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AIMS/HYPOTHESIS: Autoimmune attack against the insulin-producing beta cells in the pancreatic islets results in type 1 diabetes. However, despite considerable research, details of the type 1 diabetes immunopathology in situ are not fully understood mainly because of difficult access to the pancreatic islets in vivo. METHODS: Here, we used direct non-invasive confocal imaging of islets transplanted in the anterior chamber of the eye (ACE) to investigate the anti-islet autoimmunity in NOD mice before, during and after diabetes onset. ACE-transplanted islets allowed longitudinal studies of the autoimmune attack against islets and revealed the infiltration kinetics and in situ motility dynamics of fluorescence-labelled autoreactive T cells during diabetes development. Ex vivo immunostaining was also used to compare immune cell infiltrations into islet grafts in the eye and kidney as well as in pancreatic islets of the same diabetic NOD mice. RESULTS: We found similar immune infiltration in native pancreatic and ACE-transplanted islets, which established the ACE-transplanted islets as reliable reporters of the autoimmune response. Longitudinal studies in ACE-transplanted islets identified in vivo hallmarks of islet inflammation that concurred with early immune infiltration of the islets and preceded their collapse and hyperglycaemia onset. A model incorporating data on ACE-transplanted islet degranulation and swelling allowed early prediction of the autoimmune attack in the pancreas and prompted treatments to intercept type 1 diabetes. CONCLUSIONS/INTERPRETATION: The current findings highlight the value of ACE-transplanted islets in studying early type 1 diabetes pathogenesis in vivo and underscore the need for timely intervention to halt disease progression.

The transplantation of human pancreatic islets is a therapeutic possibility for a subset of type 1 diabetic patients who experience severe hypoglycemia. Pre- and post-transplantation loss in islet viability and function, however, is a major efficacy-limiting impediment. To investigate the effects of inflammation and hypoxia, the main obstacles hampering the survival and function of isolated, cultured, and transplanted islets, we conducted a comprehensive metabolomics evaluation of human islets in parallel with dynamic glucose-stimulated insulin release (GSIR) perifusion studies for functional evaluation. Metabolomics profiling of media and cell samples identified a total of 241 and 361 biochemicals, respectively. Metabolites that were altered in highly significant manner in both included, for example, kynurenine, kynurenate, citrulline, and mannitol/sorbitol under inflammation (all elevated) plus lactate (elevated) and N-formylmethionine (depressed) for hypoxia. Dynamic GSIR experiments, which capture both first- and second-phase insulin release, found severely depressed insulin-secretion under hypoxia, whereas elevated baseline and stimulated insulin-secretion was measured for islet exposed to the inflammatory cytokine cocktail (IL-1β, IFN-γ, and TNF-α). Because of the uniquely large changes observed in kynurenine and kynurenate, they might serve as potential biomarkers of islet inflammation, and indoleamine-2,3-dioxygenase on the corresponding pathway could be a worthwhile therapeutic target to dampen inflammatory effects.

We report the design and fabrication of a robust fluidic platform built out of inert plastic materials and micromachined features that promote optimized convective fluid transport. The platform is tested for perfusion interrogation of rodent and human pancreatic islets, dynamic secretion of hormones, concomitant live-cell imaging, and optogenetic stimulation of genetically engineered islets. A coupled quantitative fluid dynamics computational model of glucose stimulated insulin secretion and fluid dynamics was first utilized to design device geometries that are optimal for complete perfusion of three-dimensional islets, effective collection of secreted insulin, and minimization of system volumes and associated delays. Fluidic devices were then fabricated through rapid prototyping techniques, such as micromilling and laser engraving, as two interlocking parts from materials that are non-absorbent and inert. Finally, the assembly was tested for performance using both rodent and human islets with multiple assays conducted in parallel, such as dynamic perfusion, staining and optogenetics on standard microscopes, as well as for integration with commercial perfusion machines. The optimized design of convective fluid flows, use of bio-inert and non-absorbent materials, reversible assembly, manual access for loading and unloading of islets, and straightforward integration with commercial imaging and fluid handling systems proved to be critical for perfusion assay, and particularly suited for time-resolved optogenetics studies.

