Unravelling the structural complexity of glycolipids with cryogenic infrared spectroscopy

Glycolipids are complex glycoconjugates composed of a glycan headgroup and a lipid moiety. Their modular biosynthesis creates a vast amount of diverse and often isomeric structures, which fulfill highly specific biological functions. To date, no gold-standard analytical technique can provide a comprehensive structural elucidation of complex glycolipids, and insufficient tools for isomer distinction can lead to wrong assignments. Herein we use cryogenic gas-phase infrared spectroscopy to systematically investigate different kinds of isomerism in immunologically relevant glycolipids. We show that all structural features, including isomeric glycan headgroups, anomeric configurations and different lipid moieties, can be unambiguously resolved by diagnostic spectroscopic fingerprints in a narrow spectral range. The results allow for the characterization of isomeric glycolipid mixtures and biological applications

Interleukin-1 alpha modulates collagen gene expression in cultured synovial cells.

The effects of porcine interleukin-1 (IL-1) alpha on collagen production were studied in cultured human rheumatoid synovial cells. Addition of 0.05-5 ng of IL-1/ml into the cultures resulted in a dose-dependent decreased rate of collagen released into the medium over 24 h. To determine whether this inhibition was due to secondary action of prostaglandin E2 (PGE2) secreted in response to IL-1, cultures were incubated in presence of various inhibitors of arachidonate metabolism. Depending on the cell strains, these inhibitors were able to suppress or diminish the effect of IL-1, suggesting that PGE2 is involved in the mechanism. Depression of collagen production caused by IL-1 mainly affected type I collagen and therefore led to a change in the type I/type III collagen ratio in the extracellular medium. Steady-state levels of mRNA for types I and III procollagens were estimated by dot-blot hybridization and compared with the amounts of respective collagens produced in the same cultures. IL-1 generally increased procollagen type I mRNA, but to a variable extent, as did indomethacin (Indo). Depending on the cell strain, the combination of indo and IL-1 could elevate the mRNA level of type I procollagen compared with Indo alone. These results did not correlate with the production rate of collagen in the medium, which was diminished by exposure to IL-1. The level of mRNA for collagen type III was not greatly changed by incubation with IL-1, and a better correlation was generally observed with the amount of type III collagen found in the medium. These data suggest that an additional control mechanism at translational or post-translational level must exist, counterbalancing the stimulatory effect of IL-1 on collagen mRNA transcription. It is likely that IL-1 could modulate the production of collagen in synovial cells by an interplay of different mechanisms, some of them limiting the effect of primary elevation of the steady-state mRNA level

Intestinal Delivery of Proinsulin and IL-10 via Lactococcus lactis Combined With Low-Dose Anti-CD3 Restores Tolerance Outside the Window of Acute Type 1 Diabetes Diagnosis

A combination treatment (CT) of proinsulin and IL-10 orally delivered via genetically modified Lactococcus lactis bacteria combined with low-dose anti-CD3 (aCD3) therapy successfully restores glucose homeostasis in newly diagnosed non-obese diabetic (NOD) mice. Tolerance is accompanied by the accumulation of Foxp3+ regulatory T cells (Tregs) in the pancreas. To test the potential of this therapy outside the window of acute diabetes diagnosis, we substituted autoimmune diabetic mice, with disease duration varying between 4 and 53 days, with syngeneic islets at the time of therapy initiation. Untreated islet recipients consistently showed disease recurrence after 8.2 ± 0.7 days, while 32% of aCD3-treated and 48% of CT-treated mice remained normoglycemic until 6 weeks after therapy initiation (P < 0.001 vs. untreated controls for both treatments, P < 0.05 CT vs. aCD3 therapy). However, mice that were diabetic for more than 2 weeks before treatment initiation were less efficient at maintaining normoglycemia than those treated within 2 weeks of diabetes diagnosis, particularly in the aCD3-treated group. The complete elimination of endogenous beta cell mass with alloxan at the time of diabetes diagnosis pointed toward the significance of continuous feeding of the islet antigen proinsulin at the time of aCD3 therapy for treatment success. The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (ovalbumin) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Sustained tolerance was accompanied with a reduction of IGRP+CD8+ autoreactive T cells and an increase in insulin-reactive (InsB12–20 or InsB13–2) Foxp3+CD4+ Tregs, with a specific accumulation of Foxp3+ Tregs around the insulin-containing islet grafts after CT with proinsulin. The combination of proinsulin and IL-10 via oral Lactococcus lactis with low-dose aCD3 therapy can restore tolerance to beta cells in autoimmune diabetic mice, also when therapy is started outside the window of acute diabetes diagnosis, providing persistence of insulin-containing islets or prolonged beta cell function