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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Antigen-Specific Suppression of Experimental Autoimmune Encephalomyelitis by a Novel Bifunctional Peptide Inhibitor

Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas (N.K., H.K., T.J.S.); and Admunex Therapeutics Inc., Sunnyvale, California (L.G., T.M.)

Received March 24, 2007; accepted May 21, 2007.

	Abstract

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The objective of this study is to evaluate the activity of a novel peptide, i.e., bifunctional peptide inhibitor (BPI), which targets the immunological synapse and inhibits autoimmune responses in an antigen-specific manner. Proteolipid protein (PLP)-BPI was designed by conjugating two peptides, an encephalitogenic epitope of proteolipid protein (PLP_139–151) and an intercellular adhesion molecule-1-binding peptide derived from _L integrin (CD11a_237–246), via a spacer peptide. The therapeutic effect of PLP-BPI was studied in experimental autoimmune encephalomyelitis (EAE) in female SJL/J mice as a model for human multiple sclerosis. Mice that received i.v. injections of PLP-BPI showed significantly lower EAE disease scores and incidence than those treated with vehicle, PLP_139–151 peptide only, CD11a_237–246 peptide only, unlinked mixture of PLP_139–151, and CD11a_237–246 peptides, or other control peptides. Multiple injections of antigenic peptide can produce anaphylactic responses; interestingly, PLP-BPI-treated animals have significantly lower anaphylactic response than do the PLP_139–151-treated group. Therefore, PLP-BPI can effectively inhibit the disease severity and incidence of EAE with a lower possibility of inducing fatal anaphylaxis. These results suggest that BPI-type molecules can be used to treat different autoimmune diseases in which antigenic epitopes have been identified.

To develop a novel method to alter immune response in an antigen-specific manner, we have designed a bifunctional peptide inhibitor (BPI) that targets the immunological synapse. The BPI is made of the following three peptide portions: 1) antigen epitope peptide and 2) ICAM-1-binding peptide, which are conjugated via 3) a spacer peptide. Our group has previously discovered the ICAM-1-binding peptide, named LABL, derived from _L integrin (CD11a_237–246) (Yusuf-Makagiansar et al., 2001a,b). We have shown that LABL binds to domain-1 of ICAM-1 and that it can block T-cell adhesion to intestinal mucosal cell monolayers (Yusuf-Makagiansar and Siahaan, 2001; Yusuf-Makagiansar et al., 2001a,b) and pancreatic islet microvascular endothelium (Huang et al., 2005). Therefore, our hypothesis is that the antigen epitope peptide and LABL from a BPI molecule bind to MHC and ICAM-1, respectively, on the surface of APC. By bridging these target molecules, BPI prevents the translocation and segregation of Signal-1 and Signal-2, thereby inhibiting the immunological synapse formation (Fig. 1). In a parallel study, we have also demonstrated that glutamic acid decarboxylase (GAD)-BPI with GAD epitope for type-1 diabetes suppresses invasive insulitis and hyperglycemia in nonobese diabetic mice (J. S. Murray, S. Oney, J. E. Page, A. Kratochvil, Y. Hu, I. T. Makaglansar, J. C. Brown, N. Kobayashi, and T. J. Slahaan, submitted for publication).

View larger version (55K): [in this window] [in a new window]   Fig. 1. The hypothesis of BPI action in inhibiting immunological synapse formation. In the initial stages of T-cell activation, LFA-1/ICAM-1 and TCR/MHC-antigen complexes are formed at the center and around the central zone, respectively, at the T-cell-APC interface (a), and both pairs are translocated to form complete immunological synapse (b). In the presence of BPI, BPI binds to MHC-II and ICAM-1 on the surface of APC, and it prevents their migration (i.e., translocation and segregation) (c), which leads to inhibition of the immunological synapse formation (d).

