cOC values were measured 7.0 weeks after diagnosis. PR was defined as insulin dose-adjusted HbA1c (IDAA1c) ≤9., Results: In children with new-onset T1D with DKA (33.3%) or T2D (29.4%), Spearman's correlation coefficient revealed a positive association between the C-peptide levels and both uOC and uOC/cOC ratio. In T1D (n = 48), both higher serum C-peptide levels and low PI:C ratio were associated with higher BMI percentile ( β  = 0.02, P  = .001; β  = -0.01, P  = .02, respectively) and older age at diagnosis ( β  = 0.13, P  = .001; β  = -0.12, P  = .001, respectively). Furthermore, in children with T1D, C-peptide levels at V1 correlated with IDAA1c ≤ 9 at V1 ( P  = .04)., Conclusion: C-peptide levels are associated with a higher uOC and uOC/cOC ratio in paediatric diabetes. In new-onset T1D children, older age and higher BMI were associated with lower beta-cell stress and higher preserved function, which was predictive of PR on follow-up., Competing Interests: The authors declare no conflict of interest in relation to material presented in this study., (© 2019 The Authors. Endocrinology, Diabetes & Metabolism published by John Wiley & Sons Ltd.)

Background: Cranial radiotherapy (CRT) is an important part of brain tumor treatment, and although highly effective, survivors suffer from long-term cognitive side effects. In this study we aim to establish late-term imaging markers of CRT-induced brain injury and identify functional markers indicative of cognitive performance. Specifically, we aim to identify changes in executive function, brain metabolism, and neuronal organization., Methods: Male Sprague Dawley rats were fractionally irradiated at 28 days of age to a total dose of 30 Gy to establish a radiation-induced brain injury model. Animals were trained at 3 months after CRT using the 5-choice serial reaction time task. At 12 months after CRT, animals were evaluated for cognitive and imaging changes, which included positron emission tomography (PET) and magnetic resonance imaging (MRI)., Results: Cognitive deficit with signs of neuroinflammation were found at 12 months after CRT in irradiated animals. CRT resulted in significant volumetric changes in 38% of brain regions as well as overall decrease in brain volume and reduced gray matter volume. PET imaging showed higher brain glucose uptake in CRT animals. Using MRI, irradiated brains had an overall decrease in fractional anisotropy, lower global efficiency, increased transitivity, and altered regional connectivity. Cognitive measurements were found to be significantly correlated with six image features that included myelin integrity and local organization of the neural network., Conclusions: These results demonstrate that CRT leads to late-term morphological changes, reorganization of neural connections, and metabolic dysfunction. The correlation between imaging markers and cognitive deficits can be used to assess late-term side effects of brain tumor treatment and evaluate efficacy of new interventions., (© The Author(s) 2019. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Beta-cell dedifferentiation as shown by cellular colocalization of insulin with glucagon and/or vimentin, and decreased expression of MAFA and/or urocortin3 has been suggested to contribute to metabolic decompensation in type 2 diabetes, and was recently described postimplantation in islet allotransplant patients. Dysglycaemia and diabetes mellitus are often encountered preoperatively in patients undergoing pancreatectomy and islet autotransplantation (PIAT). In this series of case reports, we document variation in islet phenotypic identity in three patients with chronic pancreatitis (CP) without diabetes or significant insulin resistance who subsequently underwent PIAT. Pancreas histology was examined using colocalization of endocrine hormones, mesenchymal and pan-endocrine markers in islets, and the relative expression of MAFA and urocortin3 in insulin-expressing cells as compared to that of nondiabetic and type 2 diabetic donors. We present results of pre- and posttransplant clinical metabolic testing. Varying degrees of islet-cell dedifferentiation are identified in nondiabetic patients with CP at the time of PIAT, and may need further investigation., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Objectives: The objective of this study was to assess vitamin D status of US non-pregnant adults using a standardised assay across 15 mL/min/1.73 m 2 increments of kidney function, report the use of dietary supplements containing vitamin D and assess relationships between vitamin D and markers of bone resorption., Design: This study is a cross-sectional evaluation., Setting: The study is from the US National Health and Nutrition Evaluation Survey in 2001-2012., Participants: The participants were non-institutionalised, non-pregnant adults, age ≥20 years., Primary and Secondary Outcome Measures: The primary outcome measure was serum 25OHD evaluated using liquid chromatography-tandem mass spectroscopy traceable to international reference standards. Secondary outcome measures were use of dietary supplements containing vitamin D and the serum intact parathyroid hormone and bone-specific alkaline phosphatase in a subset of participants., Results: The median 25OHD concentration in 27 543 US non-pregnant adults was 25.7 ng/mL (range, 2.2-150.0 ng/mL). Vitamin D supplements were used by 38.0%; mean (SE)=757 (43) international units/day. The range of 25OHD concentration across groups, stratified by kidney function, was 23.0-28.1 ng/mL. The lowest concentration of 25OHD observed was in people with higher kidney function (23.0 ng/mL for estimated glomerular filtration rate >105 mL/min/1.73 m 2 ). Only 24% of people not taking a dietary supplement had a 25OHD concentration >30 ng/mL. Serum intact parathyroid hormone inversely correlated with 25OHD within all kidney function groups. Bone-specific alkaline phosphatase was also negatively associated with 25OHD concentration., Conclusions: These data indicate that 25OHD concentrations and supplement use may be suboptimal in a significant proportion of the population, across all kidney function levels. The response of bone resorption markers further suggests that 25OHD levels could be improved. Together, these data support a re-evaluation of the 25OHD concentration associated with health in adults., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Acute radiation syndrome (ARS) affects morbidity and mortality dependent on the amount of body exposed. We propose the use of echocardiography (EC) to differentiate between survivors and non-survivors by measuring changes in cardiac function (CF) and pulmonary arterial function (PAF). We also investigate the role of rheology in our observed changes. Methods and Results: Rats were irradiated to the whole body (WB) or partial body with two-legs shielded (2LS) at a lethal dose of 7.5Gy. EC and magnetic resonance imaging were performed, and rheological measurements conducted. Only 2LS survived past 12-days post-exposure and their CF and PAR were not significantly different from baseline. WB was significantly different from both baseline and 2LS in stroke volume ( P < 0.05), velocity time integral (VTI; P < 0.05) and pulmonary artery acceleration time (PAAT; P < 0.05). Differences were identified as early as six-days post-exposure, where VTI and PAAT were significantly ( P < 0.05) decreased in WB versus baseline but only PAAT was different from 2LS. Blood viscosity was significantly lower in the WB versus baseline and 2LS ( P < 0.0001). WB exhibited a significant rise in dense red blood cells versus baseline ( P < 0.01) and 2LS ( P < 0.01). Cell-free hemoglobin, a contributor to pulmonary artery hypertension and vasculopathy, was significantly elevated in WB vs. sham. Conclusions: Non-invasive and readily available imaging can be used to identify critically affected victims. Our findings point to heart failure as one possible cause of death in WB exposed animals, potentially exacerbated by rheological, hemolytic, and pulmonary factors, and the importance of developing radiomitigators against cardiac ARS mortality.

