Pulmonary Administration of Soluble Antigen Arrays Is Superior to Antigen in Treatment of Experimental Autoimmune Encephalomyelitis.

Antigen-specific immunotherapy has been used to hyposensitize patients to allergens and offers an enticing approach for attenuating autoimmune diseases. Applying antigen-specific immunotherapy to mucosal surfaces such as the lungs may engage unique immune response pathways to improve efficacy. Pulmonary delivery of soluble antigen arrays (SAgAs) was explored in mice with experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model. SAgAs were designed to impede immune response to autoantigen epitopes and are composed of a hyaluronan backbone with peptides PLP139-151 (proteolipid protein) and LABL, a disease-causing proteolipid peptide epitope and an intracellular cell-adhesion molecule-1 ligand, respectively. Pulmonary instillation of SAgAs decreased disease score, improved weight gain, and decreased incidence of disease in EAE mice compared to pulmonary delivery of hyaluronic acid polymer, LABL, or PLP. Interestingly, treating with PLP alone also showed some improvement. Splenocytes from SAgA-treated animals showed increased interferon-gamma levels, and interleukin-6 (IL-6) and IL-17 were elevated in SAgA-treated animals compared to PLP treatments. IL-10, IL-2, and tumor necrosis factor-alpha levels showed no significant difference, yet trends across all cytokines suggested SAgAs induced a very different immune response compared to treatment with PLP alone. This work suggests that codelivery of peptide components is essential when treating EAE via pulmonary instillation, and the immune response may have shifted toward immune tolerance.

IFN-ß Facilitates Neuroantigen-Dependent Induction of CD25+ FOXP3+ Regulatory T Cells That Suppress Experimental Autoimmune Encephalomyelitis.

This study introduces a flexible format for tolerogenic vaccination that incorporates IFN-ß and neuroantigen (NAg) in the Alum adjuvant. Tolerogenic vaccination required all three components, IFN-ß, NAg, and Alum, for inhibition of experimental autoimmune encephalomyelitis (EAE) and induction of tolerance. Vaccination with IFN-ß + NAg in Alum ameliorated NAg-specific sensitization and inhibited EAE in C57BL/6 mice in pretreatment and therapeutic regimens. Tolerance induction was specific for the tolerogenic vaccine Ag PLP178-191 or myelin oligodendrocyte glycoprotein (MOG)35-55 in proteolipid protein- and MOG-induced models of EAE, respectively, and was abrogated by pretreatment with a depleting anti-CD25 mAb. IFN-ß/Alum-based vaccination exhibited hallmarks of infectious tolerance, because IFN-ß + OVA in Alum-specific vaccination inhibited EAE elicited by OVA + MOG in CFA but not EAE elicited by MOG in CFA. IFN-ß + NAg in Alum vaccination elicited elevated numbers and percentages of FOXP3+ T cells in blood and secondary lymphoid organs in 2D2 MOG-specific transgenic mice, and repeated boosters facilitated generation of activated CD44high CD25+ regulatory T cell (Treg) populations. IFN-ß and MOG35-55 elicited suppressive FOXP3+ Tregs in vitro in the absence of Alum via a mechanism that was neutralized by anti-TGF-ß and that resulted in the induction of an effector CD69+ CTLA-4+ IFNAR+ FOXP3+ Treg subset. In vitro IFN-ß + MOG-induced Tregs inhibited EAE when transferred into actively challenged recipients. Unlike IFN-ß + NAg in Alum vaccines, vaccination with TGF-ß + MOG35-55 in Alum did not increase Treg percentages in vivo. Overall, this study indicates that IFN-ß + NAg in Alum vaccination elicits NAg-specific, suppressive CD25+ Tregs that inhibit CNS autoimmune disease. Thus, IFN-ß has the activity spectrum that drives selective responses of suppressive FOXP3+ Tregs.

