Mass spectrometry measurement of a therapeutic peptide for use in multiple sclerosis.

Multiple sclerosis is an autoimmune disease of the central nervous system believed to be mediated by pathogenic T lymphocytes. We have developed a next-generation therapy in which cells secrete specific therapeutic molecules to silence these aberrant T cells. We have shown that fibroblasts, transduced to secrete a myelin basic protein-derived peptide, abrogate disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis, which we hypothesized using a low-zone tolerance mechanism. To determine the efficacy (or not) of this therapy in humans, we must ensure that patients receive comparable doses of therapeutic peptide. To this end, we have used liquid chromatography coupled to tandem mass spectrometry to detect a tryptic peptide, derived from the secreted therapeutic product, at nanomolar concentrations. Success depended on growing the transduced fibroblasts in defined PC-1 medium in the presence of a cocktail of protease inhibitors.

Vitamin A status affects the development of diabetes and insulitis in BB rats.

BB/Wor rats develop autoimmune diabetes mellitus with many features in common with human insulin-dependent diabetes mellitus. Since retinoids are known to have effects on insulin secretion and immune function, these studies were designed to investigate the effects of retinoid deficiency on diabetes in BB/Wor rats and to identify a role for retinoid status in the pathogenesis of autoimmune diabetes mellitus. Litters of diabetes-prone (DP) and diabetes-resistant (DR) BB/Wor rats were divided at weaning and fed a diet either (1) devoid of retinoids and leading to clinical deficiency at approximately 60 days of age (A-def diet)-following 10 days of clinical deficiency, rats on the A-def diet were changed to a diet containing 2 microg/g retinoic (A-def/RA diet); (2) containing 2 microg/g retinoic acid but deficient in retinol (RA diet); or (3) replete in retinol with 4 microg/g retinyl palmitate (RP diet). Rats receiving RP or RA diets were pair-fed to rats on the A-def/RA diet. Diabetes by 120 days of age was greatly reduced (P < .01) in DP rats that received the A-def/RA diet (four of 27) or RA diet (four of 29) versus the RP diet (13 of 31). Insulitis progressed with age in nondiabetic DP rats receiving the RP diet (P < .02) or RA diet (P < .05), but not the A-def/RA diet (P > .22). Insulin secretion was measured in perfused pancreas of nondiabetic rats after age 120 days and correlated negatively with insulitis (P < .05). DP rats receiving the RP diet had reduced insulin secretion as compared with other DP and DR rats (P < .05). In DR rats, retinoid status had no effects on insulitis through 120 days of age or on insulin secretion after 120 days of age. In conclusion, retinol deficiency reduces diabetes and insulitis in DP BB/Wor rats, and retinoic acid can at least partly substitute for retinol in the development of insulitis.

Dissection of cross-reactivities using a panel of H-2Ld alloreactive T cell hybridomas.

In order to explore the role which class I structure plays in alloreactivity, we have generated Ld-reactive T cell hybridomas by fusion of a dm2 anti-BALB/cJ MLR with the BW5147 cell line and examined their stimulation by the following class I molecules (alpha 1/alpha 2/alpha 3): Lq, Dq, dm1, Ld/Ld/Dd, Lq/Dq/Ld, and Q10/Q10/Ld. We found that their specificities differed in their patterns of cross-reactions and were reasonably representative of those present in the bulk population of MLR-generated CTLs. Ld/Ld/Dd and Q10/Q10/Ld stimulated the majority of the hybridomas, Lq and dm1 were recognized by over half of the panel, and Lq/Dq/Ld stimulated only modestly, while Dq was not recognized by any hybridoma. Correlation of these observed reactivities with class I structure suggests that putative TCR contact residues may play a significant role in recognition when compared to the polymorphic amino acid residues which control pocket specificity and peptide binding. Specifically, Lq and Dq possess very similar or identical pockets, in contrast to those of dm1 and Q10. However, Q10 has identical TCR contact residues to Ld, both on the alpha 1 and alpha 2 alpha helices, unlike Dq which is mismatched on both helices. Lq and dm1 are mismatched compared to Ld on only one helix. Thus, a molecular rationale for the cross-reactions observed in this study involves the direct participation of residues of class I molecules in allorecognition.

Phosphatidyl inositol-linked forms of a murine class I MHC molecule expressed on Chinese hamster ovary cells retain peptide binding capability and alloreactivity.

A gene encoding a phosphatidyl inositol-linked form of the murine class I MHC molecule H-2Kd was constructed and the protein expressed in Chinese hamster ovary cells together with murine or human beta 2-microglobulin (beta 2m). The resulting lipid-linked class I heterodimers can be efficiently converted into a soluble form by treatment of transfected cells with a phospholipase. Cells expressing Kd heterodimers were characterized with respect to heavy chain levels at the cell surface, peptide binding, and recognition by Kd-specific antibodies and alloreactive cytotoxic T cells. All transfectants bound a 3H-labeled Kd-restricted nonamer peptide, although more peptide bound to cells expressing the Kd/human beta 2m combination, perhaps because of a greater number of empty molecules at the cell surface. A dissociation constant of 5 x 10(-8) M derived by Scatchard analysis is within the range expected for interactions of peptides with class I MHC molecules. Alloreactive cytotoxic T cells which recognize wild-type Kd on murine cells lysed the hamster cells expressing lipid-linked Kd without regard to the species of the beta 2m light chain. These results indicated that the engineered lipid-linked Kd molecule is expressed at the cell surface, is recognized by antibodies and T cells, and functions to bind peptide.