Modeling the Ternary Complex TCR-Vβ/CollagenII(261–273)/HLA-DR4 Associated with Rheumatoid Arthritis

Background

It is known that genetic predisposition to rheumatoid arthritis (RA) is associated with the MHC class II allele HLA-DR4 and that residues 261–273 of type II collagen (huCollp261) represent an immunodominant T cell epitope restricted by the DR4 molecule. Despite recent advances in characterization of MHC and T cell receptor (TCR) contacts to this epitope, the atomic details of TCR/huCollp261/HLA-DR4 ternary complex are not known.

Methodology/Principal Findings

Here we have used computational modeling to get insight into this interaction. A three-dimensional model of the TCR Vβ domain from a DR4⁺ patient affected by RA has been derived by homology modeling techniques. Subsequently, the structure of the TCR Vβ domain in complex with huCollp261/HLA-DR4 was obtained from a docking approach in conjunction with a filtering procedure based on biochemical information. The best complex from the docking experiments was then refined by 20 ns of molecular dynamics simulation in explicit water. The predicted model is consistent with available experimental data. Our results indicate that residues 97–101 of CDR3β are critical for recognition of huCollp261/HLA-DR4 by TCR. We also show that TCR contacts on p/MHC surface affect the conformation of the shared epitope expressed by DR alleles associated with RA susceptibility.

Conclusions/Significance

This work presents a three-dimensional model for the ternary complex TCR-Vβ/collagenII(261–273)/HLA-DR4 associated with rheumatoid arthritis that can provide insights into the molecular mechanisms of self reactivity.     

Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance

Cellular therapy for myocardial injury has improved ventricular function in both animal and clinical studies, though the mechanism of benefit is unclear. This study was undertaken to examine the effects of cellular injection after infarction on myocardial elasticity. Coronary artery ligation of Lewis rats was followed by direct injection of human mesenchymal stem cells (MSCs) into the acutely ischemic myocardium. Two weeks postinfarct, myocardial elasticity was mapped by atomic force microscopy. MSC-injected hearts near the infarct region were twofold stiffer than myocardium from noninfarcted animals but softer than myocardium from vehicle-treated infarcted animals. After 8 wk, the following variables were evaluated: MSC engraftment and left ventricular geometry by histological methods, cardiac function with a pressure-volume conductance catheter, myocardial fibrosis by Masson Trichrome staining, vascularity by immunohistochemistry, and apoptosis by TdT-mediated dUTP nick-end labeling assay. The human cells engrafted and expressed a cardiomyocyte protein but stopped short of full differentiation and did not stimulate significant angiogenesis. MSC-injected hearts showed significantly less fibrosis than controls, as well as less left ventricular dilation, reduced apoptosis, increased myocardial thickness, and preservation of systolic and diastolic cardiac function. In summary, MSC injection after myocardial infarction did not regenerate contracting cardiomyocytes but reduced the stiffness of the subsequent scar and attenuated postinfarction remodeling, preserving some cardiac function. Improving scarred heart muscle compliance could be a functional benefit of cellular cardiomyoplasty.     

Ouabain-insensitive salt and water movements in duck red cells. II. The role of chloride in the volume response

This paper describes the effect of external chloride on the typical swelling response induced in duck red cells by hypertonicity or norepinephrine. Lowering chloride inhibits swelling and produces concomitant changes in net movements of sodium and potassium in ouabain-treated cells, which resemble the effect of lowering external sodium or potassium. Inhibition is the same whether chloride is replaced with gluconate or with an osmotic equivalent of sucrose. Since changes in external chloride also cause predictable changes in cell chloride, pH, and water, these variables were systematically investigated by varying external pH along with chloride. Lowering pH to 6.60 does not abolish the response if external chloride levels are normal, although the cells are initially swollen due to the increased acidity. Cells deliberately preswollen in hypotonic solutions with appropriate ionic composition can also respond to norepinephrine by further swelling. These results rule out initial values of cell water, chloride, and pH as significant variables affecting the response. Initial values of the chloride equilibrium potential do have marked effect on the direction and rate of net water movement. If chloride is lowered by replacement with the permeant anion, acetate, E(Cl) is unchanged and a normal response to norepinephrine, which is inhibited by furosemide, is observed. Increasing internal sodium by the nystatin technique also inhibits the response. A theory is developed which depicts that the cotransport carrier proposed in the previous paper (W.F. Schmidt and T.J. McManus. 1977b. J. Gen. Physiol. 70:81-97) moves in response to the net electrochemical potential difference driving sodium and potassium across the membrane. Predictions of this theory fit the data for both cations and anions.     

Production of stable bispecific IgG1 by controlled Fab-arm exchange

The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of >95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques.