4S-fluorination of ProB29 in insulin lispro slows fibril formation.

Recombinant insulin is a life-saving therapeutic for millions of patients affected by diabetes mellitus. Standard mutagenesis has led to insulin variants with improved control of blood glucose; for instance, the fast-acting insulin lispro contains two point mutations that suppress dimer formation and expedite absorption. However, insulins undergo irreversible denaturation, a process accelerated for the insulin monomer. Here we replace ProB29 of insulin lispro with 4R-fluoroproline, 4S-fluoroproline, and 4,4-difluoroproline. All three fluorinated lispro variants reduce blood glucose in diabetic mice, exhibit similar secondary structure as measured by CD, and rapidly dissociate from the zinc- and resorcinol-bound hexamer upon dilution. Notably, however, we find that 4S-fluorination of ProB29 delays the formation of undesired insulin fibrils that can accumulate at the injection site in vivo and can complicate insulin production and storage. These results demonstrate how subtle molecular changes achieved through non-canonical amino acid mutagenesis can improve the stability of protein therapeutics.

A scalable human islet 3D-culture platform maintains cell mass and function long-term for transplantation.

Present-day islet culture methods provide short-term maintenance of cell viability and function, limiting access to islet transplantation. Attempts to lengthen culture intervals remain unsuccessful. A new method was developed to permit the long-term culture of islets. Human islets were embedded in polysaccharide 3D-hydrogel in cell culture inserts or gas-permeable chambers with serum-free CMRL 1066 supplemented media for up to 8 weeks. The long-term cultured islets maintained better morphology, cell mass, and viability at 4 weeks than islets in conventional suspension culture. In fact, islets cultured in the 3D-hydrogel retained ß cell mass and function on par with freshly isolated islets in vitro and, when transplanted into diabetic mice, restored glucose balance similar to fresh islets. Using gas-permeable chambers, the 3D-hydrogel culture method was scaled up over 10-fold and maintained islet viability and function, although the cell mass recovery rate was 50%. Additional optimization of scale-up methods continues. If successful, this technology could afford flexibility and expand access to islet transplantation, especially single-donor islet-after-kidney transplantation.

A subcutaneous pancreatic islet transplantation platform using a clinically applicable, biodegradable Vicryl mesh scaffold - an experimental study.

Pancreatic islet transplantation into the liver is an effective treatment for type 1 diabetes but has some critical limitations. The subcutaneous site is a potential alternative transplant site, requiring minimally invasive procedures and allowing frequent graft monitoring; however, hypoxia is a major drawback. Our previous study without scaffolding demonstrated post-transplant graft aggregation in the subcutaneous site, which theoretically exacerbates lethal intra-graft hypoxia. In this study, we introduce a clinically applicable subcutaneous islet transplantation platform using a biodegradable Vicryl mesh scaffold to prevent aggregation in a diabetic rat model. Islets were sandwiched between layers of clinically proven Vicryl mesh within thrombin-fibrin gel. In vitro, the mesh prevented islet aggregation and intra-islet hypoxia, which significantly improved islet viability. In vivo rat syngeneic islet transplantations into a prevascularized subcutaneous pocket demonstrated that the mesh significantly enhanced engraftment, as measured by assays for graft survival and function. Histological examination at 6 weeks showed well-vascularized grafts sandwiched in a flat shape between the mesh layers. The biodegradable mesh was fully absorbed by three months, which alleviated chronic foreign body reaction and fibrosis, and supported long-term graft maintenance. This simple graft shape modification approach is an effective and clinically applicable strategy for improved subcutaneous islet transplantation.

Synthesis and evaluation of 18F-PTTCO-Cys40-Exendin-4 for PET imaging of ectopic insulinomas in rodents.

