Celiac and superior mesenteric ganglia removal improves glucose tolerance and reduces pancreas islet size.

The sympathetic nervous system is crucial for the regulation of visceral organ function. For instance, the activation of the sympathetic nervous system promotes glycogenolysis in the liver and modulates glucagon and insulin release from the pancreas, thereby raising blood glucose levels. A decrease in sympathetic nerve activity has the opposite effect. Although such acute effects of sympathetic activity changes have been studied, their long-term outcomes have not been previously examined. In this study, we removed the celiac/superior mesenteric ganglia, where sympathetic postganglionic neurons innervating pancreas and liver locate, and examined its effects on glucose homeostasis and islet size several weeks after surgery. Consistent with the reduction in gluconeogenesis, glucose tolerance improved in gangliectomized mice. However, contrary to our expectation that the inhibition of pancreatic function by sympathetic nerves would be relieved with gangliectomy, insulin or C-peptide release did not increase. Examining the size distribution of pancreatic islets, we identified that the gangliectomy led to a size reduction in large islets and a decrease in the proportion of α and ß cells within each islet, as analyzed by immunostaining for insulin and glucagon, respectively. These results indicate that the absence of sympathetic nerve activity reduces the size of the pancreatic islets within a few weeks to reinstate the homeostatic mechanism of blood glucose levels.

Elucidating T cell dynamics and molecular mechanisms in syngeneic and allogeneic islet transplantation through single-cell RNA sequencing.

Islet transplantation is a promising therapy for diabetes treatment. However, the molecular underpinnings governing the immune response, particularly T-cell dynamics in syngeneic and allogeneic transplant settings, remain poorly understood. Understanding these T cell dynamics is crucial for enhancing graft acceptance and managing diabetes treatment more effectively. This study aimed to elucidate the molecular mechanisms, gene expression differences, biological pathway alterations, and intercellular communication patterns among T-cell subpopulations after syngeneic and allogeneic islet transplantation. Using single-cell RNA sequencing, we analyzed cellular heterogeneity and gene expression profiles using the Seurat package for quality control and dimensionality reduction through t-SNE. Differentially expressed genes (DEGs) were analyzed among different T cell subtypes. GSEA was conducted utilizing the HALLMARK gene sets from MSigDB, while CellChat was used to infer and visualize cell-cell communication networks. Our findings revealed genetic variations within T-cell subpopulations between syngeneic and allogeneic islet transplants. We identified significant DEGs across these conditions, highlighting molecular discrepancies that may underpin rejection or other immune responses. GSEA indicated activation of the interferon-alpha response in memory T cells and suppression in CD4+ helper and γδ T cells, whereas TNFα signaling via NFκB was particularly active in regulatory T cells, γδ T cells, proliferating T cells, and activated CD8+ T cells. CellChat analysis revealed complex communication patterns within T-cell subsets, notably between proliferating T cells and activated CD8+ T cells. In conclusion, our study provides a comprehensive molecular landscape of T-cell diversity in islet transplantation. The insights into specific gene upregulation in xenotransplants suggest potential targets for improving graft tolerance. The differential pathway activation across T-cell subsets underscores their distinct roles in immune responses posttransplantation.

Supragel-mediated efficient generation of pancreatic progenitor clusters and functional glucose-responsive islet-like clusters.

Although several synthetic hydrogels with defined stiffness have been developed to facilitate the proliferation and maintenance of human pluripotent stem cells (hPSCs), the influence of biochemical cues in lineage-specific differentiation and functional cluster formation has been rarely reported. Here, we present the application of Supragel, a supramolecular hydrogel formed by synthesized biotinylated peptides, for islet-like cluster differentiation. We observed that Supragel, with a peptide concentration of 5 mg/mL promoted spontaneous hPSCs formation into uniform clusters, which is mainly attributable to a supporting stiffness of ∼1.5 kPa as provided by the Supragel matrix. Supragel was also found to interact with the hPSCs and facilitate endodermal and subsequent insulin-secreting cell differentiation, partially through its components: the sequences of RGD and YIGSR that interacts with cell membrane molecules of integrin receptor. Compared to Matrigel and suspension culturing conditions, more efficient differentiation of the hPSCs was also observed at the stages 3 and 4, as well as the final stage toward generation of insulin-secreting cells. This could be explained by 1) suitable average size of the hPSCs clusters cultured on Supragel; 2) appropriate level of cell adhesive sites provided by Supragel during differentiation. It is worth noting that the Supragel culture system was more tolerance in terms of the initial seeding densities and less demanding, since a standard static cell culture condition was sufficient for the entire differentiation process. Our observations demonstrate a positive role of Supragel for hPSCs differentiation into islet-like cells, with additional potential in facilitating germ layer differentiation.