	Materials and Methods

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Mice. SJL/J (H-2^s) female mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and they were housed under specific pathogen-free conditions at the Association for Assessment and Accreditation of Laboratory Animal Care-approved facility in the University of Kansas. All protocols involving live mice were approved by the university's Institutional Animal Care and Use Committee.

Peptide Synthesis. Peptides used in the present study are listed in Table 1. The peptides were synthesized with 9-fluorenylmethyloxycarbonyl-protected amino acid chemistry on appropriate polyethylene glycol-polystyrene resins (Applied Biosystems, Foster City, CA) using the automated peptide synthesis system (Pioneer; Perceptive Biosystems, Framingham, MA). Cleavage of the peptides from the resin and removal of the protecting groups from the side chain were carried out using trifluoroacetic acid with scavengers. The crude peptides were purified by reversed-phase high-performance liquid chromatography using a C18 column with a gradient of solvent A [95%/5%, H₂O (0.1% trifluoroacetic acid)/acetonitrile] and solvent B (100% acetonitrile). The purity of the peptide was analyzed by analytical high-performance liquid chromatography using an analytical C18 column. The identity of the synthesized peptide was confirmed by matrix-assisted laser desorption ionization/time of flight mass spectrometry.

Induction of EAE and Therapeutic Study. Five- to 7-week-old SJL/J female mice were immunized s.c. with 200 µgofPLP_139–151 in a 0.2-ml emulsion composed of equal volumes of phosphate-buffered saline (PBS) and complete Freund's adjuvant (CFA) containing killed Mycobacterium tuberculosis strain H37RA (at final concentration of 4 mg/ml; Difco, Detroit, MI). The PLP_139–151/CFA was administered to regions above the shoulder and the flanks (total of four sites; 50 µl at each injection site). In addition, 200 ng of pertussis toxin (List Biological Laboratories Inc., Campbell, CA) was injected i.p. on the day of immunization (day 0) and 2 days postimmunization. The mice received i.v. injections of either vehicle (PBS), PLP-BPI, or various control peptides (100 nmol/mouse, unless otherwise mentioned) on days 4, 7, 10, and 14. Disease progression was evaluated blindly by the same observer using a clinical scoring as follows: 0, no clinical signs of the disease; 1, tail weakness or limp tail; 2, paraparesis (weakness or incomplete paralysis of one or two hind limbs); 3, paraplegia (complete paralysis of two hind limbs); 4, paraplegia with forelimb weakness or paralysis; and 5, moribund (mice were euthanized once they were found to be moribund). Body weight was also measured daily.

Induction and Monitoring of Anaphylaxis. Mice received s.c. immunization with PLP_139–151/CFA on day 0, and i.p. injection of pertussis toxin on the day of immunization and 2 days postimmunization. Four to 5 weeks later, the mice were divided into groups; to avoid the effect of their disease histories, all the groups had a very similar set of mice based on the average highest disease score, the average cumulative disease score, and the average day of disease onset. Then, the mice received i.v. injections of either PLP_139–151, PLP-BPI, or unlinked mixture of PLP_139–151 and LABL (100 nmol/mouse). Incidence of anaphylactic response was judged by death occurring within 30 min or by the characteristic signs of immediate hypersensitivity, such as piloerection; prostration; erythema of the tail, ears, and footpads; shallow breathing; and lethargy, observed within a few minutes after peptide injection. Any mice that became moribund or that did not recover from anaphylactic signs were euthanized.

