Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) can be induced in SJL/J mice by immunization with spinal cord homogenate and adjuvant. The specific Ag(s) responsible for acute disease and subsequent relapses in this model is unknown. Myelin basic protein (BP), an encephalitogenic peptide of BP (BP 87-99), and proteolipid protein (PLP) can each induce R-EAE in SJL/J mice, and a peptide of PLP (PLP 139-151) has been reported to induce acute EAE. To determine the encephalitogens in cord-immunized mice with R-EAE, the in vitro proliferative responses of lymph node cells (LNC) and central nervous system mononuclear cells to BP, BP peptides, and PLP peptides were examined during acute EAE and during relapses. LNC responded only to PLP peptides 139-151 and 141-151 and did not respond to BP or its peptides during acute or chronic disease. Central nervous system mononuclear cells also preferentially responded to PLP 139-151 and 141-151 during acute and relapsing disease. A PLP 139-151 peptide-specific Th cell line was selected from LNC of cord-immunized donors. Five million peptide-specific line cells transferred severe relapsing demyelinating EAE to naive recipients. We conclude that PLP peptide 139-151 is the major encephalitogen for R-EAE in cord-immunized SJL/J mice. We demonstrate for the first time that Th cells specific for this peptide are sufficient to transfer relapsing demyelinating EAE. The predominance of a PLP immune response rather than a BP response in SJL/J mice suggests that genetic background may determine the predominant myelin Ag response in human demyelinating diseases such as multiple sclerosis.     

I-domain-antigen conjugate (IDAC) for delivering antigenic peptides to APC: synthesis, characterization, and in vivo EAE suppression

The objectives of this work are to characterize the identity of I-domain-antigen conjugate (IDAC) and to evaluate the in vivo efficacy of IDAC in suppressing experimental autoimmune encephalomyelitis (EAE) in mouse model. The hypothesis is that the I-domain delivers PLP(139-151) peptides to antigen-presenting cells (APC) and alters the immune system by simultaneously binding to ICAM-1 and MHC-II, blocking immunological synapse formation. IDAC was synthesized by derivatizing the lysine residues with maleimide groups followed by conjugation with PLP-Cys-OH peptide. Conjugation with PLP peptide does not alter the secondary structure of the protein as determined by CD. IDAC suppresses the progression of EAE, while I-domain and GMB-I-domain could only delay the onset of EAE. As a positive control, Ac-PLP-BPI-NH(2)-2 can effectively suppress the progress of EAE. The number of conjugation sites and the sites of conjugations in IDAC were determined using tryptic digest followed by LC-MS analysis. In conclusion, conjugation of I-domain with an antigenic peptide (PLP) resulted in an active molecule to suppress EAE in vivo.     

Amelioration of established experimental autoimmune encephalomyelitis by an MHC anchor-substituted variant of proteolipid protein 139-151

Murine experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated autoimmune disorder directed against myelin proteins within the CNS. We propose that variant peptides containing amino acid substitutions at MHC anchor residues will provide a unique means to controlling the polyclonal autoimmune T cell response. In this study, we have identified an MHC variant of proteolipid protein (PLP) 139-151 (145D) that renders PLP(139-151)-specific T cell lines anergic in vitro, as defined by a significant reduction in proliferation and IL-2 production following challenge with wild-type peptide. In vivo administration of 145D before challenge with PLP(139-151) results in a significant reduction in disease severity and incidence. Importantly, we demonstrate the ability of an MHC variant peptide to ameliorate established EAE. An advantage to this treatment is that the MHC variant peptide does not induce an acute hypersensitivity reaction. This is in contrast to previous work in the PLP(139-151) model demonstrating that anaphylactic shock resulting in death occurs upon rechallenge with the encephalitogenic peptide. Taken together, these data demonstrate the effectiveness of MHC anchor-substituted peptides in the treatment of EAE and suggest their utility in the treatment of other autoimmune disorders.     