Type 1 diabetes mellitus following COVID-19 vaccination: a report of two cases and review of literature.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) infection, which led to the coronavirus 2019 (COVID-19) pandemic, has promoted the development of novel therapeutic agents and vaccines to combat the global spread of the virus. While the COVID-19 vaccines approved thus far have proven to be effective in clinical settings, there have been reports of autoimmune diseases occurring following vaccination, including autoimmune/inflammatory syndrome induced by adjuvant syndrome. We herein report two cases of type 1 diabetes mellitus that occurred following COVID-19 vaccination and provide a literature review. Both cases received multiple vaccinations as recommended to ensure optimal antibody titers. Moreover, the HLA associated with susceptibility to type 1 diabetes was prototypic in both cases. This indirect evidence suggests that the COVID-19 vaccines may be implicated in the pathogenesis of type 1 diabetes. Further case reports to establish a clearer understanding of a potential association are warranted. Supplementary Information: The online version contains supplementary material available at 10.1007/s13340-024-00695-9.

Human research islet cell culture outcomes at the Alberta Diabetes Institute IsletCore.

Human islets from deceased organ donors have made important contributions to our understanding of pancreatic endocrine function and continue to be an important resource for research studies aimed at understanding, treating, and preventing diabetes. Understanding the impacts of isolation and culture upon the yield of human islets for research is important for planning research studies and islet distribution to distant laboratories. Here, we examine islet isolation and cell culture outcomes at the Alberta Diabetes Institute (ADI) IsletCore (n = 197). Research-focused isolations typically have a lower yield of islet equivalents (IEQ), with a median of 252,876 IEQ, but a higher purity (median 85%) than clinically focused isolations before culture. The median recovery of IEQs after culture was 75%, suggesting some loss. This was associated with a shift toward smaller islet particles, indicating possible islet fragmentation, and occurred within 24 h with no further loss after longer periods of culture (up to 136 h). No overall change in stimulation index as a measure of islet function was seen with culture time. These findings were replicated in a representative cohort of clinical islet preparations from the Clinical Islet Transplant Program at the University of Alberta. Thus, loss of islets occurs within 24 h of isolation, and there is no further impact of extended culture prior to islet distribution for research.

Ginseng extract improves pancreatic islet injury and promotes ß-cell regeneration in T2DM mice.

Introduction: Panax ginseng C. A. Mey. (Araliaceae; Ginseng Radix et Rhizoma), a traditional plant commonly utilized in Eastern Asia, has demonstrated efficacy in treating neuro-damaging diseases and diabetes mellitus. However, its precise roles and mechanism in alleviating type 2 diabetes mellitus (T2DM) need further study. The objective of this study is to explore the pharmacological effects of ginseng extract and elucidate its potential mechanisms in protecting islets and promoting ß-cell regeneration. Methods: The T2DM mouse model was induced through streptozotocin combined with a high-fat diet. Two batches of mice were sacrificed on the 7th and 28th days following ginseng extract administration. Body weight, fasting blood glucose levels, and glucose tolerance were detected. Morphological changes in the pancreatic islets were examined via H & E staining. Levels of serum insulin, glucagon, GLP-1, and inflammatory factors were measured using ELISA. The ability of ginseng extract to promote pancreatic islet ß-cell regeneration was evaluated through insulin & PCNA double immunofluorescence staining. Furthermore, the mechanism behind ß-cells regeneration was explored through insulin & glucagon double immunofluorescence staining, accompanied by immunohistochemical staining and western blot analyses. Results and Discussion: The present research revealed that ginseng extract alleviates symptoms of T2DM in mice, including decreased blood glucose levels and improved glucose tolerance. Serum levels of insulin, GLP-1, and IL-10 increased following the administration of ginseng extract, while levels of glucagon, TNF-α, and IL-1ß decreased. Ginseng extract preserved normal islet morphology, increased nascent ß-cell population, and inhibited inflammatory infiltration within the islets, moreover, it decreased α-cell proportion while increasing ß-cell proportion. Mechanistically, ginseng extract might inhibit ARX and MAFB expressions, increase MAFA level to aid in α-cell to ß-cell transformation, and activate AKT-FOXM1/cyclin D2 to enhance ß-cell proliferation. Our study suggests that ginseng extract may be a promising therapy in treating T2DM, especially in those with islet injury.

