Effect of plasma exchange in accelerating natalizumab clearance and restoring leukocyte function.

BACKGROUND: Accelerating the clearance of therapeutic monoclonal antibodies (mAbs) from the body may be useful to address uncommon but serious complications from treatment, such as progressive multifocal leukoencephalopathy (PML). Treatment of PML requires immune reconstitution. Plasma exchange (PLEX) may accelerate mAb clearance, restoring the function of inhibited proteins and increasing the number or function of leukocytes entering the CNS. We evaluated the efficacy of PLEX in accelerating natalizumab (a therapy for multiple sclerosis [MS] and Crohn disease) clearance and alpha4-integrin desaturation. Restoration of leukocyte transmigratory capacity was evaluated using an in vitro blood-brain barrier (ivBBB). METHODS: Twelve patients with MS receiving natalizumab underwent three 1.5-volume PLEX sessions over 5 or 8 days. Natalizumab concentrations and alpha4-integrin saturation were assessed daily throughout PLEX and three times over the subsequent 2 weeks, comparing results with the same patients the previous month. Peripheral blood mononuclear cell (PBMC) migration (induced by the chemokine CCL2) across an ivBBB was assessed in a subset of six patients with and without PLEX. RESULTS: Serum natalizumab concentrations were reduced by a mean of 92% from baseline to 1 week after three PLEX sessions (p < 0.001). Although average alpha4-integrin saturation was not reduced after PLEX, it was reduced to less than 50% when natalizumab concentrations were below 1 mug/mL. PBMC transmigratory capacity increased 2.2-fold after PLEX (p < 0.006). CONCLUSIONS: Plasma exchange (PLEX) accelerated clearance of natalizumab, and at natalizumab concentrations below 1 mug/mL, desaturation of alpha4-integrin was observed. Also, CCL2-induced leukocyte transmigration across an in vitro blood-brain barrier was increased after PLEX. Therefore, PLEX may be effective in restoring immune effector function in natalizumab-treated patients.

Clinical and immune responses correlate in glatiramer acetate therapy of multiple sclerosis.

Glatiramer acetate (GA) treatment for relapsing remitting multiple sclerosis (RRMS) leads to decreased GA-specific proliferative responses and a Th2 cytokine shift. To study a possible correlation between immunological and clinical responses to GA therapy, we prospectively followed RRMS patients clinically, by magnetic resonance imaging and by primary immunological assays. Fluctuation of GA-specific proliferative responses was significantly lower in treatment responders than in untreated patients, and GA-specific proliferative responses were increased during relapses. These associations suggest a possible causal relationship between immunological and clinical responses to GA therapy. Primary proliferation assays may thus be a useful marker for treatment response.

In vitro evidence that subcutaneous administration of glatiramer acetate induces hyporesponsive T cells in patients with multiple sclerosis.

Glatiramer acetate (GA; Copaxone) is a random sequence polypeptide used in the treatment of relapsing remitting multiple sclerosis (RR MS). We have recently demonstrated that prior to treatment, GA induces proliferation of resting T cells and is not cross-reactive with myelin antigens. Daily GA injections induce a significant loss of this GA responsiveness, which is associated with the induction of highly cross-reactive Th2-type T cells potentially capable of suppressing inflammatory responses. The mechanism of action by which GA induces T cell nonresponsiveness leading to T cell receptor degeneracy in patients with RR MS is unknown. Here, we examined the effects of daily GA administration on the induction of T cell hyporesponsiveness. The frequency of GA-reactive T cells in peripheral blood of seven patients with RR MS was measured by limiting dilution analysis prior to and during 6 months of treatment. In addition, a model in which GA-reactive T cells were stimulated in vitro was developed to better characterize the selection of T cell populations over time. In vivo treatment with GA induced a decrease in GA-reactive T cell frequencies and hyporesponsiveness of CD4(+) T cell reactivity to GA in vitro that was only partially reversed by the addition of IL-2. These data suggest that T cell peripheral tolerance to GA was achieved in vivo during treatment. Thus, our in vitro data suggest that the underlying changes in GA-reactive CD4(+) T cell reactivity could be explained by the induction of T cell anergy and clonal elimination.

Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues.

The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance to the organism by expelling excess copper. Two related human ATPase genes, ATP7A (EC 3.6.1.36) and ATP7B (EC 3.6.1.36), have been cloned as the loci of mutations causing Menkes and Wilson diseases, diseases of copper metabolism. Many mutations in these genes have been identified in patients. Since it has not yet been possible to purify the human copper ATPases, it has proved difficult to test the impact of mutations on ATPase function. Some mutations occur in highly conserved sequence motifs, suggesting that their effect on function can be tested with a homologous enzyme. Here, we used the E. hirae CopB ATPase to investigate the impact of such mutations on enzyme function in vivo and in vitro. The Menkes disease mutation of Cys-1000-->Arg, changing the conserved Cys-Pro-Cys ('CPC') motif, was mimicked in CopB. The corresponding Cys-396-->Ser CopB ATPase was unable to restore copper resistance in a CopB knock-out mutant in vivo. The purified mutant ATPase still formed an acylphosphate intermediate, but possessed no detectable ATP hydrolytic activity. The most frequent Wilson disease mutation, His-1069-->Gln, was introduced into CopB as His-480-->Gln (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purified H480Q CopB formed an acylphosphate intermediate and retained a small, but significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are suggested for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively.

Human and murine CD4 T cell reactivity to a complex antigen: recognition of the synthetic random polypeptide glatiramer acetate.

The capacity of glatiramer acetate (GA), a random copolymer of alanine, lysine, glutamic acid, and tyrosine to stimulate primary in vitro human and murine T cell proliferation was examined. PBMCs isolated from healthy humans and relapsing remitting multiple sclerosis patients and spleen cells from inbred strains of mice, expressing different H-2 haplotypes, were used as sources of non-GA-primed lymphocytes. GA functioned as a universal Ag, inducing dose-dependent proliferation of all non-GA-primed human and murine T cell populations tested. Moreover, GA stimulated PBMCs derived ex vivo from human cord blood, strongly suggesting that GA can activate both naive and memory T cells. The human T cell proliferative responses to GA were HLA class II DR-restricted by virtue of the ability of anti-class II Ab to inhibit T cell proliferation, and the demonstration that individual GA specific human T cell clones were HLA class II DR-restricted by either restriction element but not both. Furthermore, GA-reactive T cells secreted Th0 cytokines and expressed a diverse repertoire of TCR. Limiting dilution analysis indicated that the T cell precursor frequency among the healthy human adults tested ranged from 1:5,000 to 1:125,000. Given that all of the T cell populations tested were isolated from non-GA-primed donors, it appears that virtually all humans and murine strains contain significant numbers of T cell populations cross-reactive with GA. These findings may explain the recent clinical finding that daily s.c. administration of GA ameliorates the progression of multiple sclerosis.

Glatiramer acetate (Copaxone) induces degenerate, Th2-polarized immune responses in patients with multiple sclerosis.

We examined the effect of glatiramer acetate, a random copolymer of alanine, lysine, glutamic acid, and tyrosine, on antigen-specific T-cell responses in patients with multiple sclerosis (MS). Glatiramer acetate (Copaxone) functioned as a universal antigen, inducing proliferation, independent of any prior exposure to the polymer, in T-cell lines prepared from MS or healthy subjects. However, for most patients, daily injections of glatiramer acetate abolished this T-cell response and promoted the secretion of IL-5 and IL-13, which are characteristic of Th2 cells. The surviving glatiramer acetate-reactive T cells exhibited a greater degree of degeneracy as measured by cross-reactive responses to combinatorial peptide libraries. Thus, it appears that, in some individuals, in vivo administration of glatiramer acetate induces highly cross-reactive T cells that secrete Th2 cytokines. To our knowledge, glatiramer acetate is the first agent that suppresses human autoimmune disease and alters immune function by engaging the T-cell receptor. This compound may be useful in a variety of autoimmune disorders in which immune deviation to a Th2 type of response is desirable.