Background: While cranial radiation therapy (CRT) is an effective treatment, healthy areas surrounding irradiation sites are negatively affected. Frontal lobe functions involving attention, processing speed, and inhibition control are impaired. These deficits appear months to years after CRT and impair quality of life. Exercise has been shown to rejuvenate the brain and aid in recovery post-injury through its effects on neurogenesis and cognition., Methods: We developed a juvenile rodent CRT model that reproduces neurocognitive deficits. Next, we utilized the model to test whether exercise ameliorates these deficits. Fischer rats (31 days old) were irradiated with a fractionated dose of 4 Gy × 5 days, trained and tested at 6, 9, and 12 months post-CRT using 5-choice serial reaction time task. After testing, fixed rat brains were imaged using diffusion tensor imaging and immunohistochemistry., Results: CRT caused early and lasting impairments in task acquisition, accuracy, and latency to correct response, as well as causing stunting of growth and changes in brain volume and diffusion. Exercising after irradiation improved acquisition, behavioral control, and processing speed, mitigated the stunting of brain size, and increased brain fiber numbers compared with sedentary CRT values. Further, exercise partially restored global connectome organization, including assortativity and characteristic path length, and while it did not improve the specific regional connections that were lowered by CRT, it appeared to remodel these connections by increasing connectivity between alternate regional pairs., Conclusions: Our data strongly suggest that exercise may be useful in combination with interventions aimed at improving cognitive outcome following pediatric CRT.

Background: Osteocalcin (OC), a hormone secreted by osteoblasts, improves beta-cell function in vitro and in vivo. We aimed to understand the relationship between OC and hemoglobin A1c (HbA1c) in pediatric diabetes., Methods: Children (n = 70; mean [SD] age = 11.8 years [3.1]; 34.3% non-Hispanic white, 46.3% Hispanic, 14.9% African-American, 4.5% other) newly diagnosed with diabetes (69.1% type 1 diabetes [T1D], 30.9% type 2 diabetes [T2D]) were studied. We collected clinical data at diagnosis and first clinical visit (V1) 9 weeks later (interquartile range [IQR] = 7.9-12.0). Serum undercarboxylated OC (uOC) and carboxylated OC (cOC) were measured 7.0 weeks (IQR 4.3-8.9) after diagnosis., Results: Mean [SD] uOC was 20.3 (19.6) ng/mL, cOC 29.7 [13.7] ng/mL and u/cOC 0.68 [0.81]. uOC, cOC, or u/cOC were not different by gender, race/ethnicity, age, diabetes type, BMI percentile, or random C-peptide, glucose or HbA1c at diagnosis. However, among 61 children with V1 within 4 months of diagnosis, uOC was higher in those with V1 HbA1c < 7.5% (HbA1c < 58 mmol/mol) (uOC=33.1 [22.0]) compared with children with HbA1c ≥ 7.5% (uOC=17.4 [2.3], P = .0004). The difference was larger among patients with T2D (34.6 and 4.7 ng/mL, respectively, P = .0001) than T1D (32.2 and 19.3, P = .0169), and in males (36.1 and 17.4, P = .018) than females (27.6 and 17.3, P = .072). Analysis for u/cOC were similar while there were no differences in cOC. uOC was inversely correlated with HbA1c at V1 (Spearman's rho = -0.29, P = .02)., Conclusion: Our findings suggest that serum uOC is inversely related to HbA1c shortly after diagnosis of pediatric diabetes. This potentially modifiable factor of glucose metabolism warrants further studies., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Radiation therapy (RT) causes functional and transcriptomic changes in the brain; however, most studies have been carried out in normal rodent brains. Here, the long-term effect of irradiation and tumor presence during radiation was investigated., Methods and Materials: Male Wistar rats ∼7 weeks old were divided into 3 groups: sham implant, RT+sham implant, and RT+tumor implant (C6 glioma). Hypofractionated irradiation (8 or 6 Gy/day for 5 days) was localized to a 1-cm strip of cranium starting 5 days after implantation, resulting in complete tumor regression and prolonged survival. Biopsy of tissue was performed in the implant area 65 days after implantation. RNA was hybridized to GeneChip Rat Exon 1.0 ST array. Data were analyzed using significant analysis of microarrays and ingenuity pathway analysis. 1 H magnetic resonance spectroscopy ( 1 H-MRS) imaging was performed in the implantation site 65 to 70 days after implantation using a 9.4 T Biospec magnetic resonance imaging scanner with a quadrature rat brain array. Immunohistochemical staining for astrogliosis, HMG-CoA synthase 2, γ-aminobutyric acid (GABA) and taurine was performed at ∼65 days after implantation., Results: Eighty-four genes had a false discovery rate <3.5%. We compared RT+tumor implant with RT+sham implant animals. The tumor presence affected networks associated with cancer/cell morphology/tissue morphology. 1 H-MRS showed significant reduction in taurine levels (P<.04) at the implantation site in both groups. However, the RT+tumor group also showed significant increase in levels of neurotransmitter GABA (P=.02). Hippocampal taurine levels were only significantly reduced in the RT+tumor group (P=.03). HMG-CoA synthase 2, GABA and taurine levels were confirmed using staining. Glial fibrillary acidic protein staining demonstrated a significant increase in inflammation that was heightened in the RT+tumor group., Conclusions: Our data indicate that tumor presence during radiation significantly affects long-term functional transcriptomics landscape and neurotransmitter levels at the tumor implantation site/normal tissue, accompanied by increased inflammation (astrogliosis)., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Although a large proportion of patients with type 2 diabetes (T2D) accumulate misfolded aggregates composed of the islet amyloid polypeptide (IAPP), its role in the disease is unknown. Here, we show that pancreatic IAPP aggregates can promote the misfolding and aggregation of endogenous IAPP in islet cultures obtained from transgenic mouse or healthy human pancreas. Islet homogenates immunodepleted with anti-IAPP-specific antibodies were not able to induce IAPP aggregation. Importantly, intraperitoneal inoculation of pancreatic homogenates containing IAPP aggregates into transgenic mice expressing human IAPP dramatically accelerates IAPP amyloid deposition, which was accompanied by clinical abnormalities typical of T2D, including hyperglycemia, impaired glucose tolerance, and a substantial reduction on β cell number and mass. Finally, induction of IAPP deposition and diabetic abnormalities were also induced in vivo by administration of IAPP aggregates prepared in vitro using pure, synthetic IAPP. Our findings suggest that some of the pathologic and clinical alterations of T2D might be transmissible through a similar mechanism by which prions propagate in prion diseases., (© 2017 Mukherjee et al.)