Lowering an ER stress-regulated long noncoding RNA protects mice from diabetes and isolated pancreatic islets from cell death.

We investigated the role of the endoplasmic reticulum (ER) stress-regulated long noncoding RNA (lncRNA) lncMGC in pancreatic islets and the pathology of type 1 diabetes (T1D), as well as the potential of lncMGC-based therapeutics. In vivo, blood glucose levels (BGLs) and HbA1c were significantly lower in lncMGC-knockout (KO)-streptozotocin (STZ)-treated diabetic mice compared to wild-type STZ. Antisense oligonucleotides (GapmeR) targeting lncMGC significantly attenuated insulitis and BGLs in T1D NOD mice compared to GapmeR-negative control (NC). GapmeR-injected T1D Akita mice showed significantly lower BGLs compared to Akita-NC mice. hlncMGC-GapmeR lowered BGLs in partially humanized lncMGC (hlncMGC)-STZ mice compared to NC-injected mice. CHOP (ER stress regulating transcription factor) and lncMGC were upregulated in islets from diabetic mice but not in lncMGC-KO and GapmeR-injected diabetic mice, suggesting ER stress involvement. In vitro, hlncMGC-GapmeR increased the viability of isolated islets from human donors and hlncMGC mice and protected them from cytokine-induced apoptosis. Anti-ER stress and anti-apoptotic genes were upregulated, but pro-apoptotic genes were down-regulated in lncMGC KO mice islets and GapmeR-treated human islets. Taken together, these results show that a GapmeR-targeting lncMGC is effective in ameliorating diabetes in mice and also preserves human and mouse islet viability, implicating clinical translation potential.

Exploring the central region of amylin and its analogs aggregation: the influence of metal ions and residue substitutions.

Human amylin (hIAPP) is found in the form of amyloid deposits within the pancreatic cells of nearly all patients diagnosed with type 2 diabetes mellitus (T2DM). However, rat amylin (rIAPP) and pramlintide - hIAPP analogs - are both non-toxic and non-amyloidogenic. Their primary sequences exhibit only slight variations in a few amino acid residues, primarily concentrated in the central region, spanning residues 20 to 29. This inspired us to study this fragment and investigate the impact on the aggregation properties of substituting residues within the central region of amylin and its analogs. Six fragments derived from amylin have undergone comprehensive testing against various metal ions by implementing a range of analytical techniques, including Nuclear Magnetic Resonance (NMR) spectroscopy, Thioflavin T (ThT) assays, Atomic Force Microscopy (AFM), and cytotoxicity assays. These methodologies serve to provide a thorough understanding of how the substitutions and interactions with metal ions impact the aggregation behavior of amylin and its analogs.

Cleavage of protein kinase c δ by caspase-3 mediates pro-inflammatory cytokine-induced apoptosis in pancreatic islets.