The immunopathology of chronic experimental allergic encephalomyelitis induced in rabbits with bovine proteolipid protein

The role of myelin proteolipid apoprotein (PLP) in the central nervous system (CNS) immune response of rabbits has been investigated by analyzing the immunopathology of chronic experimental allergic encephalomyelitis (EAE) induced by sensitization with PLP. Clinical disease occurred in seven out of nine rabbits sensitized with bovine PLP and monitored for up to 6 mo. Positive delayed hypersensitivity skin test reactions to PLP occurred in all but one of the PLP-sensitized animals. All PLP-sensitized animals had meningeal and CNS parenchymal inflammation that correlated with disease severity. Serial blood samples were stained with a panel of antibodies to rabbit T and B cells, as well as Ia, and large and small mononuclear cell populations were analyzed by flow cytometry. Peripheral leukocyte population staining did not correlate with clinical signs or sensitization to PLP. Cryostat CNS tissue sections were stained with the same set of antibodies by using an immunoperoxidase technique, and positive cells and vessels were counted. T cells and macrophages were numerous and in equal numbers in perivascular parenchymal inflammatory infiltrates, whereas B cells were less numerous (p less than 0.001). T cells also diffusely infiltrated the parenchyma. Most perivascular inflammatory cells and many scattered parenchymal cells were Ia+; Ia vascular expression was increased over controls (p less than 0.001), and also correlated with disease severity. The immunopathology of this chronic EAE model is the same as that of whole CNS tissue- and myelin basic protein-induced EAE in other species, and is similar to that of multiple sclerosis. Cellular immune responses to PLP may therefore contribute to systemic and in situ responses in CNS tissue demyelinating diseases.     

Peptide determinants of myelin proteolipid protein (PLP) in autoimmune demyelinating disease: A review

This article reviews recent advances in understanding the role of myelin proteolipid protein (PLP) in autoimmune demyelination. It is drawn largely from work published within the last years and discusses the immunology of PLP in the historical context of what has been learned from extensive studies on the immune response to myelin basic protein (MBP). Despite the, fact that PLP is the major protein constituent of mammalian myelin, its role in autoimmune demyelination has not been widely recognized. The lack of understanding about the immunology of PLP is a direct result of the biochemical characteristics of the protein. PLP is a highly hydrophobic membrane protein with limited aqueous solubility. The hydrophobicity of PLP has thwarted, immunologic studies of the intact protein. Recent work has circumvented the technical obstacles of studying the intact protein by using soluble synthetic PLP peptides. This approach has rapidly resulted in a more definitive understanding of the immune response to PLP. Presently, the data indicate that:i) PLP is a major central nervous system (CNS) specific encephalitogen;ii) CD4+T cell reactivity to discrete PLP peptide determinants can mediate the development of acute chronic relapsing, and chronic progressive experimental autoimmune encephalomyelitis (EAE); andiii) T cell reactivity to multiple PLP determinants occurs in patients with multiple sclerosis (MS), the major human CNS demyleinating disease.Special Issue dedicated to Dr. Majorie B. Lees.     

Fine specificity of CD4+ T cell responses to the dominant encephalitogenic PLP 139-151 peptide in SJL/J mice

PLP 139-151(S) is the major encephalitogenic epitope of PLP in the SJL/J mouse. CD4⁺ T cells specific for PLP 139-151(S) induce a relapsing-remitting form of EAE which is similar to the human demyelinating disease MS in both clinical course and histopathology. We are interested in events involved in activation of autoreactive T cells and how to specifically regulate these immune response to both prevent and treat ongoing demyelinating disease. In the current study, we examined the effect of both amino acid substitutions and deletions in the native PLP 139-151(S) peptide to identify which residues are critical for immunogenicity and encephalitogenicity. Conservative and nonconservative substitutions at position 145 diminished or completely destroyed the encephalitogenic potential of the peptide without affecting the ability to recall a proliferative response in lymph node T cells primed with the native PLP 139-151(S) peptide indicating an interesting dichotomy between ability to induce T cell proliferation and ability to induce active clinical disease. In addition, tryptophan at position 144 was identified as a critical TCR contact site as a peptide containing an alanine for tryptophan at this position (A144) primed a unique population of T cells which did not cross react with the native PLP 139-151(S). In addition, A144 was unable to stimulate PLP 139-151(S)-specific T cells in vitro or to induce active relapsing EAE in vivo. The significance of these results to the potential development of new strategies for preventing and treating T cell-mediated autoimmune diseases is discussed.Special issue dedicated to Dr. Majorie B. Lees.     