Transplantation of pancreatic islets or stem cell derived insulin secreting cells is an attractive treatment strategy for diabetes. However, islet transplantation is associated with several challenges including function-loss associated with dispersion and limited vascularization as well as the need for continuous immunosuppression. To overcome these limitations, here we present a novel 3D printed and functionalized encapsulation system for subcutaneous engraftment of islets or islet like cells. The devices were 3D printed with polylactic acid and the surfaces treated and patterned to increase the hydrophilicity, cell attachment, and proliferation. Surface treated encapsulation systems were implanted with growth factor enriched platelet gel, which helped to create a vascularized environment before loading human islets. The device protected the encapsulated islets from acute hypoxia and kept them functional. The adaptability of the encapsulation system was demonstrated by refilling some of the experimental groups transcutaneously with additional islets., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The abscopal effect is the response to radiation at sites that are distant from the irradiated site of an organism, and it is thought to play a role in bone marrow (BM) recovery by initiating responses in the unirradiated bone marrow. Understanding the mechanism of this effect has applications in treating BM failure (BMF) and BM transplantation (BMT), and improving survival of nuclear disaster victims. Here, we investigated the use of multimodality imaging as a translational tool to longitudinally assess bone marrow recovery. We used positron emission tomography/computed tomography (PET/CT), magnetic resonance imaging (MRI) and optical imaging to quantify bone marrow activity, vascular response and marrow repopulation in fully and partially irradiated rodent models. We further measured the effects of radiation on serum cytokine levels, hematopoietic cell counts and histology. PET/CT imaging revealed a radiation-induced increase in proliferation in the shielded bone marrow (SBM) compared to exposed bone marrow (EBM) and sham controls. T 2 -weighted MRI showed radiation-induced hemorrhaging in the EBM and unirradiated SBM. In the EBM and SBM groups, we found alterations in serum cytokine and hormone levels and in hematopoietic cell population proportions, and histological evidence of osteoblast activation at the bone marrow interface. Importantly, we generated a BMT mouse model using fluorescent-labeled bone marrow donor cells and performed fluorescent imaging to reveal the migration of bone marrow cells from shielded to radioablated sites. Our study validates the use of multimodality imaging to monitor bone marrow recovery and provides evidence for the abscopal response in promoting bone marrow recovery after irradiation.

Positron emission tomography using 18F-Fluro-deoxy-glucose (18F-FDG) is a useful tool to detect regions of inflammation in patients. We utilized this imaging technique to investigate the kinetics of gastrointestinal recovery after radiation exposure and the role of bone marrow in the recovery process. Male Sprague-Dawley rats were either sham irradiated, irradiated with their upper half body shielded (UHBS) at a dose of 7.5 Gy, or whole body irradiated (WBI) with 4 or 7.5 Gy. Animals were imaged using 18F-FDG PET/CT at 5, 10 and 35 days post-radiation exposure. The gastrointestinal tract and bone marrow were analyzed for 18F-FDG uptake. Tissue was collected at all-time points for histological analysis. Following 7.5 Gy irradiation, there was a significant increase in inflammation in the gastrointestinal tract as indicated by the significantly higher 18F-FDG uptake compared to sham. UHBS animals had a significantly higher activity compared to 7.5 Gy WBI at 5 days post-exposure. Animals that received 4 Gy WBI did not show any significant increase in uptake compared to sham. Analysis of the bone marrow showed a significant decrease of uptake in the 7.5 Gy animals 5 days post-irradiation, albeit not observed in the 4 Gy group. Interestingly, as the metabolic activity of the gastrointestinal tract returned to sham levels in UHBS animals it was accompanied by an increase in metabolic activity in the bone marrow. At 35 days post-exposure both gastrointestinal tract and bone marrow 18F-FDG uptake returned to sham levels. 18F-FDG imaging is a tool that can be used to study the inflammatory response of the gastrointestinal tract and changes in bone marrow metabolism caused by radiation exposure. The recovery of the gastrointestinal tract coincides with an increase in bone marrow metabolism in partially shielded animals. These findings further demonstrate the relationship between the gastrointestinal syndrome and bone marrow recovery, and that this interaction can be studied using non-invasive imaging modalities., Competing Interests: The authors have declared that no competing interests exist.