In type 1 diabetes (T1D), autoreactive immune cells infiltrate the pancreas and secrete pro-inflammatory cytokines that initiate cell death in insulin producing islet ß-cells. Protein kinase C δ (PKCδ) plays a role in mediating cytokine-induced ß-cell death; however, the exact mechanisms are not well understood. To address this, we utilized an inducible ß-cell specific PKCδ KO mouse as well as a small peptide inhibitor of PKCδ. We identified a role for PKCδ in mediating cytokine-induced ß-cell death and have shown that inhibiting PKCδ protects pancreatic ß-cells from cytokine-induced apoptosis in both mouse and human islets. We determined that cytokines induced nuclear translocation and activity of PKCδ and that caspase-3 cleavage of PKCδ may be required for cytokine-mediated islet apoptosis. Further, cytokine activated PKCδ increases activity both of pro-apoptotic Bax with acute treatment and JNK with prolonged treatment. Overall, our results suggest that PKCδ mediates cytokine-induced apoptosis via nuclear translocation, cleavage by caspase-3, and upregulation of pro-apoptotic signaling in pancreatic ß-cells. Combined with the protective effects of PKCδ inhibition with δV1-1, the results of this study will aid in the development of novel therapies to prevent or delay ß-cell death and preserve ß-cell function in T1D.

Inhibition of YIPF2 Improves the Vulnerability of Oligodendrocytes to Human Islet Amyloid Polypeptide.

Excessive secretion of human islet amyloid polypeptide (hIAPP) is an important pathological basis of diabetic encephalopathy (DE). In this study, we aimed to investigate the potential implications of hIAPP in DE pathogenesis. Brain magnetic resonance imaging and cognitive scales were applied to evaluate white matter damage and cognitive function. We found that the concentration of serum hIAPP was positively correlated with white matter damage but negatively correlated with cognitive scores in patients with type 2 diabetes mellitus. In vitro assays revealed that oligodendrocytes, compared with neurons, were more prone to acidosis under exogenous hIAPP stimulation. Moreover, western blotting and co-immunoprecipitation indicated that hIAPP interfered with the binding process of monocarboxylate transporter (MCT)1 to its accessory protein CD147 but had no effect on the binding of MCT2 to its accessory protein gp70. Proteomic differential analysis of proteins co-immunoprecipitated with CD147 in oligodendrocytes revealed Yeast Rab GTPase-Interacting protein 2 (YIPF2, which modulates the transfer of CD147 to the cell membrane) as a significant target. Furthermore, YIPF2 inhibition significantly improved hIAPP-induced acidosis in oligodendrocytes and alleviated cognitive dysfunction in DE model mice. These findings suggest that increased CD147 translocation by inhibition of YIPF2 optimizes MCT1 and CD147 binding, potentially ameliorating hIAPP-induced acidosis and the consequent DE-related demyelination.

Antidiabetic effect of combined extract of Coccinia grandis and Blumea balsamifera on streptozotocin-nicotinamide induced diabetic rats.

BACKGROUND: Coccinia grandis and Blumea balsamifera are two medicinal plants that have been known to have good antidiabetic properties. Combining these two plant extracts may generate a greater effect that can increase efficacy and decrease the dose. OBJECTIVE: This research investigated the antidiabetic activity of the combination of C. grandis and B. balsamifera leaves extracts on experimental diabetic rats. MATERIALS AND METHODS: The dried leaves of C. grandis and B. balsamifera were powdered and macerated with ethanol 70% (v/v). A diabetic condition in male Wistar albino rats was generated by intraperitoneal injection of a single dose of streptozotocin (65 mg/kg) followed by nicotinamide (110 mg/kg). Diabetes-confirmed rats were then given glibenclamide (4.5 mg/kg), C. grandis extract (300 mg/kg), B. balsamifera extract (150 mg/kg), and the combined extracts with a dose ratio of 1:1, 1:3, and 3:1. The treatment was performed for 28 days and fasting blood glucose was tested once a week. The pancreas and liver organs were taken on day 29 for antioxidant, histological, and immunohistochemical assessment. RESULTS: Among all the extracts, the combined extract with a ratio of 1:3 showed the greatest glucose lowering effect. This combination also lowered malondialdehyde levels while increasing superoxide dismutase and catalase levels in the pancreas and liver organs. Histological examination showed this combination regenerated the islet of Langerhans. It also increased pancreatic insulin expression in immunohistochemical evaluation. CONCLUSION: This study revealed that the combined extracts of C. grandis and B. balsamifera exhibited enhanced antidiabetic activity via ameliorating oxidative stress, regenerating ß-cells, and increasing insulin expression.