Characterization of T-Cell Subpopulation. SJL/J mice were immunized with PLP_139–151/CFA and pertussis toxin as described above. On days 4, 7, 10, and 14, the mice were injected i.v. with either PLP-BPI (100 nmol/mouse) or PBS. On day 15, when 50% of PBS-treated mice showed clinical signs of EAE, the mice were euthanized, and lymphocytes were isolated from the spleen by centrifugation over lymphocyte separation medium (MP Biomedicals, Solon, OH). The cells (1 x 10⁵ cells) were stimulated with mitomycin (30 µg/ml for 30 min)-treated syngeneic splenocytes (1 x 10⁶ cells) and PLP_139–151 (20 µg/ml) for 0, 48, or 72 h. The cells were then treated with 50 ng/ml ionomycin and 500 ng/ml phorbol myristate acetate for 4 h and with 10 µg/ml brefeldin A for the last 2 h. Next, the cells were stained with various anti-mouse antibodies followed by analysis with a flow cytometer (FACScan; BD Biosciences, Franklin Lakes, NJ). The antibodies used for surface staining were PerCP-Cy5.5-anti-CD4 (L3T4), fluorescein isothiocyanate-anti-CD25 (7D4), fluorescein isothiocyanate-anti-CD49b (DX5), or biotinylated anti-TGF-1 followed by PE-streptavidin, and the antibodies used for intracellular staining were PE-anti-IL-4 (BVD4–1D11), PE-anti-IL-10 (JES5–16E3), or PE-anti-IFN- (XMG1.2). The antibodies were purchased from BD Biosciences (San Jose, CA), except biotinylated anti-TGF-1, which was from R&D Systems (Minneapolis, MN).

Statistic Analysis. Statistical differences among the groups in clinical disease score on each day were analyzed by Mann-Whitney U test. Statistical differences among the groups in body weight were analyzed by one-way analysis of variance followed by Fisher's least significant difference. Statistical significance in EAE disease incidence was determined by Cox proportional-hazards regression. Comparison in T-cell subpopulations was performed with one-way analysis of variance. All analyses were performed using StatView (SAS Institute, Cary, NC).

	Results

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Suppression of EAE by PLP-BPI. In vivo therapeutic activity of PLP-BPI was evaluated in an EAE model induced in SJL/J mice. The mice showed disease signs of EAE, such as weakness or paralysis of their tail and limbs, as well as loss of body weight, which become apparent around days 8 to 12 after immunization. As shown in Fig. 2, i.v. injections of PLP-BPI inhibited the disease progression of EAE more effectively than did any other peptides tested, including PLP_139–151, LABL (CD11a_237–246), and ovalbumin (OVA)-BPI (Fig. 2). PLP-BPI-treated mice had very low EAE clinical scores throughout the study (Fig. 2A); their scores were significantly lower than those of groups treated with PBS, OVA-BPI, LABL, and PLP_{L144, R147} at the peak of the disease (days 12–17; Supplemental Table 1). The PLP-BPI-treated mice had minimum loss of body weight (Fig. 2B) compared with the other groups (days 15–19; Supplemental Table 1). In addition, 30% of the mice receiving PLP-BPI never developed EAE during the course of study; even the EAE-positive mice in this group showed delayed disease onset compared with other groups (Fig. 2C). TCR antagonist peptide PLP_{L144, R147} (Kuchroo et al., 1994), which was given on day 7 by i.p. injection (500 µg/mouse), produced the worst disease scores under this experimental condition.

View larger version (26K): [in this window] [in a new window]   Fig. 2. In vivo therapeutic study-I of PLP-BPI in mouse EAE model. SJL/J female mice were immunized s.c. with PLP139–151/CFA and injected i.p. with pertussis toxin on days 0 and 2. Then, the mice received i.v. injections of 100 nmol of the indicated peptides on days 4, 7, 10, and 14. For PLPL144, R147 treatment group, the mice received i.p. injections of 500 µg (i.e., approximately 330 nmol) PLPL144, R147 peptide on day 7 only. Disease progression was evaluated using a clinical scoring method described under Materials and Methods. A, clinical disease score. B, change in body weight. C, incidence of disease. The results are expressed as the mean + S.E. (n = 10). There are significant differences between PLP-BPI- and PBS-treated groups in clinical disease score (p < 0.05, through days 12–19), body weight (p < 0.05, through days 14–23), and disease incidence (p < 0.01). The results of other combinations of statistic comparisons are shown in Supplemental Table 1.