Clínica y diagnóstico de la enfermedad de Pelizaeus-Merzbacher: descripción de cinco casos / Clinical features and diagnosis of Pelizaeus-Merzbacher disease: five case reports

Introducción. La enfermedad de Pelizaeus-Merzbacher es un trastorno hipomielinizante raro debido a alteraciones en el gen PLP1, que lleva a un fallo de la mielinización axonal de los oligodendrocitos en el sistema nervioso central. Existen dos formas descritas según la gravedad de su presentación: connatal y clásica. Se caracteriza por hipotonía neonatal, retraso del desarrollo psicomotor, espasticidad progresiva de predominio en los miembros inferiores y nistagmo, con signos y síntomas piramidales y extrapiramidales, y la forma connatal es mucho más grave. La resonancia magnética muestra leucoencefalopatía hipomielinizante difusa, los potenciales evocados usualmente se alteran y la confirmación se realiza mediante estudio molecular del gen PLP1. Casos clínicos. Se presentan cinco pacientes pediátricos afectados, cuatro con la forma clásica y uno con la forma connatal; se describen las características clínicas, los estudios complementarios y se realiza una revisión concisa de la bibliografía. Conclusión. Esta enfermedad tiene una evolución progresiva y casi invariable, lo cual es la clave clínica para diferenciarla de otras entidades como la parálisis cerebral infantil, neuropatías periféricas, esclerosis múltiple, entre otras, además de los hallazgos característicos en las neuroimágenes. Es necesario sospechar este diagnóstico y confirmar alteraciones en el gen PLP1 con el fin de obtener una incidencia real de esta entidad, probablemente subestimada, como otras leucodistrofias (AU)

Introduction. Pelizaeus-Merzbacher disease is an infrequent hypomyelinating disorder caused by alterations in the PLP1 gene, which leads to a fault in the axonal myelination of the oligodendrocytes in the central nervous system. Two forms have been reported, according to the severity of the presentation: connatal and classic. It is characterised by neonatal hypotonia, delayed psychomotor development, progressive spasticity predominantly in the lower limbs and nystagmus, with pyramidal and extrapyramidal signs and symptoms; the connatal form is far more severe. Magnetic resonance imaging shows diffuse hypomyelinating leukoencephalopathy, evoked potentials are usually altered and confirmation is obtained through a molecular study of the PLP1 gene. Case reports. We present the cases of five paediatric patients, four of whom had the classic form and one with the connatal form. The clinical characteristics and complementary studies are described, and a concise review of the literature is carried out. Conclusion. This disease has a progressive and almost unvarying course, which is the clinical key to be able to differentiate it from other entities such as infantile cerebral palsy, peripheral neuropathies or multiple sclerosis, among others, in addition to the characteristic neuroimaging findings. It is necessary to suspect this diagnosis and confirm alterations in the PLP1 gene with the aim of obtaining a real incidence of this entity, which is probably underestimated, like other leukodystrophies (AU)

Dendritic cells and anergic type I NKT cells play a crucial role in sulfatide-mediated immune regulation in experimental autoimmune encephalomyelitis.

CD1d-restricted NKT cells can be divided into two groups: type I NKT cells use a semi-invariant TCR, whereas type II express a relatively diverse set of TCRs. A major subset of type II NKT cells recognizes myelin-derived sulfatides and is selectively enriched in the CNS tissue during experimental autoimmune encephalomyelitis (EAE). We have shown that activation of sulfatide-reactive type II NKT cells by sulfatide prevents induction of EAE. In this article, we have addressed the mechanism of regulation, as well as whether a single immunodominant form of synthetic sulfatide can treat ongoing chronic and relapsing EAE in SJL/J mice. We have shown that the activation of sulfatide-reactive type II NKT cells leads to a significant reduction in the frequency and effector function of myelin proteolipid proteins 139-151/I-A(s)-tetramer(+) cells in lymphoid and CNS tissues. In addition, type I NKT cells and dendritic cells (DCs) in the periphery, as well as CNS-resident microglia, are inactivated after sulfatide administration, and mice deficient in type I NKT cells are not protected from disease. Moreover, tolerized DCs from sulfatide-treated animals can adoptively transfer protection into naive mice. Treatment of SJL/J mice with a synthetic cis-tetracosenoyl sulfatide, but not α-galactosylceramide, reverses ongoing chronic and relapsing EAE. Our data highlight a novel immune-regulatory pathway involving NKT subset interactions leading to inactivation of type I NKT cells, DCs, and microglial cells in suppression of autoimmunity. Because CD1 molecules are nonpolymorphic, the sulfatide-mediated immune-regulatory pathway can be targeted for development of non-HLA-dependent therapeutic approaches to T cell-mediated autoimmune diseases.