Deletion of RFX6 impairs iPSC-derived islet organoid development and survival, with no impact on PDX1+/NKX6.1+ progenitors.

AIMS/HYPOTHESIS: Homozygous mutations in RFX6 lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown RFX6 variants to be linked with type 2 diabetes. Despite RFX6's known function in islet development, its specific role in diabetes pathogenesis remains unclear. Here, we aimed to understand the mechanisms underlying the impairment of pancreatic islet development and subsequent hypoplasia due to loss-of-function mutations in RFX6. METHODS: We examined regulatory factor X6 (RFX6) expression during human embryonic stem cell (hESC) differentiation into pancreatic islets and re-analysed a single-cell RNA-seq dataset to identify RFX6-specific cell populations during islet development. Furthermore, induced pluripotent stem cell (iPSC) lines lacking RFX6 were generated using CRISPR/Cas9. Various approaches were then employed to explore the consequences of RFX6 loss across different developmental stages. Subsequently, we evaluated transcriptional changes resulting from RFX6 loss through RNA-seq of pancreatic progenitors (PPs) and endocrine progenitors (EPs). RESULTS: RFX6 expression was detected in PDX1+ cells in the hESC-derived posterior foregut (PF). However, in the PPs, RFX6 did not co-localise with pancreatic and duodenal homeobox 1 (PDX1) or NK homeobox 1 (NKX6.1) but instead co-localised with neurogenin 3, NK2 homeobox 2 and islet hormones in the EPs and islets. Single-cell analysis revealed high RFX6 expression levels in endocrine clusters across various hESC-derived pancreatic differentiation stages. Upon differentiating iPSCs lacking RFX6 into pancreatic islets, a significant decrease in PDX1 expression at the PF stage was observed, although this did not affect PPs co-expressing PDX1 and NKX6.1. RNA-seq analysis showed the downregulation of essential genes involved in pancreatic endocrine differentiation, insulin secretion and ion transport due to RFX6 deficiency. Furthermore, RFX6 deficiency resulted in the formation of smaller islet organoids due to increased cellular apoptosis, linked to reduced catalase expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in RFX6-knockout PPs, EPs and islets. CONCLUSIONS/INTERPRETATION: These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1+/NKX6.1+ PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes. DATA AVAILABILITY: RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: https://doi.org/10.5281/zenodo.10656891 ).

Silicone rubber membrane devices permit islet culture at high density without adverse effects.

Introduction: Conventional culture conditions, such as in T-flasks, require that oxygen diffuse through the medium to reach the islets; in turn, islet surface area density is limited by oxygen availability. To culture a typical clinical islet preparation may require more than 20 T-175 flasks at the standard surface area density of 200 IE/cm2. To circumvent this logistical constraint, we tested islets cultured on top of silicon gas-permeable (GP) membranes which place islets in close proximity to ambient oxygen. Methods: Oxygenation of individual islets under three culture conditions, standard low-density, non-GP high density, and GP high density, were first modeled with finite element simulations. Porcine islets from 30 preparations were cultured for 2 days in devices with GP membrane bottoms or in paired cultures under conventional conditions. Islets were seeded at high density (HD, ∼4000 IE/cm2, as measured by DNA) in both GP and non-GP devices. Results: In simulations, individual islets under standard culture conditions and high density cultures on GP membranes were both well oxygenated whereas non-GP high density cultured islets were anoxic. Similarly, compared to the non-GP paired controls, islet viability and recovery were significantly increased in HD GP cultures. The diabetes reversal rate in nude diabetic mice was similar for HD GP devices and standard cultures but was minimal with non-GP HD cultures. Discussion: Culturing islets in GP devices allows for a 20-fold increase of islet surface area density, greatly simplifying the culture process while maintaining islet viability and metabolism.

