Vogt-Koyanagi-Harada patients show higher frequencies of circulating NKG2Dpos NK and NK T cells.

Vogt-Koyanagi-Harada (VKH) is an autoimmune disease characterized by inflammation in tissues that contain melanocytes. We aimed to increase the knowledge regarding immunological pathways deregulated in VKH disease. We compared the percentages of circulating natural killer (NK), NK T and T cells expressing the activatory markers: CD16, CD69, NK group 2D (NKG2D), natural cytotoxicity triggering receptor 3 (Nkp30), natural cytotoxicity triggering receptor 1 (Nkp46) and the inhibitory marker: NK group 2 member A (NKG2A) in 10 active VKH patients, 20 control subjects (CTR) and seven patients with Behçet disease (BD) by flow cytometry. Cytotoxic potential of NK cells was determined through the degranulation marker CD107a expression after contact with K562 cells by flow cytometry. Moreover, plasmatic levels of 27 cytokines were determined with a multiplex bead-based assay. VKH patients showed higher percentages of NKG2Dpos NK and NK T cells versus CTR. The cytotoxic potential of NK cells induced by K562 cells was comparable between VKH patients and CTR. Finally, higher concentrations of interleukin (IL)-4, IL-5, IL-7, IL-17 and platelet-derived growth factor-subunits B (PDGF-bb) were detected in plasma of VKH patients versus CTR. The immune profile of VKH patients was similar to that of BD patients.

Modeling and mitigation of high-concentration antibody viscosity through structure-based computer-aided protein design.

For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.

Lipocalin 24p3 Induction in Colitis Adversely Affects Inflammation and Contributes to Mortality.

Recognition of microorganism associated molecular patterns by epithelial cells elicits signaling cascades resulting in the production of host defense proteins. Lipocalin 24p3 is purported to be one such protein. 24p3 binds prokaryotic and eukaryotic siderophores and by sequestering iron laden bacterial siderophores it was believed to restrict bacterial replication. As such mice deficient for 24p3 are susceptible to systemic infections. However, it is not clear whether deficiency of 24p3 on the gut mucosa contributes to inflammation. In line with 24p3's function as a bacteriostat, it would be reasonable to assume that deficiencies in the control of intestinal flora from 24p3 absence play a role in inflammatory intestinal diseases. Surprisingly, we show 24p3 is a contributor of inflammation and 24p3 deficiency protects mice from dextran sodium sulfate (DSS)-induced colitis. 24p3 was found to be a negative regulator of platelet-derived growth factor (PDGF), which helps maintain the integrity of the gut mucosa. Neutralization of PDGF-BB abrogated resistance of 24p3 null mice to DSS confirming the direct link between 24p3 and PDGF-BB. Finally, iron handling in wild-type and 24p3-null mice upon DSS treatment also differed. In summary, differential iron levels and enhanced expression of PDGF-BB in 24p3 null mice confers resistance to DSS.