Suppression of MOG- and PLP-induced experimental autoimmune encephalomyelitis using a novel multivalent bifunctional peptide inhibitor.

Previously, bifunctional peptide inhibitors (BPI) with a single antigenic peptide have been shown to suppress experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner. In this study, a multivalent BPI (MVBMOG/PLP) with two antigenic peptides derived from myelin oligodendrocyte glycoprotein (MOG38-50) and myelin proteolipid protein (PLP139-151) was evaluated in suppressing MOG38-50- and PLP139-151-induced EAE. MVBMOG/PLP significantly suppressed both models of EAE even when there was some evidence of epitope spreading in the MOG38-50-induced EAE model. In addition, MVBMOG/PLP was found to be more effective than PLP-BPI and MOG-BPI in suppressing MOG38-50-induced EAE. Thus, the development of MVB molecules with broader antigenic targets can lead to suppression of epitope spreading in EAE.

Encephalitogenicity of complete Freund's adjuvant relative to CpG is linked to induction of Th17 cells.

For decades, CFA has been the classic adjuvant for the induction of experimental autoimmune encephalomyelitis (EAE). Its encephalitogenic activity has been originally linked to the induction of Th1 responses. CpG, which is also a potent Th1 inducer, has been suggested by some studies to be comparably encephalitogenic. In this study, using the SJL proteolipid protein (PLP) 139-151 peptide EAE model, we show that active immunizations using CFA but not CpG 1826/IFA as an adjuvant induced disease. Passive induction of EAE resulted in severe disease when cells were transferred from PLP in CFA-primed mice but resulted in only a mild, transient disease when cells originated from PLP in CpG 1826/IFA-primed mice. In accordance with these findings, immunizations using CFA but not CpG 1826/IFA as an adjuvant elicited a delayed-type hypersensitivity response. ELISPOT analysis revealed that CFA promoted the differentiation of much higher levels of PLP-specific, IL-17-secreting cells compared with CpG 1826/IFA. Both adjuvants induced comparable frequencies of PLP-specific, IFN-gamma-secreting cells and also induced Ag-specific proliferation to the same extent. The severity of EAE in PLP in CFA-immunized mice was reduced when IL-17 was neutralized in vivo, demonstrating the crucial role of this cytokine in disease induction. The data show that immunizations using the autoantigen in CpG 1826/IFA result in very low frequencies of Ag-specific IL-17 cells, suggesting a lower risk of Th17-mediated pathology when using this adjuvant.

Prophylactic and therapeutic suppression of experimental autoimmune encephalomyelitis by a novel bifunctional peptide inhibitor.

The objective was to optimize and evaluate the in vivo activities of our novel bifunctional peptide inhibitor (BPI), which alters immune response in autoimmune diseases by modulating the immunological synapse formation. Previously, we have designed PLP-BPI and GAD-BPI by conjugating myelin proteolipid protein (PLP)(139-151) and glutamic acid decarboxylase (GAD)(208-217), respectively, with CD11a(237-246) via a spacer peptide. PLP-BPI and GAD-BPI suppressed the disease progression in experimental autoimmune encephalomyelitis (EAE) and in type-1 diabetes, respectively. In this study, various PLP-BPI derivatives were synthesized and evaluated in the EAE model. Intravenous injections of PLP-BPI derivatives prevented the disease progression more efficiently than did unmodified PLP-BPI. Production of IL-17, a potent proinflammatory cytokine found commonly among MS patients, was significantly low in Ac-PLP-BPI-NH(2)-2-treated mice. Treatment given after the disease onset could dramatically ameliorate the disease. BPI induced anaphylactic responses at a lower incidence than PLP(139-151). In conclusion, PLP-BPI derivatives can effectively suppress the disease severity and morbidity of EAE by post-onset therapeutic treatment as well as prophylactic use.