View larger version (26K): [in this window] [in a new window]   Fig. 3. In vivo therapeutic study-II of PLP-BPI in mouse EAE model. PLP139–151/CFA-immunized SJL/J mice were injected i.v. with 100 nmol of indicated peptide(s) on days 4, 7, 10, and 14 postimmunization. Disease progression was evaluated. A, clinical disease score. B, change in body weight. C, incidence of disease. The results are expressed as the mean + S.E. (n = 7–8). There are significant differences between PLP-BPI- and PBS-treated groups in clinical disease score (p < 0.01, through days 12–25), body weight (p < 0.01, through days 12–25), and disease incidence (p < 0.01). The results of other combinations of statistic comparisons are shown in Supplemental Table 2.

PLP-BPI Activity in Inducing Anaphylactic Reactions. One of the most important issues in treating autoimmune diseases with multiple injections of antigen-related peptides is the possibility of induction of anaphylaxis (Pedotti et al., 2001, 2003; Liu et al., 2002; Smith et al., 2005). Thus, PLP-BPI was compared with PLP_139–151 peptide in inducing anaphylactic response. In this case, PLP_139–151-immunized mice received i.v. injection of either PLP-BPI or PLP_139–151 in the late phase of the disease (i.e., on day 35 for experiment A or on day 29 for experiment B). As shown in Table 3, PLP-BPI induced lower incidence (36% in experiment A and 39% in experiment B) than did PLP_139–151 (75% in experiment A and 84.6% in experiment B). Interestingly, i.v. injection of the unlinked mixture of PLP_139–151 and LABL caused anaphylactic responses to all the mice treated. Further study needs to be done to determine whether this is a facilitated response due to the presence of LABL peptide. Taken together, these results suggest that PLP-BPI has relatively lower possibility of inducing anaphylaxis compared with the original antigenic peptide PLP_139–151. The reduced incidence of anaphylaxis in PLP-BPI is presumably due to the structure of this molecule with a linker and another peptide moiety at the C terminus.

Effect of PLP-BPI Treatment on Regulatory T-Cell Characteristics. To identify subpopulations of CD4⁺ regulatory T cells that are responsible for PLP-BPI actions, CD4⁺CD25⁺ and CD4⁺DX5⁺ regulatory T cells were characterized with their cytokine secretion phenotypes. In PLP-BPI-treated mice, CD4⁺CD25⁺TGF-⁺, CD4⁺CD25⁺IL-4⁺, and CD4⁺CD25⁺IL-10⁺ T cells were significantly increased in the lymphocyte population isolated from the spleen (Fig. 4). CD4⁺CD25⁺IFN-⁺ cells were also significantly induced by the PLP-BPI treatment. There was no such significant difference between PLP-BPI- and PBS-treated mice in CD4⁺DX5⁺ subpopulations. These results suggest that PLP-BPI suppresses EAE by inducing TGF--, IL-4-, and IL-10- as well as IFN--producing CD4⁺CD25⁺ regulatory T cells in vivo.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Characterization of T-cell subpopulation in PLP-BPI-treated mice. Lymphocytes were isolated from the spleens of immunized mice that received i.v. injections of either PLP-BPI (100 nmol/mouse) or PBS. The pooled lymphocytes in triplicate were stimulated with mitomycin-treated syngeneic splenocytes (1:10) and PLP139–151 for 0, 48, or 72 h. After activation and cytokine production, the cells were stained with various monoclonal antibodies for flow cytometry analysis. The results are expressed as the mean + S.E. Significant differences between PBS- and PLP-BPI-treated mice are shown as **, p < 0.01 and *, p < 0.05.