Background/objectives: Our objective was to assess the sustained, low-dose and constant administration of the thyroid receptor-β (TRβ)-selective agonist GC-1 (sobetirome) from a novel nanochannel membrane device (NMD) for drug delivery. As it known to speed up metabolism, accomplish weight loss, improve cholesterol levels and possess anti-diabetic effects, GC-1 was steadily administered by our NMD, consisting of an implantable nanochannel membrane, as an alternative to conventional daily administration, which is subject to compliance issues in clinical settings., Subjects/methods: Diet-induced obese C57BL/J6 male mice were fed a very high-fat diet (VHFD) and received NMD implants subcutaneously. Ten mice per group received capsules containing GC-1 or phosphate-buffered saline (control). Weight, lean and fat mass, as well as cholesterol, triglycerides, insulin and glucose, were monitored for 24 days. After treatment, plasma levels of thyroid-stimulating hormone (TSH) and thyroxine were compared. mRNA levels of a panel of thermogenic markers were examined using real-time PCR in white adipose tissue (WAT) and brown adipose tissue (BAT). Adipose tissue, liver and local inflammatory response to the implant were examined histologically. Pancreatic islet number and β-cell area were assessed., Results: GC-1 released from the NMD reversed VHFD-induced obesity and normalized serum cholesterol and glycemia. Significant reductions in body weight and fat mass were observed within 10 days, whereas reductions in serum cholesterol and glucose levels were seen within 7 days. The significant decrease in TSH was consistent with TRβ selectivity for GC-1. Levels of transcript for Ucp1 and thermogenic genes PGC1a, Cidea, Dio2 and Cox5a showed significant upregulation in WAT in NMD-GC-1-treated mice, but decreased in BAT. Although mice treated by NMD-GC-1 showed a similar number of pancreatic islets, they exhibited significant increase in β-cell area., Conclusions: Our data demonstrate that the NMD implant achieves steady administration of GC-1, offering an effective and tightly controlled molecular delivery system for treatment of obesity and metabolic disease, thereby addressing compliance.

Diabetes is one of the most prevalent, costly, and debilitating diseases in the world. Pancreas and islet transplants have shown success in re-establishing glucose control and reversing diabetic complications. However, both are limited by donor availability, need for continuous immunosuppression, loss of transplanted tissue due to dispersion, and lack of vascularization. To overcome the limitations of poor islet availability, here, we investigate the potential of bone marrow-derived mesenchymal stem cells differentiated into islet-like insulin-producing aggregates. Islet-like insulin-producing aggregates, characterized by gene expression, are shown to be similar to pancreatic islets and display positive immunostaining for insulin and glucagon. To address the limits of current encapsulation systems, we developed a novel three-dimensional printed, scalable, and potentially refillable polymeric construct (nanogland) to support islet-like insulin-producing aggregates' survival and function in the host body. In vitro studies showed that encapsulated islet-like insulin-producing aggregates maintained viability and function, producing steady levels of insulin for at least 4 weeks. Nanogland-islet-like insulin-producing aggregate technology here investigated as a proof of concept holds potential as an effective and innovative approach for diabetes cell therapy.

Unlabelled: We present and test the use of multimodality imaging as a topological tool to map the amount of the body exposed to ionizing radiation and the location of exposure, which are important indicators of survival and recovery. To achieve our goal, PET/CT imaging with 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) was used to measure cellular proliferation in bone marrow (BM), whereas MRI using ultra-small superparamagnetic iron oxide (USPIO) particles provided noninvasive information on radiation-induced vascular damage., Methods: Animals were x-ray-irradiated at a dose of 7.5 Gy with 1 of 3 radiation schemes-whole-body irradiation, half-body shielding (HBS), or 1-leg shielding (1LS)-and imaged repeatedly. The spatial information from the CT scan was used to segment the region corresponding to BM from the PET scan using algorithms developed in-house, allowing for quantification of proliferating cells, and BM blood volume was estimated by measuring the changes in the T2 relaxation rates (ΔR2) collected from MR scans., Results: (18)F-FLT PET/CT imaging differentiated irradiated from unirradiated BM regions. Two days after irradiation, proliferation of 1LS animals was significantly lower than sham (P = 0.0001, femurs; P < 0.0001, tibias) and returned to sham levels by day 10 (P = 0.6344, femurs; P = 0.3962, tibias). The degree of shielding affected proliferation recovery, showing an increase in the irradiated BM of the femurs, but not the tibias, of HBS animals when compared with 1LS (P = 0.0310, femurs; P = 0.5832, tibias). MRI of irradiated spines detected radiation-induced BM vascular damage, measured by the significant increase in ΔR2 2 d after whole-body irradiation (P = 0.0022) and HBS (P = 0.0003) with a decreasing trend of values, returning to levels close to baseline over 10 d. Our data were corroborated using γ-counting and histopathology., Conclusion: We demonstrated that (18)F-FLT PET/CT and USPIO MRI are valuable tools in mapping regional radiation exposure and the effects of radiation on BM. Analysis of the (18)F-FLT signal allowed for a clear demarcation of exposed BM regions and elucidated the kinetics of BM recovery, whereas USPIO MRI was used to assess vascular damage and recovery., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