Neonatal induction of myelin-specific Th1/Th17 immunity does not result in experimental autoimmune encephalomyelitis and can protect against the disease in adulthood.

The neonatal immune system is believed to be biased towards T helper type 2 (Th2) immunity, but under certain conditions neonates can also develop Th1 immune responses. Neonatal Th2 immunity to myelin antigens is not pathogenic and can prevent induction of experimental autoimmune encephalomyelitis (EAE) in adulthood, but the consequences of neonatally induced Th1 immunity to self-antigens have remained unresolved. Here, we show that neonatal injection of mice with myelin antigens emulsified in complete Freund's adjuvant (CFA) induced vigorous production of IFN-gamma and IL-17, but not IL-5, consistent with myelin-specific Th1/Th17 immunity. Importantly, the myelin-specific Th1/Th17 cells persisted in the mice until adulthood without causing symptoms of EAE. Intraperitoneal, but not subcutaneous injection of neonates with myelin antigens protected against induction of EAE as adults. Intraperitoneally injected neonates showed a substantial decrease of the number and avidity of myelin-reactive Th17 cells, suggesting a decrease in IL-17 producing precursor cells as the mechanism of protection from EAE upon re-injection with myelin antigens as adults. The results could provide a rationale for the presence of autoreactive T cells found in healthy human individuals without autoimmune disease.

Dissociation of experimental allergic encephalomyelitis protective effect and allergic side reactions in tolerization with neuroantigen.

Administration of autoantigens under conditions that induce type 2 immunity frequently leads to protection from T cell-mediated autoimmune diseases. Such treatments, however, are inherently linked to the induction of IgG1 Abs and to the risk of triggering anaphylactic reactions. We studied the therapeutic benefit vs risk of immune deviation in experimental allergic encephalomyelitis of SJL mice induced by MP4, a myelin basic protein-proteolipid protein (PLP) fusion protein. MP4 administration in IFA induced type 2 T cell immunity, IgG1 Abs, and experimental allergic encephalomyelitis protection, and all three were enhanced by repeat injections. Despite high Ab titers, anaphylactic side reactions were not observed when MP4 was repeatedly injected in IFA or as soluble Ag s.c. In contrast, lethal anaphylaxis was seen after s.c. injection of soluble PLP:139-151 peptide, but not when the peptide was reinjected in IFA. Therefore, the Ab response accompanying the immune therapy constituted an anaphylactic risk factor only when the autoantigen was not retained in an adjuvant and when it was small enough to be readily disseminated within the body. Taken together, our data show that treatment regimens can be designed to boost the protective type 2 T cell response while avoiding the risk of Ab-mediated allergic side effects.

Novel synthetic amino acid copolymers that inhibit autoantigen-specific T cell responses and suppress experimental autoimmune encephalomyelitis.

Copolymer 1 (Cop 1, Copaxone [Teva Marion Partners, Kansas City, Missouri, USA]), a random amino acid copolymer of tyrosine (Y), glutamic acid (E), alanine (A), and lysine (K), reduces the frequency of relapses by 30% in relapsing-remitting multiple sclerosis (MS) patients. In the present study, novel random four-amino acid copolymers, whose design was based on the nature of the anchor residues of the immunodominant epitope of myelin basic protein (MBP) 85-99 and of the binding pockets of MS-associated HLA-DR2 (DRB1*1501), have been synthesized by solid-phase chemistry. Poly (Y, F, A, K) (YFAK) inhibited binding of the biotinylated MBP 86-100 epitope to HLA-DR2 molecules more efficiently than did either unlabeled MBP 85-99 or any other copolymer including Cop 1. Moreover, YFAK and poly (F, A, K) (FAK) were much more effective than Cop 1 in inhibition of MBP 85-99-specific HLA-DR2-restricted T cell clones. Most importantly, these novel copolymers suppressed experimental autoimmune encephalomyelitis, induced in the susceptible SJL/J (H-2(s)) strain of mice with the encephalitogenic epitope PLP 139-151, more efficiently than did Cop 1. Thus, random synthetic copolymers designed according to the binding motif of the human immunodominant epitope MBP 85-99 and the binding pockets of HLA-DR2 might be more beneficial than Cop 1 in treatment of MS.