	Discussion

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MS is one type of autoimmune disease that is mediated by CD4⁺ T cells and that is marked by inflammation and destruction of myelin in the nervous system. The activation of a specific subpopulation of T cells involves the formation of the immunological synapse (Signal-1 and Signal-2) at the interface between T cell and APC. T-cell-mediated immune responses can be altered by modulating Signal-1 using a variety of molecules such as altered peptide ligands (APL) (Kuchroo et al., 1994; Samson and Smilek, 1995; Heijmans et al., 2005; Margot et al., 2005; Stern et al., 2005), oligomerized PLP_139–151 (Falk et al., 2000), mannosylated PLP_139–151 (Luca et al., 2005), PLP-, or myelin basic protein (MBP)-derived peptide coupled onto splenocytes (Vanderlugt et al., 2000; Smith et al., 2005), aggregated Ig-PLP_139–151 chimera (Legge et al., 2000; Yu et al., 2005), and soluble MHC-PLP_139–151 construct (Wang et al., 2006). A synthetic copolymer based on four amino acids of MBP (glatiramer acetate or copaxone) is currently being used to treat MS patients; this molecule modulates Signal-1 as APL to generate Th2 immunity (Johnson et al., 1995; Neuhaus et al., 2001). Likewise, T-cell activation can be suppressed by inhibiting Signal-2 formation with anti-CD28 Fab fragment (Perrin et al., 1999), CTLA-4-Ig fusion protein (Khoury et al., 1995), anti-CD40L antibody (Gerritse et al., 1996), anti-₄ integrin (natalizumab or Tysabri) (Langer-Gould et al., 2005), and CD28-derived peptidomimetics (Srinivasan et al., 2002). Unfortunately, these treatments suppress the general immune response, which is not antigen- or disease-specific. With natalizumab, it was temporarily withdrawn from the market because several patients treated with this drug were infected by JC virus infection that causes progressive multifocal leukoencephalopathy (Langer-Gould et al., 2005). Thus, there is a need to develop molecules that can selectively suppress a subpopulation of T cells that causes the progression of the autoimmune diseases without sacrificing the defense mechanism to pathogenic infections.

Our approach is to suppress T-cell activation by simultaneously modulating Signal-1 and Signal-2. We designed a BPI molecule to target the protein components of the immunological synapse on the surface of APC. Because the BPI molecule contains a specific antigen, it is hoped that it could alter the activation of only a subpopulation of T cells that recognize the particular antigen. As a result, it could minimize the nonspecific suppression of the entire immune system. The BPI molecule is composed of three tandem portions: antigen peptide, spacer peptide, and ICAM-1-binding peptide. In this study, PLP-BPI contains a fragment of PLP (i.e., PLP_139–151) that is important for selectivity for a specific subpopulation of T cells. The hypothesis is that PLP-BPI inhibits the immunological synapse formation during the process of T-cell activation due to 1) binding of PLP_139–151 and LABL to MHC-II (I-A^s) and ICAM-1, respectively; 2) connecting and tethering MHC-II (I-A^s) and ICAM-1 molecules on the surface of APC; and 3) preventing the translocation and segregation of these molecules (Fig. 1). In a parallel study, we have demonstrated that GAD-BPI binds to MHC-II (I-A^g7) and ICAM-1 simultaneously on the surface of isolated B cells. Furthermore, a cocapping study showed that I-A^g7 and ICAM-1 molecules are colocalized in the presence of GAD-BPI but not the unlinked mixture of GAD peptide and LABL.