The osteoblast-specific hormone osteocalcin (OC) was found to regulate glucose metabolism, fat mass, and β-cell proliferation in mice. Here, we investigate the effect of decarboxylated OC (D-OC) on human β-cell function and mass in culture and in vivo using a Nonobese diabetic-severe combined immunodeficiency mouse model. We found that D-OC at dose ranges from 1.0 to 15 ng/mL significantly augmented insulin content and enhanced human β-cell proliferation of cultured human islets. This was paralleled by increased expression of sulfonylurea receptor protein; a marker of β-cell differentiation and a component of the insulin-secretory apparatus. Moreover, in a Nonobese diabetic-severe combined immunodeficiency mouse model, systemic administration of D-OC at 4.5-ng/h significantly augmented production of human insulin and C-peptide from the grafted human islets. Finally, histological staining of the human islet grafts showed that the improvement in the β-cell function was attributable to an increase in β-cell mass as a result of β-cell proliferation indicated by MKI67 staining together with the increased β-cell number and decreased α-cell number data obtained using laser scanning cytometry. Our data for the first time show D-OC-enhanced β-cell function in human islets and support future exploitation of D-OC-mediated β-cell regulation for developing useful clinical treatments for patients with diabetes.

In the event of a nuclear detonation, a considerable number of projected casualties will suffer from combined radiation exposure and burn and/or wound injury. Countermeasure assessment in the setting of radiation exposure combined with dermal injury is hampered by a lack of animal models in which the effects of interventions have been characterized. To address this need, we used two separate models to characterize wound closure. The first was an open wound model in mice to study the effect of wound size in combination with whole-body 6 Gy irradiation on the rate of wound closure, animal weight and survival (morbidity). In this model the addition of interventions, wound closure, subcutaneous vehicle injection, topical antiseptic and topical antibiotics were studied to measure their effect on healing and survival. The second was a rat closed wound model to study the biomechanical properties of a healed wound at 10 days postirradiation (irradiated with 6 or 7.5 Gy). In addition, complete blood counts were performed and wound pathology by staining with hematoxylin and eosin, trichrome, CD68 and Ki67. In the mouse open wound model, we found that wound size and morbidity were positively correlated, while wound size and survival were negatively correlated. Regardless of the wound size, the addition of radiation exposure delayed the healing of the wound by approximately 5-6 days. The addition of interventions caused, at a minimum, a 30% increase in survival and improved mean survival by ∼9 days. In the rat closed wound model we found that radiation exposure significantly decreased all wound biomechanical measurements as well as white blood cell, platelet and red blood cell counts at 10 days post wounding. Also, pathological changes showed a loss of dermal structure, thickening of dermis, loss of collagen/epithelial hyperplasia and an increased density of macrophages. In conclusion, we have characterized the effect of a changing wound size in combination with radiation exposure. We also demonstrated that the most effective interventions mitigated insensible fluid loss, which could help to define the most appropriate requirements of a successful countermeasure.

Nonalcoholic fatty liver disease, particularly its more aggressive form, nonalcoholic steatohepatitis (NASH), is associated with hepatic insulin resistance. Osteocalcin, a protein secreted by osteoblast cells in bone, has recently emerged as an important metabolic regulator with insulin-sensitizing properties. In humans, osteocalcin levels are inversely associated with liver disease. We thus hypothesized that osteocalcin may attenuate NASH and examined the effects of osteocalcin treatment in middle-aged (12-mo-old) male Ldlr(-/-) mice, which were fed a Western-style high-fat, high-cholesterol diet for 12 weeks to induce metabolic syndrome and NASH. Mice were treated with osteocalcin (4.5 ng/h) or vehicle for the diet duration. Osteocalcin treatment not only protected against Western-style high-fat, high-cholesterol diet-induced insulin resistance but substantially reduced multiple NASH components, including steatosis, ballooning degeneration, and fibrosis, with an overall reduction in nonalcoholic fatty liver disease activity scores. Further, osteocalcin robustly reduced expression of proinflammatory and profibrotic genes (Cd68, Mcp1, Spp1, and Col1a2) in liver and suppressed inflammatory gene expression in white adipose tissue. In conclusion, these results suggest osteocalcin inhibits NASH development by targeting inflammatory and fibrotic processes.

Despite the clinical success of pancreatic islet transplantation, graft function is frequently lost over time due to islet dispersion, lack of neovascularization, and loss of physiological architecture. To address these problems, islet encapsulation strategies including scaffolds and devices have been developed, which produced encouraging results in preclinical models. However, islet loss from such architectures could represent a significant limitation to clinical use. Here, we developed and characterized a novel islet encapsulation silicon device, the NanoGland, to overcome islet loss, while providing a physiological-like environment for long-term islet viability and revascularization. NanoGlands, microfabricated with a channel size ranging from 3.6 nm to 60 μm, were mathematically modeled to predict the kinetics of the response of encapsulated islets to glucose stimuli, based on different channel sizes, and to rationally select membranes for further testing. The model was validated in vitro using static and perifusion testing, during which insulin secretion and functionality were demonstrated for over 30-days. In vitro testing also showed 70-83% enhanced islet retention as compared to porous scaffolds, here simulated through a 200 μm channel membrane. Finally, evidence of in vivo viability of human islets subcutaneously transplanted within NanoGlands was shown in mice for over 120 days. In this context, mouse endothelial cell infiltration suggesting neovascularization from the host were identified in the retrieved grafts. The NanoGland represents a novel, promising approach for the autotransplantation of human islets.