Monomeric recombinant TCR ligand reduces relapse rate and severity of experimental autoimmune encephalomyelitis in SJL/J mice through cytokine switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-A(s)/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 x SJL)F(1) mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 x SJL/J)F(1) mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-beta3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.     

Synthetic Peptide dendrimers block the development and expression of experimental allergic encephalomyelitis

Multiple Ag peptides (MAPs) containing eight proteolipid protein (PLP)(139-151) peptides arranged around a dendrimeric branched lysine core were used to influence the expression and development of relapsing experimental allergic encephalomyelitis (EAE) in SJL mice. The PLP(139-151) MAPs were very efficient agents in preventing the development of clinical disease when administered after immunization with the PLP(139-151) monomeric encephalitogenic peptide in CFA. The treatment effect with these MAPs was peptide specific; irrelevant multimeric peptides such as guinea pig myelin basic protein GPBP(72-84) MAP (a dendrimeric octamer composed of the 72-84 peptide) and PLP(178-191) MAP (a dendrimeric octamer composed of the PLP(178-191) peptide) had no treatment effect on PLP(139-151)-induced EAE. PLP(139-151) MAP treatment initiated after clinical signs of paralysis also altered the subsequent course of EAE; it limited developing signs of paralysis and effectively limited the severity and number of disease relapses in MAP-treated mice over a 60-day observation period. PLP(139-151) MAP therapy initiated before disease onset acts to limit the numbers of Th17 and IFN-gamma-producing cells that enter into the CNS. However, Foxp3(+) cells entered the CNS in numbers equivalent for nontreated and PLP(139-151) MAP-treated animals. The net effect of PLP(139-151) MAP treatment dramatically increases the ratio of Foxp3(+) cells to Th17 and IFN-gamma-producing cells in the CNS of PLP(139-151) MAP-treated animals.     

Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice

PLP is the major protein constituent of central nervous system myelin. We have previously shown that SJL/J (H-2s) mice develop an acute form of EAE after immunization with PLP. The purpose of the present study was to identify an encephalitogenic determinant of PLP for SJL mice. We immunized SJL/J mice with a synthetic peptide identical to residues 130-147 QAHSLERVCHCLGKWLGH of murine PLP, a sequence having an amphipathic alpha-helical conformation. Although it did not induce disease, an overlapping peptide containing residues 139-154 HCLGKWLGHPDKFVGI was encephalitogenic. Immunization with this peptide induced severe clinical and histologic EAE in 3 of 20 mice. T cell enriched ILN cells from these mice responded specifically (3H-thymidine incorporation) to this peptide as well as to shorter analogues of this domain containing serine in place of cysteine at residues 138 and 140. Immunization with the serine-substituted PLP peptides 137-151 VSHSLGKWLGHPDKF and 139-151 HSLGKWLGHPDKF induced severe, acute EAE in 4 of 9 and 15 of 15 SJL mice, respectively, and their T cell enriched ILN cells responded not only to the analogues, but also to the native PLP sequence 139-154. These results indicate that residues 139-151 of murine PLP is an encephalitogenic determinant for SJL mice. Furthermore, like the PLP encephalitogenic domain for SWR (H-2q) mice, this determinant is also a T cell epitope with a coding sequence at the end of an exon.     

CD8+ T cells specific for cryptic apoptosis-associated epitopes exacerbate experimental autoimmune encephalomyelitis

The autoimmune immunopathology occurring in multiple sclerosis (MS) is sustained by myelin-specific and -nonspecific CD8⁺ T cells. We have previously shown that, in MS, activated T cells undergoing apoptosis induce a CD8⁺ T cell response directed against antigens that are unveiled during the apoptotic process, namely caspase-cleaved structural proteins such as non-muscle myosin and vimentin. Here, we have explored in vivo the development and the function of the immune responses to cryptic apoptosis-associated epitopes (AEs) in a well-established mouse model of MS, experimental autoimmune encephalomyelitis (EAE), through a combination of immunization approaches, multiparametric flow cytometry, and functional assays. First, we confirmed that this model recapitulated the main findings observed in MS patients, namely that apoptotic T cells and effector/memory AE-specific CD8⁺ T cells accumulate in the central nervous system of mice with EAE, positively correlating with disease severity. Interestingly, we found that AE-specific CD8⁺ T cells were present also in the lymphoid organs of unprimed mice, proliferated under peptide stimulation in vitro, but failed to respond to peptide immunization in vivo, suggesting a physiological control of this response. However, when mice were immunized with AEs along with EAE induction, AE-specific CD8⁺ T cells with an effector/memory phenotype accumulated in the central nervous system, and the disease severity was exacerbated. In conclusion, we demonstrate that AE-specific autoimmunity may contribute to immunopathology in neuroinflammation.Subject terms: Cell death and immune response, Immunological disorders     

Location of a new encephalitogenic epitope (residues 43 to 64) in proteolipid protein that induces relapsing experimental autoimmune encephalomyelitis in PL/J and (SJL x PL)F1 mice.

Synthetic peptides of proteolipid protein (PLP) were screened for their ability to induce experimental autoimmune encephalomyelitis (EAE) in SJL/J, PL/J, and (SJL x PL)F1 mice, and T cell lines were selected by stimulation of lymph node cells with PLP peptides. PLP 141-151 was found to be less encephalitogenic in SJL/J mice than PLP 139-151, due to deletion of two amino acids from the amino-terminal end. PLP 139-151 immunization induced relapsing EAE in SJL/J and F1 mice but not PL/J mice. In contrast, PLP 43-64 induced relapsing EAE in PL/J and F1 mice but not SJL/J mice. F1 T cell lines specific for either PLP 43-64 or PLP 139-151 adoptively transferred demyelinating EAE to naive F1 recipients. Haplotypes H-2s and H-2u appear to be immunologically co-dominant in F1 mice in the PLP EAE system, which differs from the H-2u dominance in F1 mice in the myelin basic protein EAE system. The identification of a PLP peptide that is encephalitogenic in PL/J mice, in addition to the previous demonstration of PLP peptides that are encephalitogenic for SWR mice (PLP 103-116) and SJL/J mice (PLP 139-151), lends support to a role for PLP as a target Ag in autoimmune demyelinating diseases.     

Competitive ELISA for the identification of 35–55 myelin oligodendrocyte glycoprotein immunodominant epitope conjugated with mannan

Analogs of immunodominant myelin peptides involved in multiple sclerosis (MS: the most common autoimmune disease) have been extensively used to modify the immune response over the progression of the disease. The immunodominant 35–55 epitope of myelin oligodendrocyte glycoprotein (MOG_35–55) is an autoantigen appearing in MS and stimulates the encephalitogenic T cells, whereas mannan polysaccharide (Saccharomyces cerevisiae) is a carrier toward the mannose receptor of dendritic cells and macrophages. The conjugate of mannan-MOG_35–55 has been extensively studied for the inhibition of chronic experimental autoimmune encephalomyelitis (EAE: an animal model of MS) by inducing antigen-specific immune tolerance against the clinical symptoms of EAE in mice. Moreover, it presents a promising approach for the immunotherapy of MS under clinical investigation. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the MOG_35–55 peptide that is conjugated to mannan. Intra- and inter-day assay experiments proved that the proposed ELISA methodology is accurate and reliable and could be used in the following applications: (i) to identify the peptide (antigen) while it is conjugated to mannan and (ii) to adequately address the alterations that the MOG_35–55 peptide may undergo when it is bound to mannan during production and stability studies.     