Comprehensive characterization of islet remodeling in development and in diabetes using mass cytometry.

The pancreatic islet is the functional and structural unit of the pancreatic endocrine portion. Islet remodeling occurs in both normal development and pathogenesis of type 1 (T1D) and type 2 diabetes (T2D). However, accurately quantifying changes in islet cellular makeup and hormone expressions poses significant challenges due to large intra- and inter-donor heterogeneity and the limited scalability of traditional methods such as immunostaining. The cytometry by time-of-flight (CyTOF) technology enables simultaneous quantification of over 30 protein markers at the single-cell resolution in a high throughput fashion. Moreover, with distinct DNA and viability markers, single live cells can be explicitly selected in CyTOF. Here, leveraging the CyTOF data generated by the Human Pancreas Analysis Program (HPAP), we characterized more than 12 million islet cells from 71 donors. Our data revealed continued age-related changes in islet endocrine cell compositions, but the maturity of endocrine cells is reached by three years of age. We also observed significant changes in beta cell numbers and key protein expressions, along with a significant increase in bihormonal cells in T1D donors. In contrast, T2D donors exhibited minimal islet remodeling events. Our data shine a light on the islet dynamics during development and diabetes pathogenesis and suggest divergent pathogenesis processes of T1D and T2D. Our comprehensive approach not only elucidates islet plasticity but also establishes a foundation for integrated CyTOF analysis in islet biology and beyond.

Reduced Nephrin Tyrosine Phosphorylation Enhances Insulin Secretion and Increases Glucose Tolerance With Age.

BACKGROUND: Nephrin is a transmembrane protein with well-established signaling roles in kidney podocytes, and a smaller set of secretory functions in pancreatic ß cells are implicated in diabetes. Nephrin signaling is mediated in part through its 3 cytoplasmic YDxV motifs, which can be tyrosine phosphorylated by high glucose and ß cell injuries. Although in vitro studies demonstrate these phosphorylated motifs can regulate ß cell vesicle trafficking and insulin release, in vivo evidence of their role in this cell type remains to be determined. METHODS: To further explore the role of nephrin YDxV phosphorylation in ß cells, we used a mouse line with tyrosine to phenylalanine substitutions at each YDxV motif (nephrin-Y3F) to inhibit phosphorylation. We assessed islet function via primary islet glucose-stimulated insulin secretion assays and oral glucose tolerance tests. RESULTS: Nephrin-Y3F mice successfully developed pancreatic endocrine and exocrine tissues with minimal structural differences. Unexpectedly, male and female nephrin-Y3F mice showed elevated insulin secretion, with a stronger increase observed in male mice. At 8 months of age, no differences in glucose tolerance were observed between wild-type (WT) and nephrin-Y3F mice. However, aged nephrin-Y3F mice (16 months of age) demonstrated more rapid glucose clearance compared to WT controls. CONCLUSION: Taken together, loss of nephrin YDxV phosphorylation does not alter baseline islet function. Instead, our data suggest a mechanism linking impaired nephrin YDxV phosphorylation to improved islet secretory ability with age. Targeting nephrin phosphorylation could provide novel therapeutic opportunities to improve ß cell function.

Pancreatic stellate cells support human pancreatic ß-cell viability in vitro and enhance survival of immunoisolated human islets exposed to cytokines.