Purpose: Damage to normal tissue is a limiting factor in clinical radiotherapy (RT). We tested the hypothesis that the presence of tumor alters the response of normal tissues to irradiation using a rat in situ brain tumor model., Methods and Materials: Intravital microscopy was used with a rat cranial window to assess the in situ effect of rat C6 glioma on peritumoral tissue with and without RT. The RT regimen included 40 Gy at 8 Gy/day starting Day 5 after tumor implant. Endpoints included blood-brain barrier permeability, clearance index, leukocyte-endothelial interactions and staining for vascular endothelial growth factor (VEGF) glial fibrillary acidic protein, and apoptosis. To characterize the system response to RT, animal survival and tumor surface area and volume were measured. Sham experiments were performed on similar animals implanted with basement membrane matrix absent of tumor cells., Results: The presence of tumor alone increases permeability but has little effect on leukocyte-endothelial interactions and astrogliosis. Radiation alone increases tissue permeability, leukocyte-endothelial interactions, and astrogliosis. The highest levels of permeability and cell adhesion were seen in the model that combined tumor and irradiation; however, the presence of tumor appeared to reduce the volume of rolling leukocytes. Unirradiated tumor and peritumoral tissue had poor clearance. Irradiated tumor and peritumoral tissue had a similar clearance index to irradiated and unirradiated sham-implanted animals. Radiation reduces the presence of VEGF in peritumoral normal tissues but did not affect the amount of apoptosis in the normal tissue. Apoptosis was identified in the tumor tissue with and without radiation., Conclusions: We developed a novel approach to demonstrate that the presence of the tumor in a rat intracranial model alters the response of normal tissues to irradiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: In this article, we investigate the role of tumor necrosis factor-alpha (TNF) in the initiation of acute damage to the blood-brain barrier (BBB) and brain tissue following radiotherapy (RT) for CNS tumors., Methods and Materials: Intravital microscopy and a closed cranial window technique were used to measure quantitatively BBB permeability to FITC-dextran 4.4-kDa molecules, leukocyte adhesion (Rhodamine-6G) and vessel diameters before and after 20-Gy cranial radiation with and without treatment with anti-TNF. Immunohistochemistry was used to quantify astrogliosis post-RT and immunofluorescence was used to visualize protein expression of TNF and ICAM-1 post-RT. Recombinant TNF (rTNF) was used to elucidate the role of TNF in leukocyte adhesion and vessel diameter., Results: Mice treated with anti-TNF showed significantly lower permeability and leukocyte adhesion at 24 and 48 h post-RT vs. RT-only animals. We observed a significant decrease in arteriole diameters at 48 h post-RT that was inhibited in TNF-treated animals. We also saw a significant increase in activated astrocytes following RT that was significantly lower in the anti-TNF-treated group. In addition, immunofluorescence showed protein expression of TNF and ICAM-1 in the cerebral cortex that was inhibited with anti-TNF treatment. Finally, administration of rTNF induced a decrease in arteriole diameter and a significant increase in leukocyte adhesion in venules and arterioles., Conclusions: TNF plays a significant role in acute changes in BBB permeability, leukocyte adhesion, arteriole diameter, and astrocyte activation following cranial radiation. Treatment with anti-TNF protects the brain's microvascular network from the acute damage following RT.

Islet cell transplantation holds great promise for treating patients with type 1 diabetes mellitus (T1DM), and for preventing unstable metabolic state commonly refereed to as brittle diabetes in patients that undergo pancreatic resection given that it is a relatively noninvasive procedure and an attractive alternative to pancreas transplantation for restoring endogenous insulin secretion. The success of recent clinical trials for allogeneic islet transplantation as well as the increasing centers that perform auto-transplantation is showing that the beta cell replacement therapy for the treatment of patients with diabetes or total pancreatectomy has been firmly established. It needs only to be improved and made more widely available to the millions of desperate patients who search for alternatives to a life of insulin injections, hypoglycemia and the risks of end-organ damage. Steady progress has been achieved in recent years in different areas in the pancreatic islet transplantation process including islet cell processing, preservation, and immune therapies that justify optimism. To implement this therapeutic approach to larger cohorts of patients that would benefit from the restoration of beta cell function requires multiple interventions and the standardization of the different stages of islet transplant process. This article will review the possible areas of intervention and the ongoing research toward this important goal.

The ability to isolate high-yield pure and viable islets from human cadaver pancreas donors is dependent on donor factor as well as isolation factors. The aim of this study was to examine factors influencing islets recovery and in vivo function with an emphasis on donor and isolation methods as well as to compare the effectiveness of Liberase, widely used in clinical islet isolation, with Serva for the isolation of pure functional islets. The results of 123 islet isolations using Liberase for digestion were compared with those of 113 isolations with Serva. Islet equivalents per gram of tissue were similar between Liberase and Serva (3620 +/- 1858 vs. 4132 +/- 2104, p < 0.2) as well as the percent purity (75 +/- 16 vs. 74 +/- 15, p < 0.9). In vivo function of islets from 71 isolations (Liberase = 45, Serva = 26) were further tested by transplantation into NOD-SCID mice following short-term culture (< 6 days, n = 71). Our data show that both Liberase- and Serva-isolated islets showed similar function results following short-term culture. These data demonstrate that there is no difference in islet yield, purity, and function between the two enzymes. However, when these 71 isolations were analyzed for in vivo function with emphasis on donor factors, cold ischemia time (12.0 +/- 5.3 vs. 15.0 +/- 5.7, p < 0.04), islet integrity (1.6 +/- 0.7 vs. 1.3 +/- 0.5, p < 0.05), and female gender were the only factors that correlated with in vivo function. We also compared the mechanical-shaking method for islets isolation with hand-shaking methods. Our results show that although there is no different in islet yield, purity, and integrity between different enzymes using the same method, hand-shaking method yields more islets with better integrity than mechanical-shaking method.

Transforming growth factor-beta1 (TGF-beta1) is a pleotropic cytokine that promotes angiogenesis and extracellular matrix protein synthesis in addition to its immunosuppressive effects. The purpose of this study is to identify optimal conditions for in vivo expression of TGF-beta1 by human islets to exploit the possible beneficial effects and minimize undesirable side effects. We transduced human islets with adenoviral vectors encoding the active form of Ad-TGF-beta1 or Ad-LacZ to test the effects of TGF-beta1 gene expression on islet in vivo function following their transplantation into a NOD-SCID mouse model. Islets were transduced with multiplicity of infection (MOI) of 20, 10, 5, and 2.5 per islet cell. At a MOI ranging from 2.5 to 20, expression of TGF-beta1 in islet supernatant persisted for 1-2 months and ranged from 153 +/- 5 to 2574 +/- 1299 pg/ml, respectively. Transduction with the lowest MOI (2.5) did not compromise the in vivo production of human C-peptide. We conclude that TGF-beta1 expression in transplanted islets does not compromise viability and that adenoviral transduction with the TGF-beta1 gene has a dose-dependent effect, with larger MOIs being deleterious. The data also indicate that in vitro culture system and the in vivo NOD-SCID model could be used successfully to evaluate the nonimmune effects of gene transduction.