A major limitation in the development of radiolabeled Exendin-4 analogues (short half-life isotopes) is an inability to efficiently and rapidly separate final products from precursors. This is important as lack of purity in the final product decreases probe efficiency. The purpose of this study was to develop a method to prepare the high-purity imaging reagent [18F] PTTCO-Cys40-Exendin-4. To accomplish this, magnetic TCO-beads were incubated with the crude product to remove unlabeled Exendin-4. In rodents pre-treatment with purified [18F] PTTCO-Cys40-Exendin-4 (~1.85 MBq) allowed precise microPET imaging of ectopic insulinomas. Moreover, analogue uptake was successfully blocked by administering non-labelled "cold" Exendin-4. Biodistribution data revealed that [18F] PTTCO-Cys40-Exendin-4 accumulated specifically in GLP-1R-enriched insulinomas in mice, confirming results obtained using miroPET. Investigation of [18F] PTTCO-Cys40-Exendin-4 as a tracer to image portal vein-transplanted pancreatic islets is proceeding in animals.

4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation.

Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of noncanonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.

Involvement of a proapoptotic gene (BBC3) in islet injury mediated by cold preservation and rewarming.

Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved postpreservation mitochondrial viability. Cold preservation resulted in decreased postisolation islet yield in both wild-type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3-KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3-KO islets from cold-preserved pancreata showed reduced HMGB1 (high-mobility group box 1 protein) expression and decreased levels of 4-hydroxynonenal (4-HNE) protein adducts, which was indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was upregulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after rewarming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/rewarming-mediated islet injury, possibly through modulating HMGB1- and oxidative stress-mediated injury to islets.

Isolated human islets require hyperoxia to maintain islet mass, metabolism, and function.

Pancreatic islet transplantation has been recognized as an effective treatment for Type 1 diabetes; however, there is still plenty of room to improve transplantation efficiency. Because islets are metabolically active they require high oxygen to survive; thus hypoxia after transplant is one of the major causes of graft failure. Knowing the optimal oxygen tension for isolated islets would allow a transplant team to provide the best oxygen environment during pre- and post-transplant periods. To address this issue and begin to establish empirically determined guidelines for islet maintenance, we exposed in vitro cultured islets to different partial oxygen pressures (pO2) and assessed changes in islet volume, viability, metabolism, and function. Human islets were cultured for 7 days in different pO2 media corresponding to hypoxia (90 mmHg), normoxia (160 mmHg), and hyerpoxia (270 or 350 mmHg). Compared to normoxia and hypoxia, hyperoxia alleviated the loss of islet volume, maintaining higher islet viability and metabolism as measured by oxygen consumption and glucose-stimulated insulin secretion responses. We predict that maintaining pre- and post-transplanted islets in a hyperoxic environment will alleviate islet volume loss and maintain islet quality thereby improving transplant outcomes.

Mechanisms of islet damage mediated by pancreas cold ischemia/rewarming.

Prolonged pancreas cold ischemia is known to negatively correlate with islet isolation outcomes, hindering successful islet transplantation to treat Type-1 Diabetes. Due to poor islet isolation outcome, pancreata with over 16 h cold ischemia are currently not considered for islet transplantation. Mechanisms involved in pancreas cold ischemia/rewarming mediated islet damage during islet isolation and culture are not well understood. Using an en bloc cold preserved rat pancreas preparation, we attempted to clarify possible mechanisms of islet death associated with prolonged pancreas cold ischemia and subsequent rewarming. Cold ischemia lasting 16 h decreased post-isolation islet yield and increased islet death during the initial 6 h of culture. Electron micrographs revealed swelling and severe disruption of cellular and mitochondrial membranes, as well as an enlarged endoplasmic reticulum (ER) in ß-cells isolated from cold preserved pancreata. Prolonged cold ischemia of the pancreas transiently activated mitogen-activated protein kinases (MAPKs) in isolated islets and increased lipid peroxidation products 4-hydroxynonenal (HNE) and heat shock protein (Hsp) 70 after culture, indicating the activation of oxidative stress signaling pathways. The islet isolation process, irrespective of pancreas cold ischemia, activated unfolded protein response (UPR), while the ER protective chaperon BiP was further upregulated by pancreas cold ischemia/rewarming. During the first 6 h of culture following islet isolation, p53 upregulated modulator of apoptosis (Puma) and caspase-3 activation were also upregulated. Our study indicates the involvement of both apoptosis and necrosis in islet death, and suggests oxidative stress and disruption of membranes are critical mechanisms mediated by pancreas cold ischemia/rewarming.