Pancreatic islet transplantation is proposed as a cure for type 1 diabetes mellitus (T1D). Despite its success in optimal regulation of glucose levels, limitations in longevity of islet grafts still require innovative solutions. Inflammatory stress post-transplantation and loss of extracellular matrix attribute to the limited ß-cell survival. Pancreatic stellate cells (PSCs), identified as pancreatic-specific stromal cells, have the potential to play a crucial role in preserving islet survival. Our study aimed to determine the effects of PSCs co-cultured with human CM ß-cells and human islets under inflammatory stress induced by a cytokine cocktail of IFN-γ, TNF-α and IL-1ß. Transwell culture inserts were utilized to assess the paracrine impact of PSCs on ß-cells, alongside co-cultures enabling direct interaction between PSCs and human islets. We found that co-culturing PSCs with human CM ß-cells and human cadaveric islets had rescuing effects on cytokine-induced stress. Effects were different under normoglycemic and hyperglycemic conditions. PSCs were associated with upregulation of ß-cell mitochondrial activity and suppression of inflammatory gene expression. The rescuing effects exist both in indirect and direct co-culture methods. Furthermore, we tested whether PSCs have rescuing effects on human islets in conventional alginate-based microcapsules and in composite microcapsules composed of alginate-pectin collagen type IV, laminin sequence RGD, Nec-1, and amino acid. PSCs partially prevented cytokine-induced stress in both systems, but beneficial effects were stronger in composite capsules. Our findings show novel effects of PSCs on islet health. Islets and PSCs coculturing or co-transplantation might mitigate the inflammation stress and improve islet transplantation outcomes.

Effects of phospholipid type and particle size on lipid nanoparticle distribution in vivo and in pancreatic islets.

Lipid nanoparticles (LNPs) have recently been used as nanocarriers in drug delivery systems for nucleic acid drugs. Their practical applications are currently primarily limited to the liver and specific organs. However, altering the type and composition ratio of phospholipids improves their distribution in organs other than the liver, such as the spleen and lungs. This study aimed to elucidate the effects of LNP components and particle size on in vivo distribution through systemic circulation to pancreatic islets to achieve better targeting of islets, which are a fundamental therapeutic target for diabetes. Fluorescence-labeled LNPs were prepared using three phospholipids: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), with particle sizes of 30-160 nm (diameter) using a microfluidic device. Baffled-structured iLiNP devices with adjusted flow-rate ratios and total flow rates were used. After the intravenous administration of LNPs to C57BL/6 J mice, the distribution of each LNP type to the major organs, including the pancreas and pancreatic islets, was compared using ex vivo fluorescence imaging and observation of pancreatic tissue sections. DSPC-LNPs- and DOPE-LNPs showed the highest distribution in the spleen and liver, respectively. In contrast, the DOPC-LNPs showed the highest distribution in the pancreas and the lowest distribution in the liver and spleen. In addition, smaller particles showed better distribution throughout the pancreas. The most significant LNP distribution in the islets was observed for DOPC-LNPs with a particle size of 160 nm. Furthermore, larger LNPs tended to be distributed in the islets, whereas smaller LNPs tended to be distributed in the exocrine glands. DOPC-LNPs were distributed in the islets at all cholesterol concentrations, with a high distribution observed at >40% cholesterol and > 3% PEG and the distribution was higher at 24 h than at 4 h. Thus, LNP composition and particle size significantly affected islet distribution characteristics, indicating that DOPC-LNPs may be a drug delivery system for effectively targeting the pancreas and islets.

ISLET-1 expression in soft tissue neoplasms reveals high sensitivity but moderate specificity for desmoplastic small round cell tumors and potential utility as a diagnostic biomarker.

ISLET-1 (ISL1) is a LIM-homeodomain transcription factor. Selective ISL1 expression is shown in neuroendocrine, non-neuroendocrine, and some soft tissue tumors including desmoplastic small round cell tumor (DSRCT). We assessed the specificity of ISL1 (clone EP283, 1:500, Cell Marque) in 288 soft tissue tumors, which included 17 DSRCTs and other histologic mimics. Positive staining threshold for ISL1 was set to >10 % of neoplastic cell nuclei at moderate intensity. ISL1 IHC was positive in 15/16 (94 %) DSRCTs with 75 % showing diffuse (>50 %) expression. ISL1 was positive in 1/10 (10 %) Ewing sarcomas (EWS), 7/13 (54 %) alveolar rhabdomyosarcoma (RMS), 14/22 (63 %) embryonal RMS, 7/14 (50 %) synovial sarcomas, 15/16 (93 %) neuroblastoma, 1/5 (20 %) Wilms tumor, 2/4 (50 %) olfactory neuroblastoma, and all 9 Merkel cell carcinomas. Other tumors, including all CIC::DUX4 sarcomas, were negative except 3/27 leiomyosarcomas, and 1 each of angiosarcoma, myxoid liposarcomas, inflammatory myofibroblastic tumor, malignant peripheral nerve sheath tumor, tenosynovial giant cell tumor, dedifferentiated LPS, and 1 ectomesenchymoma. In summary, among the soft tissue tumors tested, ISL1 is a highly sensitive but moderately specific marker for DSRCT and may be useful to distinguish from round cell mimics including EWS and CIC::DUX4 sarcomas. The oncogenic role of ISL1 in these tumors warrants further investigation.