Wound healing complications have been observed in patients receiving sirolimus (SLR). This study examined the degree and duration of delayed healing in various protocols using SLR. Sprague-Dawley rats underwent a standard midline abdominal incision and wound closure. Groups of 6 rats each were randomized to receive different doses of SLR (2 and 5 mg/kg) with or without loading dose (10 mg/kg x3 days), and with or without steroids (20 mg/kg x3 days followed by 5 mg/kg for 2 weeks). Rats were humanely killed on postoperative days 5, 10, or 15. Wound breaking force was measured using the EHMI BIAX-II instrument and tensile strength was calculated. Wounds in control animals had gradual increase in tensile strength during the 15-day observation. In contrast, high and loading doses of SLR caused reduction in wound strength until day 10, but the wounds' tensile strength became equivalent to control by day 15. The addition of steroids prolonged wound recovery with low doses of SLR until day 15 and had very profound effects on healing in high-dose SLR-treated animals (>50% reduction) that continued beyond the 2 weeks of observation. Low doses of SLR in non-steroid-treated animals had a short-term (5-day) impact on wound healing; high dose and loading doses delayed healing for 10 to 15 days. The addition of steroids had a synergistic effect on delayed wound healing, particularly in animals receiving high-dose SLR, which demonstrated prolonged wound weakness. These results may provide practical guidelines for postoperative introduction of SLR in the context of various clinical protocols.

Islet transplant faces significant challenges, mainly because of the high incidence of primary nonfunction of transplanted islets. Protocol modifications to improve the rate of islet function have included changes in pancreatic preservation and the introduction of short-term culture. Islet culture for 48 to 72 hours has become a standard part of most successful protocols for clinical islet transplantation. We have previously reported gene expression profiles associated with human pancreatic islet function. The aim of this study was to determine the change in gene expression profiles of functional islets after 2 weeks of culture in Memphis-serum free media. Human islets from four isolations were maintained in culture for 14 days in Memphis-serum free media. RNA was extracted from 10000 IEQ for analysis of the gene expression profiles using high-density Affymetrix U133A GeneChips and Genespring software. Islet function was assessed by measurements of human C-peptide at days 7 and 14 posttransplant into NOD-SCID mice. Human C-peptide levels were determined by radioimmunoassay. Our preliminary data showed that genes related to functionality, such as those directed toward insulin processing and secretion, did not vary over 14 days of culture, while genes related to exocrine pancreas and organ architecture and immune-associated genes decreased over time. The ability to maintain islets in culture is an important step toward the development of islet tissue repositories, as well as toward screening human islet preparations for additional pathogens.

Acute rejection after pancreas transplantation remains a significant problem and contributes to immunological graft loss. No clinical markers of pancreas rejection have been universally accepted. The purpose of this study was to investigate the use of genetic markers; granzyme B, perforin, and HLA-DRA in the peripheral blood of pancreas transplant recipients. These genes have been identified in renal and islet cell transplant recipients as noninvasive tools to predict acute rejection. Blood samples were collected weekly for up to 1 year posttransplant. Surveillance biopsies of the pancreas were scheduled at weeks 2, 4, 8, and 12 as part of the typical posttransplant protocol for patients with pancreas alone or pancreas after kidney transplantation. Exclusion criteria included a diagnosis of biopsy-proven chronic rejection alone, pancreatitis, or kidney rejection within 2 months after pancreas biopsy. Gene expression levels of granzyme B, perforin, and HLA-DRA were compared in patients with (n = 7) and without biopsy proven acute rejection (n = 7). Recipients with acute rejection showed increased expression of granzyme B, HLA-DRA, as well as perforin genes compared to patients without biopsy-proven rejection. In addition, we observed that elevation of these genes occurred as early as 4 weeks before the traditional biopsy diagnosis, while the recipients with no rejection showed no change in gene expression. Our data indicated that serial measurements of peripheral blood granzyme B, perforin, and HLA-DRA gene expression can be a useful tool to predict pancreas rejection in its earliest stage.

Islet culture has become a standard part of most successful protocols for clinical islet transplantation. To date, however, islets are transplanted based on crude measures of viability, purity and in vitro insulin production without adequate prior assessment of the potential for in vivo function. The purpose of this study was to define the gene expression profiles of human islets associated with in vivo function using a nonimmune NOD-scid mouse model. Human islets from eight isolations were maintained in culture for 7 to 14 days in Memphis serum-free media until transplanted. The RNA was extracted from 10,000 IEQ using RNASTAT-60. The gene expression profiles were analyzed using high-density Affymetrix U133A GeneChips and Genespring software. An aliquot of 2000 IEQ from each islet preparation was also transplanted into NOD-scid animals (n = 5) for in vivo function assessments. Islet function was assessed by measurements of human C-peptide at days 7 and 14 posttransplant. Human C-peptide levels were determined by radioimmunoassay. Gene analysis of nonfunction islets (4 of 8 islet preparations) showed high relative levels of expression of proinflammatory genes and low relative levels of genes directed toward insulin processing and secretion as well as islet integrity. Overexpression of hypoxia and proinflammatory genes may result in reduced insulin secretion and lead to islet destruction posttransplantation. Identifying and validating those genes could allow the development of a potency assay for human transplantation that would be very useful for screening human islet preparations before clinical transplant.