Sodium levels of human pancreatic donors are a critical factor for determination of islet efficacy and survival.

Organs from hypernatremia (elevated Na+) donors when used for transplantation have had dismal outcomes. However, islet isolation from hypernatremic donors for both transplantation and research applications has not yet been investigated. A retrospective analysis of in vivo and in vitro islet function studies was performed on islets isolated from hypernatremic (serum sodium levels≥160 meq/l) and normal control (serum sodium levels≤155 meq/l) donors. Twelve isolations from 32 hypernatremic and 53 isolations from 222 normal donors were randomly transplanted into diabetic NOD Scid mice. Sodium levels upon pancreas procurement were significantly elevated in the hypernatremia group (163.5±0.6 meq/l) compared with the normal control group (145.9±0.4 meq/l) (P<0.001). The postculture islet recovery rate was significantly lower in the hypernatremia (59.1±3.8%) group compared with the normal (73.6±1.8%) group (P=0.005). The duration of hypernatremia was inversely correlated with the recovery rate (r2=0.370, P<0.001). Furthermore, the percentage of successful graft function when transplanted into diabetic NOD Scid mice was significantly lower in the hypernatremia (42%) group compared with the normal control (85%) group (P<0.001). The ability to predict islet graft function posttransplantation using donor sodium levels and duration of hypernatremia was significant (ROC analysis, P=0.022 and 0.042, respectively). In conclusion, duration of donor hypernatremia is associated with reduced islet recovery postculture. The efficacy of islets from hypernatremia donors diminished when transplanted into diabetic recipients.

Mechanism of Action of Oral Salmonella-Based Vaccine to Prevent and Reverse Type 1 Diabetes in NOD Mice.

A combination therapy of preproinsulin (PPI) and immunomodulators (TGFß+IL10) orally delivered via genetically modified Salmonella and anti-CD3 promoted glucose balance in in NOD mice with recent onset diabetes. The Salmonella bacteria were modified to express the diabetes-associated antigen PPI controlled by a bacterial promoter in conjunction with over-expressed immunomodulating molecules. The possible mechanisms of action of this vaccine to limit autoimmune diabetes remained undefined. In mice, the vaccine prevented and reversed ongoing diabetes. The vaccine-mediated beneficial effects were associated with increased numbers of antigen-specific CD4+CD25+Foxp3+ Tregs, CD4+CD49b+LAG3+ Tr1-cells, and tolerogenic dendritic-cells (tol-DCs) in the spleens and lymphatic organs of treated mice. Despite this, the immune response to Salmonella infection was not altered. Furthermore, the vaccine effects were associated with a reduction in islet-infiltrating lymphocytes and an increase in the islet beta-cell mass. This was associated with increased serum levels of the tolerogenic cytokines (IL10, IL2, and IL13) and chemokine ligand 2 (CCL2) and decreased levels of inflammatory cytokines (IFNγ, GM-CSF, IL6, IL12, and TNFα) and chemokines (CXCL1, CXCL2, and CXCL5). Overall, the data suggest that the Salmonella-based vaccine modulates the immune response, reduces inflammation, and promotes tolerance specifically to an antigen involved in autoimmune diabetes.

Reversal of diabetes by an oral Salmonella-based vaccine in acute and progressive diabetes in NOD mice.