Costimulatory interactions are required for T cell activation and development of an effective immune response; hence, they are valuable therapeutic targets for immunomodulation. However, they, as all other protein-protein interactions, are difficult to target by small molecules. Here, we report the identification of novel small-molecule inhibitors of the CD40-CD40L interaction designed starting from the chemical space of organic dyes. For the most promising compounds such as DRI-C21045, activity (IC50) in the low micromolar range has been confirmed in cell assays including inhibition of CD40L-induced activation in NF-κB sensor cells, THP-1 myeloid cells, and primary human B cells as well as in murine allogeneic skin transplant and alloantigen-induced T cell expansion in draining lymph node experiments. Specificity versus other TNF-superfamily interactions (TNF-R1-TNF-α) and lack of cytotoxicity have also been confirmed at these concentrations. These novel compounds provide proof-of-principle evidence for the possibility of small-molecule inhibition of costimulatory protein-protein interactions, establish the structural requirements needed for efficient CD40-CD40L inhibition, and serve to guide the search for such immune therapeutics.

A global nontargeted longitudinal metabolomics study was carried out in male and female NOD mice to characterize the time-profile of the changes in the metabolic signature caused by onset of type 1 diabetes (T1D) and identify possible early biomarkers in T1D progressors. Metabolomics profiling of samples collected at five different time-points identified 676 and 706 biochemicals in blood and feces, respectively. Several metabolites were expressed at significantly different levels in progressors at all time-points, and their proportion increased strongly following onset of hyperglycemia. At the last time-point, when all progressors were diabetic, a large percentage of metabolites had significantly different levels: 57.8% in blood and 27.8% in feces. Metabolic pathways most strongly affected included the carbohydrate, lipid, branched-chain amino acid, and oxidative ones. Several biochemicals showed considerable (4×) change. Maltose, 3-hydroxybutyric acid, and kojibiose increased, while 1,5-anhydroglucitol decreased more than 10-fold. At the earliest time-point (6-week), differences between the metabolic signatures of progressors and nonprogressors were relatively modest. Nevertheless, several compounds had significantly different levels and show promise as possible early T1D biomarkers. They include fatty acid phosphocholine derivatives from the phosphatidylcholine subpathway (elevated in both blood and feces) as well as serotonin, ribose, and arabinose (increased) in blood plus 13-HODE, tocopherol (increased), diaminopimelate, valerate, hydroxymethylpyrimidine, and dulcitol (decreased) in feces. A combined metabolic signature based on these compounds might serve as an early predictor of T1D-progressors.

To explore the effects immune-isolating encapsulation has on the insulin secretion of pancreatic islets and to improve our ability to quantitatively describe the glucose-stimulated insulin release (GSIR) of pancreatic islets, we conducted dynamic perifusion experiments with isolated human islets. Free (unencapsulated) and hydrogel encapsulated islets were perifused, in parallel, using an automated multi-channel system that allows sample collection with high temporal resolution. Results indicated that free human islets secrete less insulin per unit mass or islet equivalent (IEQ) than murine islets and with a less pronounced first-phase peak. While small microcapsules (d ≈ 700 µm) caused only a slightly delayed and blunted first-phase insulin response compared to unencapsulated islets, larger capsules (d ≈ 1800 µm) completely blunted the first-phase peak and decreased the total amount of insulin released. Experimentally obtained insulin time-profiles were fitted with our complex insulin secretion computational model. This allowed further fine-tuning of the hormone-release parameters of this model, which was implemented in COMSOL Multiphysics to couple hormone secretion and nutrient consumption kinetics with diffusive and convective transport. The results of these GSIR experiments, which were also supported by computational modeling, indicate that larger capsules unavoidably lead to dampening of the first-phase insulin response and to a sustained-release type insulin secretion that can only slowly respond to changes in glucose concentration. Bioartificial pancreas type devices can provide long-term and physiologically desirable solutions only if immunoisolation and biocompatibility considerations are integrated with optimized nutrient diffusion and insulin release characteristics by design. This article is protected by copyright. All rights reserved

For isolated pancreatic islet cell preparations, it is important to be able to reliably assess their mass and quality, and for clinical applications, it is part of the regulatory requirement. Accurate assessment, however, is difficult because islets are spheroid-like cell aggregates of different sizes (2= 0.78, slope = 1.02). Variability and reproducibility are also improved compared to the manual method, and most of the remaining variability (CV = 8.9%) results from the rearrangement of the islet particles due to movement of the sample between counts. Characterization of the size distribution is also important, and the present digitally collected data allow more detailed analysis and coverage of a wider size range. We found again that for human islet cell preparations, a Weibull distribution function provides good description of the particle size.