To date no in vitro viability test is known to accurately predict in vivo human islet function, making transplantation into various nonimmune animal models mandatory. The diabetic mouse model has been proposed as a standard method for human islet viability assessment. However, the use of streptozotocin for diabetes induction is associated with inconsistency with respect to induction protocols and the significant mortality rate. The purpose of this study was to compare a nondiabetic NOD-scid mouse model to its diabetic counterpart in terms of predicting islet viability. Diabetes was induced in NOD-scid mice using intraperitoneal injection of streptozotocin at concentrations ranging from 100 to 200 mg/kg. Blood glucose levels were monitored for 7 to 10 days, and mice that had levels of >300 mg/dL were used in the experiment. For nondiabetic mice, blood glucose and baseline human C-peptide levels were checked after an overnight fast. Transplantation of 2000 human islet equivalent was done in both models using the same technique. Islet function was determined in the diabetic mice by return to normoglycemia for 2 consecutive days and measurement of fasting human C-peptide on days 7 and 14 posttransplant. Viability was tested in nondiabetic mice after intraperitoneal injection of glucose (2 g/kg) and the measurement of human C-peptide levels using radioimmunoassay. Titration of the streptozotocin dose from 200 to 100 mg/kg showed a significant reduction in mice mortality (40% to 10%) and an increase of diabetes induction (55% to 81%). The 23 human islet isolations tested in both models showed complete consistency of the viability results.

Background: Culturing human islets in Memphis serum-free media (M-SFM) is associated with excellent postculture recovery, in vitro function, and in vivo survival. The authors investigate the possibility of preserving islet function for extended periods (6 months) in culture and describe the in vitro and in vivo functional outcomes associated with these extended culture times., Methods: Human islets isolated from three cadaveric donor organs were cultured in M-SFM for 1, 3, or 6 months before transplantation under the kidney capsule of nonobese diabetic (NOD)-severe combined immunodeficiency (SCID) mice. In vitro function was measured by static incubation at the time of transplantation. In vivo function was assessed by measuring human insulin and C-peptide production, and by the ability of 6-month cultured islets to cure streptozotocin-induced diabetes in this mouse model., Results: Islet recovery ratios after 1 month in culture ranged from 85% to 88% and declined to 28% to 53% after 6 months of culture (P <0.01). Insulin stimulation indices did not differ among the fresh or the 6-month cultured preparations. All preparations cultured for 1 to 3 months functioned in the NOD-SCID mice. After 6 months of culture, two of the three preparations demonstrated in vivo function and were able to cure streptozotocin-induced diabetes., Conclusions: These data demonstrate that human islets can be cultured in M-SFM for extended periods and still retain in vitro and in vivo function and the ability to cure experimental diabetes. The ability to maintain islets in culture for prolonged periods is an important step toward the development of islet tissue repositories and distribution centers.

Purpose: To elucidate the brain molecular response to irradiation. The expression of the intercellular adhesion molecule (ICAM-1) and tumour necrosis factor-alpha (TNF-alpha) in the mouse brain was compared after single-dose and fractionated whole-brain irradiation., Materials and Methods: Mice received a single dose of 2, 10 or 20 Gy or a fractionated dose (2 Gy day(-1)) of 10, 20 or 40 Gy. ICAM-1, and TNF-alpha mRNA expression were quantified by the highly sensitive real-time polymerase chain reaction technique. Expression of ICAM-1 protein was quantified by dual-labelled monoclonal antibody assay., Results: After a 20-Gy single dose, there was an increase in ICAM-1 and TNF-alpha mRNA levels (14- and 11-fold, respectively) as well as a significant increase in the level of ICAM-1 protein (p=0.0243). The expression of ICAM-1 and TNF-alpha mRNA increased at the end of the 40-Gy fractionated regimen (3.55- and 2.30-fold, respectively)., Conclusions: The molecular response of the brain to single-dose irradiation was rapid, while its response to fractionated irradiation was slow. This finding is consistent with clinical observations and could be of use when designing strategies to mitigate radiation sequelae.

Background: Difficulties with recovering and preserving pancreatic islets have hampered progress in islet transplantation. In previous in vitro studies, our laboratory successfully demonstrated that using serum-free medium for prolonged pancreatic islet culture allows postculture recovery ratios greater than those obtained with standard media with sustained in vitro islet function. The goal of this study was to determine whether culturing of islets in a modified serum-free medium (M-SFM) would sustain function in vivo., Methods: Islets were isolated from pancreata procured from 12 cadaveric organ donors and cultured in the M-SFM for up to 2 months, cryopreserved at -70 degrees C within 1-3 days of isolation for 2 months, or placed in short-term culture (3-5 days) before their transplantation under the kidney capsule of nonobese diabetic-severe combined immunodeficient mice (n=4-7 per group/time point). In vivo islet function was assessed by measuring the production of human insulin and C-peptide over a period of 3-15 months., Results: After extended culture of islets in M-SFM for 1 or 2 months, transplanted islets maintained their viability, and in some instances in vivo function improved when compared with short-term cultured islets transplanted from the same preparation (P<0.01). Improvement was particularly evident for islets cultured for 1 month. Furthermore, when compared with cryopreserved preparations, early function (postoperative day 7) of islets from 1-month culture preparations was statistically better (P<0.05). Prolonged culture in M-SFM had no significant impact on long-term function, inasmuch as cultured islets functioned for more than 120 days., Conclusion: These data demonstrate that prolonged islet culture in M-SFM sustained viability and function, and in some instances had a positive effect on in vivo islet function, particularly in the 1-month cultures. No negative effect on long-term in vivo function was demonstrated in this study. Confirmation in clinical models utilizing extended (1-2 months) islet culture in M-SFM could significantly enhance islet transplantation by allowing the identification of best-matched recipients, pretransplantation recipient conditioning, and possible pretransplantation islet modifications to promote engraftment and prolonged graft function.