Type 1 diabetes (T1D)-associated hyperglycemia develops, in part, from loss of insulin-secreting beta cells. The degree of glycemic dysregulation and the age at onset of disease can serve as indicators of the aggressiveness of the disease. Tracking blood glucose levels in prediabetic mice may demonstrate the onset of diabetes and, along with animal age, also presage disease severity. In this study, an analysis of blood glucose levels obtained from female NOD mice starting at 4 weeks until diabetes onset was undertaken. New onset diabetic mice were orally vaccinated with a Salmonella-based vaccine towards T1D-associated preproinsulin combined with TGFß and IL10 along with anti-CD3 antibody. Blood glucose levels were obtained before and after development of disease and vaccination. Animals were classified as acute disease if hyperglycemia was confirmed at a young age, while other animals were classified as progressive disease. The effectiveness of the oral T1D vaccine was greater in mice with progressive disease that had less glucose excursion compared to acute disease mice. Overall, the Salmonella-based vaccine reversed disease in 60% of the diabetic mice due, in part, to lessening of islet inflammation, improving residual beta cell health, and promoting tolerance. In summary, the age of disease onset and severity of glucose dysregulation in NOD mice predicted response to vaccine therapy. This suggests a similar disease categorization in the clinic may predict therapeutic response.

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.

Biological hypoxia in pre-transplant human pancreatic islets induces transplant failure in diabetic mice.

Evaluating the quality of isolated human islets before transplantation is crucial for predicting the success in treating Type 1 diabetes. The current gold standard involves time-intensive in vivo transplantation into diabetic immunodeficient mice. Given the susceptibility of isolated islets to hypoxia, we hypothesized that hypoxia present in islets before transplantation could indicate compromised islet quality, potentially leading to unfavorable outcomes. To test this hypothesis, we analyzed expression of 39 hypoxia-related genes in human islets from 85 deceased donors. We correlated gene expression profiles with transplantation outcomes in 327 diabetic mice, each receiving 1200 islet equivalents grafted into the kidney capsule. Transplantation outcome was post-transplant glycemic control based on area under the curve of blood glucose over 4 weeks. In linear regression analysis, DDIT4 (R = 0.4971, P < 0.0001), SLC2A8 (R = 0.3531, P = 0.0009) and HK1 (R = 0.3444, P = 0.0012) had the highest correlation with transplantation outcome. A multiple regression model of 11 genes increased the correlation (R = 0.6117, P < 0.0001). We conclude that assessing pre-transplant hypoxia in human islets via gene expression analysis is a rapid, viable alternative to conventional in vivo assessments. This approach also underscores the importance of mitigating pre-transplant hypoxia in isolated islets to improve the success rate of islet transplantation.

Trefoil factor 2 expressed by the murine pancreatic acinar cells is required for the development of islets and for beta cell function during aging.

Exocrine-to-endocrine crosstalk in the pancreas is crucial to maintain beta cell function. However, the molecular mechanisms underlying this crosstalk are largely undefined. Trefoil factor 2 (Tff2) is a secreted factor known to promote the proliferation of beta cells in vitro, but its physiological role in vivo in the pancreas is unknown. Also, it remains unclear which pancreatic cell type expresses Tff2 protein. We therefore created a mouse model with a conditional knockout of Tff2 in the murine pancreas. We find that the Tff2 protein is preferentially expressed in acinar but not ductal or endocrine cells. Tff2 deficiency in the pancreas reduces beta cell mass on embryonic day 16.5. However, homozygous mutant mice are born without a reduction of beta cells and with acinar Tff3 compensation by day 7. When mice are aged to 1 year, both male and female homozygous and male heterozygous mutants develop impaired glucose tolerance without affected insulin sensitivity. Perifusion analysis reveals that the second phase of glucose-stimulated insulin secretion from islets is reduced in aged homozygous mutant compared to controls. Collectively, these results demonstrate a previously unknown role of Tff2 as an exocrine acinar cell-derived protein required for maintaining functional endocrine beta cells in mice.

One-Step Automatic Radiosynthesis and Evaluation of [18F]TM-30089 as GPR44 Radiotracer.