tRNA-derived fragments in T lymphocyte-beta cell crosstalk and in type 1 diabetes pathogenesis in NOD mice.

AIMS/HYPOTHESIS: tRNAs play a central role in protein synthesis. Besides this canonical function, they were recently found to generate non-coding RNA fragments (tRFs) regulating different cellular activities. The aim of this study was to assess the involvement of tRFs in the crosstalk between immune cells and beta cells and to investigate their contribution to the development of type 1 diabetes. METHODS: Global profiling of the tRFs present in pancreatic islets of 4- and 8-week-old NOD mice and in extracellular vesicles released by activated CD4+ T lymphocytes was performed by small RNA-seq. Changes in the level of specific fragments were confirmed by quantitative PCR. The transfer of tRFs from immune cells to beta cells occurring during insulitis was assessed using an RNA-tagging approach. The functional role of tRFs increasing in beta cells during the initial phases of type 1 diabetes was determined by overexpressing them in dissociated islet cells and by determining the impact on gene expression and beta cell apoptosis. RESULTS: We found that the tRF pool was altered in the islets of NOD mice during the initial phases of type 1 diabetes. Part of these changes were triggered by prolonged exposure of beta cells to proinflammatory cytokines (IL-1ß, TNF-α and IFN-γ) while others resulted from the delivery of tRFs produced by CD4+ T lymphocytes infiltrating the islets. Indeed, we identified several tRFs that were enriched in extracellular vesicles from CD4+/CD25- T cells and were transferred to beta cells upon adoptive transfer of these immune cells in NOD.SCID mice. The tRFs delivered to beta cells during the autoimmune reaction triggered gene expression changes that affected the immune regulatory capacity of insulin-secreting cells and rendered the cells more prone to apoptosis. CONCLUSIONS/INTERPRETATION: Our data point to tRFs as novel players in the crosstalk between the immune system and insulin-secreting cells and suggest a potential involvement of this novel class of non-coding RNAs in type 1 diabetes pathogenesis. DATA AVAILABILITY: Sequences are available from the Gene Expression Omnibus (GEO) with accession numbers GSE242568 and GSE256343.

Diabetes current and future translatable therapies.

Diabetes is one of the major diseases and concerns of public health systems that affects over 200 million patients worldwide. It is estimated that 90% of these patients suffer from diabetes type 2, while 10% present diabetes type 1. This type of diabetes and certain types of diabetes type 2, are characterized by dysregulation of blood glycemic levels due to the total or partial depletion of insulin-secreting pancreatic ß-cells. Different approaches have been proposed for long-term treatment of insulin-dependent patients; amongst them, cell-based approaches have been the subject of basic and clinical research since they allow blood glucose level sensing and in situ insulin secretion. The current gold standard for insulin-dependent patients is on-demand exogenous insulin application; cell-based therapies aim to remove this burden from the patient and caregivers. In recent years, protocols to isolate and implant pancreatic islets from diseased donors have been developed and tested in clinical trials. Nevertheless, the shortage of donors, along with the need of immunosuppressive companion therapies, have pushed researchers to focus their attention and efforts to overcome these disadvantages and develop alternative strategies. This review discusses current tested clinical approaches and future potential alternatives for diabetes type 1, and some diabetes type 2, insulin-dependent patients. Additionally, advantages and disadvantages of these discussed methods.