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Pancreatic beta-cell death is a major factor in the pathogenesis of type 1 diabetes (T1D), but straightforward methods to measure beta-cell loss in humans are lacking, underlining the need for novel biomarkers. Using studies in INS-1 cells, human islets, diabetic mice, and serum samples of subjects with T1D at different stages, we have identified serum miR-204 as an early biomarker of T1D-associated beta-cell loss in humans. MiR-204 is a highly enriched microRNA in human beta-cells, and we found that it is released from dying beta-cells and detectable in human serum. We further discovered that serum miR-204 was elevated in children and adults with T1D and in autoantibody-positive at-risk subjects but not in type 2 diabetes or other autoimmune diseases and was inversely correlated with remaining beta-cell function in recent-onset T1D. Thus, serum miR-204 may provide a much needed novel approach to assess early T1D-associated human beta-cell loss even before onset of overt disease.

Diabetes is characterized by a loss of β-cell mass, and a greater understanding of the transcriptional mechanisms governing β-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA–binding protein 3 (SSBP3) regulates the genes necessary for β-cell function, but few proteins are known to interact with this complex in β-cells. To identify additional components, here we performed SSBP3 reverse–cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse β-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for β-cell function. We observed that Rnf20 and Rnf40 depletion reduces β-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate β-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.

Background Evidence suggests that GABA may reduce pancreatic inflammation, protect β-cells from autoimmune destruction, and potentiate the regeneration of new β-cells in the setting of type 1 diabetes mellitus (T1DM). The enzyme GAD, also expressed in human pancreatic β-cells, is an antigenic target of reactive T cells. We hypothesized that treatment of children with recent onset T1DM with GABA or combination GABA with GAD will preserve β-cell function and ameliorate autoimmune dysregulation. Methods This is a one-year, prospective, randomized, double-blind, placebo-controlled trial. Ninety-nine patients aged 4–18 years with newly diagnosed T1DM are randomized into three treatment groups: 1) oral GABA twice daily in addition to two injections of recombinant GAD enzyme, 2) oral GABA plus placebo GAD injections, or 3) placebo GABA and placebo GAD. Patients are evaluated at baseline and months 1, 5, 8 and 12. Mixed meal tolerance testing is performed at all but the 8-month visit. Laboratory studies will assess indices of beta and alpha cell function, glycemic control, immunophenotyping, and diabetes-related autoantibodies. Results The primary outcome is the effect on pancreatic β-cell function as measured by meal-stimulated c-peptide secretion compared between the treatment groups before and after one year of treatment. Secondary outcomes include: 1) fasting and meal stimulated glucagon and proinsulin levels, 2) response in insulin usage by participants, 3) indices of immune cell function, and 4) effect on autoantibodies GAD65, ICA512, and ZnT8. Conclusions: This trial will determine the safety and efficacy of GABA and combination GABA/GAD therapy to delay T1DM progression in children.

The bone structure is very dynamic and continuously adapts its geometry to external stimuli by modeling and remodeling the mineralized tissue. In vivo microCT-based time-lapse morphometry is a powerful tool to study the temporal and spatial dynamics of bone (re)modeling. Here an advancement in the methodology to detect and quantify site-specific differences in bone (re)modeling of 12-week-old BALB/c nude mice is presented. We describe our method of quantifying new bone surface interface readouts and how these are influenced by bone curvature. This method is then used to compare bone surface (re)modeling in mice across different anatomical regions to demonstrate variations in the rate of change and spatial gradients thereof. Significant differences in bone (re)modeling baseline parameters between the metaphyseal and epiphyseal are shown, as well as cortical and trabecular bone of the distal femur and proximal tibia. These results are validated using conventional static in vivo microCT analysis. Finally, the insights from these new baseline values of physiological bone (re)modeling were used to evaluate pathological bone (re)modeling in a pilot breast cancer bone metastasis model. The method shows the potential to be suitable to detect early pathological events and track their spatio-temporal development in both cortical and trabecular bone. This advancement in (re)modeling surface analysis and defined baseline parameters according to distinct anatomical regions will be valuable to others investigating various disease models with site-distinct local alterations in bone (re)modeling.ER

Swimming microorganisms often experience complex environments in their natural habitat. The same is true for microswimmers in envisioned biomedical applications. The simple aqueous conditions typically studied in the lab differ strongly from those found in these environments and often exclude the effects of small volume confinement or the influence that external fields have on their motion. In this work, we investigate magnetically steerable microswimmers, specifically magnetotactic bacteria, in strong spatial confinement and under the influence of an external magnetic field. We trap single cells in micrometer-sized microfluidic chambers and track and analyze their motion, which shows a variety of different trajectories, depending on the chamber size and the strength of the magnetic field. Combining these experimental observations with simulations using a variant of an active Brownian particle model, we explain the variety of trajectories by the interplay between the wall interactions and the magnetic torque. We also analyze the pronounced cell-to-cell heterogeneity, which makes single-cell tracking essential for an understanding of the motility patterns. In this way, our work establishes a basis for the analysis and prediction of microswimmer motility in more complex environments.

Genome-wide association studies have identified ICOSLG, which encodes the inducible costimulator ligand (ICOSLG or ICOSL) as a susceptibility locus for inflammatory bowel disease. ICOSL has been implicated in the enhancement of pattern recognition receptor signaling in dendritic cells, induction of IL-10 production by CD4 T cells, and the generation of high-affinity antibodies to specific antigens—all of which can potentially explain its involvement in gastrointestinal inflammation. Here, we show that murine ICOSL deficiency results in significant enrichment of IL-10–producing CD4 T cells particularly in the proximal large intestine. Transient depletion of IL-10–producing cells from adult ICOSL-deficient mice induced severe colonic inflammation that was prevented when mice were first treated with metronidazole. ICOSL-deficient mice displayed reduced IgA and IgG antibodies in the colon mucus and impaired serum antibody recognition of microbial antigens, including flagellins derived from mucus-associated bacteria of the Lachnospiraceae family. Confirming the synergy between ICOSL and IL-10, ICOSL deficiency coupled with CD4-specific deletion of the Il10 gene resulted in juvenile onset colitis that was impeded when pups were fostered by ICOSL-sufficient dams. In this setting, we found that both maternally acquired and host-derived antibodies contribute to the life anti-commensal antibody repertoire that mediates this protection in early life. Collectively, our findings reveal a partnership between ICOSL-dependent anti-commensal antibodies and IL-10 in adaptive immune regulation of the microbiota in the large intestine. Furthermore, we identify ICOSL deficiency as an effective platform for exploring the functions of anti-commensal antibodies in host–microbiota mutualism.

Currently, no oral medications are available for type 1 diabetes (T1D). While our recent randomized placebo-controlled T1D trial revealed that oral verapamil had short-term beneficial effects, their duration and underlying mechanisms remained elusive. Now, our global T1D serum proteomics analysis identified chromogranin A (CHGA), a T1D-autoantigen, as the top protein altered by verapamil and as a potential therapeutic marker and revealed that verapamil normalizes serum CHGA levels and reverses T1D-induced elevations in circulating proinflammatory T-follicular-helper cell markers. RNA-sequencing further confirmed that verapamil regulates the thioredoxin system and promotes an anti-oxidative, anti-apoptotic and immunomodulatory gene expression profile in human islets. Moreover, continuous use of oral verapamil delayed T1D progression, promoted endogenous beta-cell function and lowered insulin requirements and serum CHGA levels for at least 2 years and these benefits were lost upon discontinuation. Thus, the current studies provide crucial mechanistic and clinical insight into the beneficial effects of verapamil in T1D.