Recently, a G-protein coupled receptor 44 (GPR44) was discovered to play a significant role in the process of inflammation-related diseases, including cancer and diabetes. However, the precise role of GPR44 has yet to be fully elucidated. Currently, there is a strong and urgent need for the development of GPR44 radiotracers as a non-invasive methodology to explore the exact mechanism of GPR44 on inflammation-related diseases and monitor the progress of therapy. TM-30089 is a potent GPR44 antagonist that exhibits a high specificity and selectivity for GPR44. Its structure contains a fluorine nuclide, which could potentially be replaced with 18F. In the present study, we successfully took a highly effective synthesis strategy that pretreated the unprotected carboxylic acid group of the precursor and developed a feasible one-step automatic radiosynthesis strategy for [18F]TM-30089 with a high radiochemical purity and a good radiochemical yield. We further evaluated this radiotracer using mice models implanted with 1.1 B4 cell lines (GPR44-enriched cell lines) and human islets (high GPR44 expression), respectively. The results revealed the persistent and specific uptake of [18F]TM-30089 in GPR44 region, indicating that [18F]TM-30089 is a promising candidate for targeting GPR44. Further evaluation is ongoing.

Methylcellulose colony assay and single-cell micro-manipulation reveal progenitor-like cells in adult human pancreatic ducts.

Progenitor cells capable of self-renewal and differentiation in the adult human pancreas are an under-explored resource for regenerative medicine. Using micro-manipulation and three-dimensional colony assays we identify cells within the adult human exocrine pancreas that resemble progenitor cells. Exocrine tissues were dissociated into single cells and plated into a colony assay containing methylcellulose and 5% Matrigel. A subpopulation of ductal cells formed colonies containing differentiated ductal, acinar, and endocrine lineage cells, and expanded up to 300-fold with a ROCK inhibitor. When transplanted into diabetic mice, colonies pre-treated with a NOTCH inhibitor gave rise to insulin-expressing cells. Both colonies and primary human ducts contained cells that simultaneously express progenitor transcription factors SOX9, NKX6.1, and PDX1. In addition, in silico analysis identified progenitor-like cells within ductal clusters in a single-cell RNA sequencing dataset. Therefore, progenitor-like cells capable of self-renewal and tri-lineage differentiation either pre-exist in the adult human exocrine pancreas, or readily adapt in culture.

Critical Considerations in Bioluminescence Imaging of Transplanted Islets: Dynamic Signal Change in Early Posttransplant Phase and Signal Absorption by Tissues.

OBJECTIVES: In pancreatic islet transplantation studies, bioluminescence imaging enables quantitative and noninvasive tracking of graft survival. Amid the recent heightened interest in extrahepatic sites for islet and stem cell-derived beta-like cell transplantations, proper understanding the nature of bioluminescence imaging in these sites is important. METHODS: Islets isolated from Firefly rats ubiquitously expressing luciferase reporter gene in Lewis rats were transplanted into subcutaneous or kidney capsule sites of wild-type Lewis rats or immunodeficient mice. Posttransplant changes of bioluminescence signal curves and absorption of bioluminescence signal in transplantation sites were examined. RESULTS: The bioluminescence signal curve dynamically changed in the early posttransplantation phase; the signal was low within the first 5 days after transplantation. A substantial amount of bioluminescence signal was absorbed by tissues surrounding islet grafts, correlating to the depth of the transplanted site from the skin surface. Grafts in kidney capsules were harder to image than those in the subcutaneous site. Within the kidney capsule, locations that minimized depth from the skin surface improved the graft detectability. CONCLUSIONS: Posttransplant phase and graft location/depth critically impact the bioluminescence images captured in islet transplantation studies. Understanding these parameters is critical for reducing experimental biases and proper interpretation of data.

Micropyramid-patterned, oxygen-permeable bottomed dish for high density culture of pancreatic islets.

