Modulation of naïve mesenchymal stromal cells by extracellular vesicles derived from insulin-producing cells: an in vitro study.

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.

High variability in short and long-term recovery kinetic of blood cell count and blood chemistry in a partial body irradiation mouse model.

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 LD50. To determine the effect of shielding on blood cell counts and chemistry, animals were either WBI at 6 Gy (LD2230) or 6 Gy PBI with one leg shielding (LD030). 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.

Osteocalcin protects islet identity in low-density lipoprotein receptor knockout mice on high-fat diet.

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 ß-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 ß-cell identity resulting from MetS. Osteocalcin (OC), an insulin-sensitizing protein secreted by bone, shows promising impact on ß-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 %ß-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 %ß 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 ß-cell mitochondria fluorescence intensity were noted with HFD, but largely prevented with OC administration. OC supplementation here suggests a benefit to ß-cell identity in LDLr-/- mice and offers intriguing clinical implications for countering metabolic syndrome.

Pretransplant HOMA-ß Is Predictive of Insulin Independence in 7 Patients With Chronic Pancreatitis Undergoing Islet Autotransplantation.

Islet and ß-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-ß) 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 ß-cell function metrics, including pretransplant and 6-mo posttransplant HOMA-ß 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 ß-cell maturity markers. Results: Pre-PIAT, HOMA-ß (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-ß values. Preoperative low serological ß-cell function was displayed by concurrent evidence of ß-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-ß assessment before PIAT for estimating posttransplant insulin independence.

Human pancreatic microenvironment promotes ß-cell differentiation via non-canonical WNT5A/JNK and BMP signaling.

In vitro derivation of pancreatic ß-cells from human pluripotent stem cells holds promise as diabetes treatment. Despite recent progress, efforts to generate physiologically competent ß-cells are still hindered by incomplete understanding of the microenvironment's role in ß-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 ß-cell differentiation. Further, we uncover unique factors that guide in vitro development of endocrine progenitors, with WNT5A markedly improving human ß-cell differentiation. WNT5A initially acts through the non-canonical (JNK/c-JUN) WNT signaling and cooperates with Gremlin1 to inhibit the BMP pathway during ß-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 ß-cells for translational applications.

Cognitive and Imaging Differences After Proton and Photon Whole Brain Irradiation in a Preclinical Model.

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 18F-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.

From Mesenchymal Stromal/Stem Cells to Insulin-Producing Cells: Immunological Considerations.

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-L1 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.

NF-κB Blockade by NEMO Binding Domain Peptide Ameliorates Inflammation and Neurobehavioral Sequelae After Cranial Radiation Therapy in Juvenile Mice.

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.

Serum C-peptide and osteocalcin levels in children with recently diagnosed diabetes.

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 (ß = 0.02, P = .001; ß = -0.01, P = .02, respectively) and older age at diagnosis (ß = 0.13, P = .001; ß = -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.

Differentiation of Heterogeneous Radiation Exposure Using Hematology and Blood Chemistry.

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, LD100/30) or partial-body irradiation (PBI), as follows: one leg shielded (1LS, LD0/30), two legs shielded (2LS, LD0/30) or the upper half of the body shielded (UHS, LD0/30). 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.