Minireview: Autoimmune responses to myelin proteolipid protein

The authors present a brief historical sketch of the development of our understanding of immune responses to myelin proteolipid protein (PLP) and the acceptance of PLP as a potent antigen in the induction of experimental allergic encephalomyelitis (EAE). The distinct characteristics of the PLP molecule that may contribute to complex immune responses to this protein are reviewed and these responses are compared with those to MBP, both in the pathology of EAE and at the level of the T cell. Recent evidence demonstrating differences between T cell responses to PLP and MBP is reviewed. Finally, the potential contribution of immune responses to PLP in human diseases, particularly mutiple sclerosis (MS), that have been identified to date are then summarized.For the authors to write a review on PLP and its role in EAE without Marjorie is like their sailing a ship without a captain, compass or rudder. This review is largely based on work and ideas generated in Marjorie's laboratory, but it was prepared without her input. Consequently, it lacks her meticulous reflection on the structure of each of its sentences and on the use of each word. Papers written with Marjorie are usually honed to near perfection late into the evening at her kitchen table in Newton, where food, ideas, and warmth abound, and where her very patient and accommodating husband Sidney and a demanding but lovable canine are close at hand. Writing this essay gave the authors a chance to recognize our scientific forebears, to consider where we are at this point and to contemplate our future directions in studying immune responses to PLP. We are, indeed, very grateful and indebted to Marjorie for her generous personal and scientific support, wise guidance, inspiration, strength, energy and, most importantly, friendship. Marjorie, we thank you, you are our role model, and we affectionately anticipate many more years of continued collaboration with you.Abbreviations used in this paper CNS central nervous system - EAE experimental allergic encephalomyelitis - MBP myelin basic protein - MHC major histocompatibility complex - MOG myelin oligodendrocyte cyte glycoprotein - MS multiple sclerosis - PLP myelin proteolipid protein - PNS peripheral nervous system - TcR T cell receptor Special issue dedicated to Dr. Marjorie B. Lees.     

Human amniotic epithelial cells suppress relapse of corticosteroid-remitted experimental autoimmune disease

Background aimsMultiple sclerosis (MS) is considered to be a T-cell–mediated disease. Although MS remits with corticosteroid treatment, the disease relapses on discontinuation of therapy. Human amniotic epithelial cells (hAEC) from the placenta are readily accessible in large quantities and have anti-inflammatory properties. Previously we reported that hAEC given near disease onset ameliorated clinical signs and decreased myelin oligodendrocyte glycoprotein (MOG)-specific immune responses in MOG-induced experimental autoimmune encephalomyelitis (EAE), an experimental MS model.MethodsTo examine the therapeutic effect of hAEC in a clinically relevant setting, we first treated MOG peptide–induced EAE mice with a corticosteroid, prednisolone, in drinking water to induce remission. hAEC were then infused intravenously into the remitted mice. Anti-MOG antibodies in serum were detected by enzyme-linked immunoassay. Splenocyte proliferation was assessed by ³H-thymidine incorporation. Immune cell subpopulations in spleens and lymph nodes and secreted cytokines in splenocyte culture were quantified by flow cytometry. Central nervous system histology was examined with the use of hematoxylin and eosin, Luxol fast blue and immunostaining.ResultsWith cessation of prednisolone treatment, hAEC delayed EAE relapse for 7 days, and, after another 7 days, largely remitted disease in six of eight responder mice. Splenocyte proliferation was suppressed, anti-MOG_35–55 antibodies in serum were decreased and interleukin-2 and interleukin-5 production by splenocytes were elevated after hAEC treatment. In the central nervous system, hAEC-treated mice had decreased demyelination and fewer macrophages in the inflammatory infiltrates. hAEC treatment also increased CD4⁺CD25⁺FoxP3⁺ regulatory T cells in inguinal lymph nodes.ConclusionsThese data demonstrate that the therapeutic effects of hAEC after corticosteroid treatment in an MS model probably are the consequence of peripheral immunoregulation. We suggest that hAEC may have potential as a cell therapy for remitted MS.     

Oligodendrocyte-specific protein peptides induce experimental autoimmune encephalomyelitis in SJL/J mice.

