Cell-based gene therapy experiments in murine experimental autoimmune encephalomyelitis.

With the ultimate goal of developing a novel treatment for multiple sclerosis (MS), we have developed a cell-based gene therapy protocol for the treatment of murine experimental autoimmune encephalomyelitis (EAE), a powerful animal model for MS. We have determined that transduced fibroblasts secreting encephalogenic epitopes, when injected into mice with EAE, cause a striking abrogation of disease. Both myelin basic protein (MBP) and proteolipid protein mini-gene constructs expressed in syngeneic fibroblast cells were capable of ameliorating ongoing EAE induced by MBP protein. These experiments are crucial since they suggest that not all encephalogenic epitopes need be secreted for the control of disease. We also demonstrate the success of this protocol when transduced syngeneic, and most importantly, allogeneic cells are sequestered within an implantable chamber. Furthermore, we find that through modifying antigen expression by changing the signal sequence of the mini-gene construct, we were able to significantly reduce the dose of cells required for treatment. These improvements to the mini-gene delivery system are critical for the eventual translation of our protocol to the clinic.

The ability of peptides to induce cytotoxic T cells in vitro does not strongly correlate with their affinity for the H-2Ld molecule: implications for vaccine design and immunotherapy.

The hypothesis that the ability of a peptide to bind to a class I molecule correlates with its immunogenicity is controversial. In this paper we have measured the affinity constants of nine synthetic peptides, which have been previously identified as binding to H-2L(d) molecules, and have determined their immunogenicity in an in vitro cytotoxic T lymphocyte (CTL) induction assay. We find that six peptides bind with high affinity (K(a) > 10(7)/M); of these, four are of viral origin but only two elicit potent CTLs, one is a self peptide which is not immunogenic, while the sixth is of bacterial origin and also does not generate effective CTLs. Two peptides bind with intermediate affinity (K(a) > 10(6)/M); one of these elicits a moderate CTL response, while the other, a tumor-derived epitope, is highly immunogenic. Intriguingly, the peptide with lowest affinity (p2Ca) is exceedingly effective at eliciting CTLs. The efficacy of peptides with modest affinity for their restriction elements appears to correlate well with the CTL precursor frequency. We have also examined intrinsic parameters of some of the peptides such as solubility and stability. Taken together, our results underscore the relevance of factors other than affinity which affect immunogenicity and which may be critical in the design of peptide-based vaccines as well as tumor immunotherapy approaches.

Identification of intraocular lens-adsorbed proteins in mammalian in vitro and in vivo systems.

OBJECTIVE: Mammalian in vitro and in vivo systems were used to study the protein-adsorbing potential of intraocular lenses (IOLs). METHODS: Intraocular lenses composed of polymethyl methacrylate optics with polypropylene haptics were incubated in rabbit plasma for 3 hours (in vitro grouping) or implanted in rabbit eyes for 48 hours (in vivo grouping). Lens-adsorbed proteins from both experimental groupings were eluted with sodium dodecyl sulfate and identified by Western Blot analyses. RESULTS: The adsorbed protein layer was composed of at least six different proteins: albumin, complement C3 fragments, IgG, fibrinogen/fibrin (as a fibrin clot in vivo), fibronectin, and transferrin. Of the identified components, albumin, IgG, fibronectin, and fibrinogen were the predominant protein species on the in vitro IOLs, while fibronectin and fibrin were on the in vivo IOLs. CONCLUSIONS: The composition of the protein layer has the potential to alter the biological property of IOLs.

Complement activation by surface modified poly(methyl methacrylate) intraocular lenses.

The complement activation potential of surface modified (passivated) poly(methyl methacrylate) (PMMA) intraocular lenses (IOLs) with polypropylene loops was compared to that of standard PMMA IOLs with polypropylene loops. Both lens types were incubated in human sera for six hours and then the C3a and C5a levels were measured by radioimmunoassay. Sera incubated with either IOL type generated significantly higher levels of C3a and C5a than control sera incubated without any IOL. The amount of C3a and C5a generated by the passivated PMMA IOLs was comparable to the levels generated by the standard PMMA IOLs. The results of this study show that surface passivated PMMA IOLs with polypropylene loops activated complement to the same level as standard PMMA IOLs with polypropylene loops.