In the present study, injections of PLP-BPI via the i.v. route dramatically inhibited the disease progression in the mouse EAE model (Figs. 2 and 3; Table 2). Comparison of PLP-BPI with other peptides (i.e., VP2-BPI, PLP-BPI_sLABL, and the mixture of PLP_139–151 and LABL) suggests the necessity of 1) both PLP_139–151 and LABL peptides and 2) linking these two peptides in the same molecule. Although the mechanism of how PLP-BPI works has not yet been elucidated, it is clear that the unique structure of PLP-BPI contributes to its activity. The populations of TGF--, IL-4-, and IL-10-producing CD4⁺CD25⁺ T cells were significantly increased in PLP-BPI-treated mice, suggesting that the Th2 cytokine-producing CD4⁺CD25⁺ regulatory T cells are in part responsible for the BPI action (Fig. 4). This is consistent with previous reports that indicate the involvement of Th2 cytokine-producing CD4⁺CD25⁺ regulatory T cells in resistance to and recovery from EAE (Reddy et al., 2004; Yu et al., 2005; Zhang et al., 2006). Furthermore, because there is a dramatic increase in IFN--producing CD4⁺CD25⁺ T cells, it is possible that PLP-BPI treatment altered the characteristics of cytokine producers from "strictly Th1-like" phenotype to "balanced Th1-plus Th2-like" cells. As we predicted, treatment with PLP_139–151, PLP-BPI_sLABL, or PLP_139–151 plus LABL also showed moderate activity in lowering the clinical disease scores. This could be due to the presence of the antigenic determinant PLP_139–151 in the formulation because systemic administration of soluble antigen or antigenic peptides has previously been shown to induce immune tolerance in animal models (Karin et al., 1994; Liblau et al., 1996; Bercovici et al., 1999; Wildbaum et al., 2002). It has been suggested that the induction of immune tolerance involves elicitation of regulatory T cells, peripheral deletion of CD4⁺ T cells, and TCR desensitization (Liblau et al., 1996; Bercovici et al., 1999; Wildbaum et al., 2002). Unfortunately, the TCR antagonist peptide PLP_{L144, R147} (Kuchroo et al., 1994), which we intended to use as a positive control, worsened the disease. Differences in experimental conditions such as timing of peptide injection may account for this discrepancy. It also seems that treatment with LABL peptide alone exacerbates the disease. This might be, in part, because of nonspecific inhibition of LFA-1/ICAM-1 interactions. Treatment with anti-LFA-1 monoclonal antibody augments disease severity and mortality in EAE mice (Welsh et al., 1993; Rose et al., 1999).

There are several possible mechanisms of action of PLP-BPI in suppressing the progression of EAE. First, the PLP-BPI simultaneously binds to MHC-II and ICAM-1 on the surface of APC and blocks their translocation during binding of T cells to APC. A second possible mechanism is that the PLP_139–151 fragment binds to MHC-II (Signal-1) and the linker and LABL peptide serve only as a steric hindrance for the clustering of Signal-1; thus, it inhibits the formation of the immunological synapse. The third possibility is that the LABL peptide fragment binds to ICAM-1, and the PLP_139–151 peptide fragment acts as a steric hindrance that blocks the clustering of Signal-2. Finally, a combination of these three possible mechanisms may operate for the activity of PLP-BPI in suppressing EAE. In our observations, both PLP-BPI and PLP-BPI_sLABL showed better EAE-suppressing activity than PLP_139–151 alone (Figs. 2 and 3). Furthermore, PLP-polyG was less effective in suppressing EAE than PLP-BPI_sLABL. This presumably is due to a lower steric hindrance from the polyglycine at the C terminus of PLP-polyG than the scrambled LABL fragment in PLP-BPI_sLABL (data not shown). This indicates that the steric hindrance is one of the important factors in the BPI functions. BPI may also alter the immunological synapse formation using the third mechanism; although LABL without any additional polypeptide (LABL alone) worsened the disease somewhat in some mice, LABL with nonrelated peptide (OVA-BPI) or LABL with I-A^s-binding peptide (VP2-BPI) did not change the EAE severity dramatically. Thus, steric hindrance from additional polypeptide to LABL may avoid the exacerbating effect. Nevertheless, PLP-BPI is a much better EAE suppressor than any of these peptides (i.e., OVA-BPI and VP2-BPI). These results imply that PLP-BPI action is antigen-specific, and the possible BPI side effects, which may come from nonspecific inhibition of LFA-1/ICAM-1 interaction by LABL portion, could be excluded.