Oligodendrocyte-specific protein (OSP) is a recently isolated and cloned, 207-aa, hydrophobic, four-transmembrane protein found in CNS myelin. It represents approximately 7% of total myelin protein. The OSP cDNA sequence has no significant homology with previously reported genes, but the predicted protein structure suggests that OSP is a CNS homologue of peripheral myelin protein-22. We previously reported the presence of anti-OSP Abs in the cerebrospinal fluid of relapsing-remitting multiple sclerosis (MS) patients, but not control patient groups. In this study, we tested the ability of a panel of 20-mer peptides with 10-aa overlaps, representing the sequence of murine OSP, to induce experimental autoimmune encephalomyelitis (EAE), an animal model for MS. SJL mice challenged with murine OSP peptides 52-71, 82-101, 102-121, 142-161, 182-201, and 192-207 exhibited clinical EAE. OSP:52-71 elicited severe relapsing-remitting EAE in some individuals. All other encephalitogenic peptides elicited, at most, a loss of tail tonicity from which the mice most often completely recovered. Mononuclear cell infiltrates and focal demyelination characteristic of EAE were evident. T cell proliferative responses were seen with all encephalitogenic peptides except 142-161 and 182-201. OSP peptides 72-91 and 132-151 did not cause clinical EAE, but did elicit robust proliferative responses. B10.PL and PL/J mice challenged with the same OSP peptide doses as SJL mice did not exhibit clinical EAE. These results in the SJL EAE model, together with the results from MS patient clinical samples, make OSP a promising candidate for autoantigenic involvement in MS.     

Parental MHC molecule haplotype expression in (SJL/J x SWR)F1 mice with acute experimental allergic encephalomyelitis induced with two different synthetic peptides of myelin proteolipid protein.

To determine if the Ag that induces an autoimmune disease influences parental MHC haplotype molecule expression in situ in MHC heterozygotes, acute experimental allergic encephalomyelitis (EAE) was induced with different encephalitogenic peptides in (SJL/J x SWR)F1 mice. The mice were sensitized with either a synthetic peptide corresponding to mouse myelin proteolipid protein (PLP) residues 103-116 YKTTICGKGLSATV which induces EAE in SWR (H-2q), but not SJL/J (H-2s) mice or a synthetic peptide corresponding to PLP residues 139-151 HCLGKWLGHPDKF which is encephalitogenic in SJL/J but not SWR mice. Mice were killed when they were moribund or at 30 days after sensitization. Twelve of 18 F1 mice given PLP peptide 103-116 and 12 of 17 mice given PLP peptide 139-151 developed EAE within 2 to 3 wk after sensitization. Cryostat sections of brain samples from F1 and parental mice were immunostained with a panel of mAb identifying H-2s and H-2q class I and II MHC molecules. In brains of controls, class I MHC molecules were expressed on choroid plexus, endothelial cells, and microglia whereas class II MHC molecules were absent. In EAE lesions, class I and II MHC molecules were present on inflammatory and parenchymal cells, but the degree of parental haplotype molecule expression did not vary with the different peptide Ag tested. Thus, in (SJL/J x SWR)F1 mice, myelin PLP peptides 103-116 and 139-151 are co-dominant Ag with respect to clinical and histologic disease and parental haplotype MHC molecule expression. We propose a unifying hypothesis consistent with these results and previous observations of differential Ia expression in (responder x non-responder)F1 guinea pigs. We suggest that MHC molecules may bind locally derived peptide Ag in inflammatory sites and that these interactions influence levels of MHC haplotype molecules on APC.     

Synthesis of palmitoyl-thioester T-cell epitopes of myelin proteolipid protein (PLP). Comparison of two thiol protecting groups (StBu and Mmt) for on-resin acylation.

In order to test the effect of thiopalmitoylation on the encephalitogenic properties of two proteolipid protein (PLP) T-cell epitopes, we have studied the on-resin S-palmitoylation of peptides, synthesized using the Fmoc/tBu strategy. The use of two Cys protecting groups was investigated: the tert-butylsulfenyl (StBu) and the methoxytrityl (Mmt). Our studies show that the ease of deprotection of the thiol protected with StBu was sequence dependent. The deprotection of Cys(StBu) was difficult in the case of the two peptides PLP(104-117) and PLP(139-151). Neither of the two Cys(StBu) (Cys108 and Cys140, respectively) could be deprotected with tributylphosphine. Beta-mercaptoethanol was only efficient for the deprotection of Cys(StBu)140 at 85 degrees C and at 135 degrees C for Cys108. The two palmitoylated peptides could be obtained in good yield starting from Cys protected with Mmt. Our conclusion is that the Mmt group is the more versatile protecting group of the thiol for use in the on-resin synthesis of thiopalmitoylated peptides.     