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death. Here we introduce a culture dish incorporating a micropyramid-patterned surface to prevent the unsolicited fusion and oxygen-permeable bottom for optimal oxygen environment. A 400µm-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400µm-base and 200µm-height was fabricated to apply to the 24-well plate format. The micropyramid pattern separated the individual pancreatic islets to prevent the fusion of multiple islets. This platform supported the high oxygen demand of islets at high seeding density at 260 islet equivalents cm-2, a 2-3-fold higher seeding density compared to the conventional islet culture used in a preparation for the clinical islet transplantations, demonstrating improved islet morphology, metabolism and function in a 4 d-culture. Transplantation of these islets into immunodeficient diabetic mice exhibited significantly improved engraftment to achieve euglycemia compared to islets cultured in the conventional culture wells. Collectively, this simple design modification allows for high-density cultures of three-dimensional cell spheroids to improve the viability and function for an array of investigational and clinical replacement tissues.

Biodistribution of Intra-Arterial and Intravenous Delivery of Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles in a Rat Model to Guide Delivery Strategies for Diabetes Therapies.

Umbilical cord mesenchymal stem cell-derived extracellular vesicles (UC-MSC-EVs) have become an emerging strategy for treating various autoimmune and metabolic disorders, particularly diabetes. Delivery of UC-MSC-EVs is essential to ensure optimal efficacy of UC-MSC-EVs. To develop safe and superior EVs-based delivery strategies, we explored nuclear techniques including positron emission tomography (PET) to evaluate the delivery of UC-MSC-EVs in vivo. In this study, human UC-MSC-EVs were first successfully tagged with I-124 to permit PET determination. Intravenous (I.V.) and intra-arterial (I.A.) administration routes of [124I]I-UC-MSC-EVs were compared and evaluated by in vivo PET-CT imaging and ex vivo biodistribution in a non-diabetic Lewis (LEW) rat model. For I.A. administration, [124I]I-UC-MSC-EVs were directly infused into the pancreatic parenchyma via the celiac artery. PET imaging revealed that the predominant uptake occurred in the liver for both injection routes, and further imaging characterized clearance patterns of [124I]I-UC-MSC-EVs. For biodistribution, the uptake (%ID/gram) in the spleen was significantly higher for I.V. administration compared to I.A. administration (1.95 ± 0.03 and 0.43 ± 0.07, respectively). Importantly, the pancreas displayed similar uptake levels between the two modalities (0.20 ± 0.06 for I.V. and 0.24 ± 0.03 for I.A.). Therefore, our initial data revealed that both routes had similar delivery efficiency for [124I]I-UC-MSC-EVs except in the spleen and liver, considering that higher spleen uptake could enhance immunomodulatory application of UC-MSC-EVs. These findings could guide the development of safe and efficacious delivery strategies for UC-MSC-EVs in diabetes therapies, in which a minimally invasive I.V. approach would serve as a better delivery strategy. Further confirmation studies are ongoing.

Oral Salmonella msbB Mutant as a Carrier for a Salmonella-Based Vaccine for Prevention and Reversal of Type 1 Diabetes.

A therapy that includes an oral vaccine for type 1 diabetes (T1D) using live attenuated Salmonella MvP728 (ΔhtrA/ΔpurD), cytokines (IL10 and TGFß) and preproinsulin (PPI) antigen in combination with a sub-therapeutic dose of anti-CD3 mAb was developed by our team. The vaccine combination therapy reduced insulitis and prevented and reversed diabetes in non-obese diabetic (NOD) mice. Here, we show the effectiveness of an alternative Salmonella mutant (ΔmsbB) as a carrier strain, which is anticipated to have lower risks of an inflammatory response and septicemia as a result of modification in the lipopolysaccharide (LPS) via detoxification of lipid A. This mutant strain proved to have highly reduced pathogenic side effects. Salmonella strain ΔmsbB expressed autoantigens and in combination with cytokines and anti-CD3 mAb, successfully prevented and reversed T1D to levels comparable to the previously used carrier strain ΔhtrA/ΔpurD. Additionally, the Salmonella msbB mutant resulted in higher rates of host cell infection. These results further demonstrate the potential of an oral Salmonella-based combined therapy in the treatment of early T1D.