Accelerated turnover of MHC class II molecules in nonobese diabetic mice is developmentally and environmentally regulated in vivo and dispensable for autoimmunity.

The H2-A(g7) (A(g7)) MHC class II (MHCII) allele is required for type 1 diabetes (T1D) in NOD mice. A(g7) not only has a unique peptide-binding profile, it was reported to exhibit biochemical defects, including accelerated protein turnover. Such defects were proposed to impair Ag presentation and, thus, self-tolerance. Here, we report measurements of MHCII protein synthesis and turnover in vivo. NOD mice and BALB/c controls were labeled continuously with heavy water, and splenic B cells and dendritic cells were isolated. MHCII molecules were immunoprecipitated and digested with trypsin. Digests were analyzed by liquid chromatography/mass spectrometry to quantify the fraction of newly synthesized MHCII molecules and, thus, turnover. MHCII turnover was faster in dendritic cells than in B cells, varying slightly between mouse strains. Some A(g7) molecules exhibited accelerated turnover in B cells from young, but not older, prediabetic female NOD mice. This acceleration was not detected in a second NOD colony with a high incidence of T1D. Turnover rates of A(g7) and H2-A(d) were indistinguishable in (NOD × BALB/c) F1 mice. In conclusion, accelerated MHCII turnover may occur in NOD mice, but it reflects environmental and developmental regulation, rather than a structural deficit of the A(g7) allele. Moreover, this phenotype wanes before the onset of overt T1D and is dispensable for the development of autoimmune diabetes. Our observations highlight the importance of in vivo studies in understanding the role of protein turnover in genotype/phenotype relationships and offer a novel approach for addressing this fundamental research challenge.

Effect of Rotation on Scaffold Motion and Cell Growth in Rotating Bioreactors.

Efficient use of different bioreactor designs to improve cell growth in three-dimensional scaffolds requires an understanding of their mechanism of action. To address this for rotating wall vessel bioreactors, fluid and scaffold motion were investigated experimentally at different rotation speeds and vessel fill volumes. Low cost bioreactors with single and dual axis rotation were developed to investigate the effect of these systems on human osteoblast proliferation in free floating and constrained collagen-glycosaminoglycan porous scaffolds. A range of scaffold motions (free fall, periodic oscillation, and orbital motion) were observed at the rotation speeds and vessel fluid/air ratios used, with 85% fluid fill and an outer vessel wall velocity of ∼14 mm s-1 producing a scaffold in a free fall state. The cell proliferation results showed that after 14 and 21 days of culture, this combination of fluid fill and speed of rotation produced significantly greater cell numbers in the scaffolds than when lower or higher rotation speeds (p < 0.002) or when the chamber was 60% or 100% full (p < 0.01). The fluid flow and scaffold motion experiments show that biaxial rotation would not improve the mass transfer of medium into the scaffold as the second axis of rotation can only transition the scaffold toward oscillatory or orbital motion and, hence, reduce mass transport to the scaffold. The cell culture results confirmed that there was no benefit to the second axis of rotation with no significant difference in cell proliferation either when the scaffolds were free floating or constrained (p > 0.05).