IL-10 plays an important role in the homeostatic regulation of the autoreactive repertoire in naive mice

We have previously shown that naive SJL (H-2(s)) mice, which are highly susceptible to myelin proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), have a very high frequency (1/20,000 CD4 T cells) of PLP(139-151)-reactive T cells in the naive repertoire. In this study, we examine the function of this endogenous PLP(139-151)-reactive repertoire in vivo and find that this repertoire encompasses the precursors of pathogenic T cells. Because SJL mice do not develop spontaneous EAE, we have explored the mechanisms that keep this autopathogenic repertoire in check and prevent the development of spontaneous autoimmunity. We crossed IL-4 and IL-10 deficiency onto the SJL background and analyzed the roles of these two immunoregulatory cytokines in regulating the size and effector function of the endogenous PLP(139-151)-reactive repertoire and development of autoimmune disease. We find that IL-10 is important in the homeostatic regulation of the endogenous PLP(139-151)-reactive repertoire in that it both limits the size of the repertoire and prevents development of effector autoaggressive T cells. SJL IL-10(-/-) mice with high numbers of PLP(139-151)-specific precursors in the repertoire did not develop spontaneous EAE, but when they were injected with pertussis toxin, they showed atypical clinical signs of EAE with small numbers of typical mononuclear cell infiltrates predominantly in the meninges. EAE could be inhibited by prior tolerization of the mice with soluble PLP(139-151) peptide. These findings indicate that IL-10 may contribute to the regulation of the endogenous autoimmune repertoire.     

Ceramide synthase 6 plays a critical role in the development of experimental autoimmune encephalomyelitis

Ceramides are mediators of apoptosis and inflammatory processes. In an animal model of multiple sclerosis (MS), the experimental autoimmune encephalomyelitis (EAE) model, we observed a significant elevation of C(16:0)-Cer in the lumbar spinal cord of EAE mice. This was caused by a transiently increased expression of ceramide synthase (CerS) 6 in monocytes/macrophages and astroglia. Notably, this corresponds to the clinical finding that C(16:0)-Cer levels were increased 1.9-fold in cerebrospinal fluid of MS patients. NO and TNF-α secreted by IFN-γ-activated macrophages play an essential role in the development of MS. In murine peritoneal and mouse-derived RAW 264.7 macrophages, IFN-γ-mediated expression of inducible NO synthase (iNOS)/TNF-α and NO/TNF-α release depends on upregulation of CerS6/C(16:0)-Cer. Downregulation of CerS6 by RNA interference or endogenous upregulation of C(16:0)-Cer mediated by palmitic acid in RAW 264.7 macrophages led to a significant reduction or increase in NO/TNF-α release, respectively. EAE/IFN-γ knockout mice showed a significant delay in disease onset accompanied by a significantly less pronounced increase in CerS6/C(16:0)-Cer, iNOS, and TNF-α compared with EAE/wild-type mice. Treatment of EAE mice with l-cycloserine prevented the increase in C(16:0)-Cer and iNOS/TNF-α expression and caused a remission of the disease. In conclusion, CerS6 plays a critical role in the onset of MS, most likely by regulating NO and TNF-α synthesis. CerS6 may represent a new target for the inhibition of inflammatory processes promoting MS development.     

Production of stable isotope enriched antimicrobial peptides in Escherichia coli: An application to the production of a 15N-enriched fragment of lactoferrin

A method is described for the production of recombinant isotopically enriched peptides in E. coli. Peptides are produced in high yield as fusion proteins with ketosteroid isomerase which form insoluble inclusion bodies. This insoluble form allows easy purification, stabilizes the peptide against degradation and prevents bactericidal activity of the peptide. Cyanogen bromide cleavage released peptide which was conjugated with alkylamines to form lipopeptide. An important advantage of this system is that it allows production of peptides that are toxic to bacteria, which we have demonstrated on a dodecapeptide based on residues 21–31 of human bactericidal protein lactoferrin.