One potential problem that arises when treating autoimmune diseases with multiple injections of antigen-related peptides is the possibility of inducing anaphylactic shock. Despite the fact that peptides are thought to be less antigenic and safer than whole proteins due to their relatively small size, anaphylactic reactions to several peptides have been clearly described (Pedotti et al., 2001, 2003; Liu et al., 2002; Smith et al., 2005). The development of MBP-derived APL for treatment of MS has been suspended because of hypersensitivity reactions in patients in phase II clinical trials (Bielekova et al., 2000; Kappos et al., 2000). In humans, the mechanism of anaphylactic response is considered to be antigen-induced cross-linking of IgE bound to FcRI on mast cells, which leads to mast cell degranulation and release of various inflammatory mediators, such as histamine and cytokines. In the present study, we observed that i.v. injection of PLP_139–151 at 4 to 5 weeks postimmunization induces anaphylactic reactions in the mice at a high incidence. Surprisingly, similar i.v. injections of PLP-BPI cause anaphylactic reactions to much fewer mice (Table 3). This lower incidence of anaphylaxis for PLP-BPI is probably due to covalent linking between PLP_139–151 and LABL, not simply the presence of LABL peptide, since the mixture of PLP_139–151 and LABL induced anaphylactic shock in all the treated mice. Involvement of LFA-1/ICAM-1-mediated heterotypic aggregation of activated T cells to mast cells is implicated in augmenting FcRI cross-linking, mast cell degranulation, and histamine release (Inamura et al., 1998). Thus, inhibition of LFA-1/ICAM-1 interactions by LABL peptide at the site of IgE antigen recognition might be, in part, the reason for the lower anaphylactic potential of PLP-BPI.

In conclusion, PLP-BPI effectively inhibited the disease severity and incidence of EAE. Although anaphylaxis was not completely suppressed by PLP-BPI treatment, it seems that PLP-BPI is safer than the parental antigenic peptide alone (PLP_139–151) in terms of inducing anaphylactic reactions. The detailed mechanisms of PLP-BPI actions, including the antigen-specific immunosuppressing activity and the relatively lower potential to induce anaphylaxis, are still being investigated. Further studies for improving the structure and the sequence of BPI and for optimizing the treatment dose and schedule of BPI will hopefully provide a more efficient and less toxic method of BPI-based immunotherapy. Finally, the concept of BPI molecules can be applied to treat different autoimmune diseases in which antigenic epitopes have been identified.

	Acknowledgements

 
We express our appreciation to Dr. Byron Gajewski (The University of Kansas Medical Center) for valuable suggestions in statistic analyses and to Nancy Harmony for proofreading the manuscript.

	Footnotes

 
This work is supported by National Institutes of Health Grant R01-AI-063002 and General Research Funds from The University of Kansas (to T.J.S.) and by a postdoctoral fellowship (to N.K.) from the American Heart Association, Heartland Affiliate.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.123257.

ABBREVIATIONS: MS, multiple sclerosis; APC, antigen presenting cell; TCR, T-cell receptor; MHC, major histocompatibility complex; LFA, lymphocyte function-associated antigen; ICAM, intercellular adhesion molecule; BPI, bifunctional peptide inhibitor; GAD, glutamic acid decarboxylase; PLP, proteolipid protein; EAE, experimental autoimmune encephalomyelitis; PBS, phosphate-buffered saline; CFA, complete Freund's adjuvant; TGF, transforming growth factor; PE, phycoerythrin; IFN, interferon; OVA, ovalbumin; IL, interleukin; APL, altered peptide ligand; MBP, myelin basic protein; Th, T-helper.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Teruna J. Siahaan, Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave, Lawrence, KS 66047-3729. E-mail: siahaan{at}ku.edu

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