Willem M. Kühtreiber

Low levels of C-peptide have clinical significance for established Type 1 diabetes.

To determine whether the low C‐peptide levels (< 50 pmol/l) produced by the pancreas for decades after onset of Type 1 diabetes have clinical significance.

Strategic internal covalent cross-linking of TNF produces a stable TNF trimer with improved TNFR2 signaling

BackgroundSoluble TNF superfamily (TNFSF) ligands are less stable and less active than their transmembrane (tm) analogues. This is a problem for the therapeutic use of recombinant TNFSF ligands in diverse diseases including cancer and autoimmunity. Creating TNFSF ligand analogues with improved targeting of their respective receptors is important for research and therapeutic purposes.FindingsCovalent internal cross-linking of TNF monomers by double mutations, S95C/G148C, results in stable trimers with improved TNFR2 function. The resulting mutein induced the selective death of autoreactive CD8 T cells in type-1 diabetic patients and demonstrates targeted proliferation and expansion of human CD4 Tregs.ConclusionsStable TNF trimers, created by internal covalent cross-linking, show improved signaling. The high structural homology within the TNF superfamily provides an opportunity to extend internal cross-linking to other TNF superfamily proteins to produce active trimers with improved stability and receptor signaling, and with potential applications for cancer, autoimmunity, infections, and transplantation.

Novel Automated Blood Separations Validate Whole Cell Biomarkers

Background Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples. Methods and Findings To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficolls uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes. Conclusions Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials.

Long-term reduction in hyperglycemia in advanced type 1 diabetes: the value of induced aerobic glycolysis with BCG vaccinations

Mycobacterium are among the oldest co-evolutionary partners of humans. The attenuated Mycobacterium bovis Bacillus Calmette Guérin (BCG) strain has been administered globally for 100 years as a vaccine against tuberculosis. BCG also shows promise as treatment for numerous inflammatory and autoimmune diseases. Here, we report on a randomized 8-year long prospective examination of type 1 diabetic subjects with long-term disease who received two doses of the BCG vaccine. After year 3, BCG lowered hemoglobin A1c to near normal levels for the next 5 years. The BCG impact on blood sugars appeared to be driven by a novel systemic and blood sugar lowering mechanism in diabetes. We observe a systemic shift in glucose metabolism from oxidative phosphorylation to aerobic glycolysis, a state of high glucose utilization. Confirmation is gained by metabolomics, mRNAseq, and functional assays of cellular glucose uptake after BCG vaccinations. To prove BCG could induce a systemic change to promote accelerated glucose utilization and impact blood sugars, murine data demonstrated reduced blood sugars and aerobic induction in non-autoimmune mice made chemically diabetic. BCG via epigenetics also resets six central T-regulatory genes for genetic re-programming of tolerance. These findings set the stage for further testing of a known safe vaccine therapy for improved blood sugar control through changes in metabolism and durability with epigenetic changes even in advanced Type 1 diabetes.Diabetes: Repurposing a classic tuberculosis vaccineIn patients with long-term type 1 diabetes, the tuberculosis vaccine BCG lowers blood sugar levels to near-normal after three years. Denise Faustman and her team from Massachusetts General Hospital and Harvard Medical School investigated a cohort of type 1 diabetics that received two doses of BCG before being monitored over eight years. After three years, vaccine-treated patients lowered their HbA1c levels—a diabetes biomarker reflecting average blood sugar over 8–12 weeks—by over 10%. This reduction increased to 18% in the fourth year, after which HbA1c levels remained low up to the final year of monitoring. The researchers report that the BCG vaccine appeared to reset diabetes-implicated parts of the immune system and, through a novel mechanism, shift glucose metabolism to lower blood sugar to healthy levels. Future studies will further classify BCG’s benefits in diabetes.

Disposable No Longer: The Spleen Holds a Reservoir of Stem Cells

Regenerative medicine is coming to recognize the value of a stem cell population that is unique to the spleen. This splenic stem cell population can robustly facilitate adult end organ regeneration and it expresses a key embryonic transcription factor, Hox11, which regulates organogenesis in diverse vertebrate species. This review article discusses the identification of this Hox11 stem cell population for its therapeutic potential in the pancreas, salivary glands, heart, bone and cranial neurons for organ regeneration. It also discusses the deleterious effects of Hox11 stem cells in cancer, in which malignant cells revert to a Hox11 phenotype, and in certain forms of autoimmunity, in which Hox11 lineages of cells may contribute to abnormal development of end organs.

Early- Versus Late-Onset Type 1 Diabetes: Two Different Pathophysiological Subtypes with Implications for Therapy

Insulin, as measured by C-peptide, is produced for decades after onset of type 1 diabetes, and even very low levels of C-peptide have clinical significance. In this chapter we show that two distinct pathophysiological subtypes of type 1 diabetic subjects can be distinguished. Early-onset diabetic subjects (≤20 years) have rapid loss of C-peptide, whereas late-onset diabetic subjects (>20 years) have slower C-peptide declines over decades. Early-onset diabetics have significantly lower levels of persistent autoreactive CD8+ T cells than do late-onset diabetic subjects. In late-onset disease, robust production of autoreactive T-cells occurs even in the absence of C-peptide. Metabolomics analysis reveals frequent differences between the two subtypes of subjects in the levels of amino acids, carbohydrates, cofactors, lipids, peptides, and xenobiotics. There are statistically significant differences related to protective islet functions, islet health, development, blood sugar control, and regulation of exocrine pancreas function. Taken together these findings suggest that pancreas pathobiology, as well as durability of abnormal T-cell response should be considered in immune targeting treatments. Therapies aimed at immune defects alone are likely to work best in late-onset diabetics. Therapies aimed at islet cell preservation in early-onset diabetic subjects likely have greater efficacy if administered shortly after disease onset.

Possible Transient Benefits of Epstein Barr Virus Infection in Three Subjects with Established Type 1 Diabetes

Tumor necrosis factor (TNF) is a novel immunotherapy for type I diabetes because it selectively kills insulin autoreactive T-cells, which enables recovery of insulin production by pancreatic islet cells. The TNF inducer Bacillus Calmette-Guerin (BCG) also has therapeutic value by activating innate immunity that beneficially modifies the course of type 1 diabetes (T1D) in the same manner as TNF. Epstein Barr Virus (EBV) infection is also an inducer of TNF. This observational study was undertaken to determine whether EBV has the same beneficial effects as BCG in a recent clinical trial. We describe three cases of long-term diabetic subjects with active mononucleosis that were followed for 15 weeks to determine the impact of EBV infection on established T1D. In comparison to non-EBVinfected long-term diabetics, EBV transiently diminished the autoimmune response in two of three cases. EBV infection triggered rapid increase of circulating insulin-B autoreactive T-cells whose striking loss of CD8 marker indicated that the cells were injured or apoptotic. EBV infection also caused a transient surge in the secretion of C-peptide, a marker for pancreatic insulin secretion. EBV acts like BCG in eliciting innate immunity and beneficially modifying the course of TID.