Pertussis toxin modulates the immune response to neuroantigens injected in incomplete Freund's adjuvant: induction of Th1 cells and experimental autoimmune encephalomyelitis in the presence of high frequencies of Th2 cells.

Pertussis toxin (PT) has been widely used to facilitate the induction of experimental autoimmune encephalomyelitis (EAE) in rodents. It has been suggested that this microbial product promotes EAE by opening up the blood-brain barrier and thereby facilitates the migration of pathogenic T cells to the CNS. However, PT has other biological effects that could contribute to its activity in EAE, such as enhancing the cytokine production by T cells and induction of lymphocytosis. In this work, we investigated the effects of PT on the pathogenicity, cytokine differentiation, and clonal sizes of neuroantigen-reactive T cells in EAE in mice. Our results show that PT prevented the protection from EAE conferred by injection of PLPp139-151 in IFA and induced high frequencies of peptide-specific Th1 cells and disease. Interestingly, the mice developed EAE despite the simultaneous vigorous clonal expansion of PLPp139-151-specific Th2 cells. The data indicate that the Th2 cells in this model neither were protective against EAE nor promoted the disease. Furthermore, the results suggested that the effects of the toxin on neuroantigen-reactive T cells were promoted by the PT-induced activation of APCs in lymphoid tissues and the CNS. Together, the results suggest that microbial products, such as PT, could contribute to the initiation of autoimmune disease by modulating the interaction between the innate and adaptive immune system in the response to self Ags.

Production of a recombinant hybrid molecule of cholera toxin-B-subunit and proteolipid-protein-peptide for the treatment of experimental encephalomyelitis.

Mucosal administration of experimental autoimmune encephalomyelitis (EAE)-specific autoantigens can reduce the onset of disease. To examine whether cholera toxin-B-subunit (CTB)-conjugated EAE-specific T-cell epitope can reduce development of the autoimmune disease in mice, we produced a recombinant hybrid molecule of CTB fusion protein linked with proteolipid-protein (PLP)-peptide139-151(C140S) at levels up to 0.1 gram per liter culture media in Bacillus brevis as a secretion-expression system. Amino acid sequencing and GM1-receptor binding assay showed that this expression system produced a uniformed recombinant hybrid protein. EAE was induced in SJL/J mice by systemic administration with the PLP-peptide. When nasally immunized 5 times with 70 microg rCTB PLP-peptide hybrid protein, mice showed a significantly suppressed development of ongoing EAE and an inhibition of both the PLP-peptide-specific delayed-type hypersensitivity (DTH) responses and leukocyte infiltration into the spinal cord. In contrast, all mice given the PLP-peptide alone or the PLP-peptide with the free form of CTB did not suppress the development of EAE and DTH responses. These results suggest that nasal treatment with the recombinant B. brevis-derived hybrid protein of CTB and autoantigen peptide could prove useful in the control of multiple sclerosis.

Multi-modal antigen specific therapy for autoimmunity.

Peripheral tolerance, represents an attractive strategy to down-regulate previously activated T cells and suppress an ongoing disease. Herein, immunoglobulins (Igs) were used to deliver self and altered self peptides for efficient peptide presentation without costimulation to test for modulation of experimental allergic encephalomyelitis (EAE). Accordingly, the encephalitogenic proteolipid protein (PLP) sequence 139-151 (referred to as PLP1) and an altered form of PLP1 known as PLP-LR were genetically expressed on Igs and the resulting Ig-PLP1 and Ig-PLP-LR were tested for efficient presentation of the peptides and for amelioration of ongoing EAE. Evidence is presented indicating that Ig-PLP1 as well as Ig-PLP-LR given in saline to mice with ongoing clinical EAE suppresses subsequent relapses. However, aggregation of both chimeras allows crosslinking of Fcgamma receptors (FcgammaRs) and induction of IL-10 production by APCs but does not promote the up-regulation of costimulatory molecules. Consequently, IL-10 displays bystander suppression and synergizes with presentation without costimulation to drive effective modulation of EAE. As Ig-PLP1 is more potent than Ig-PLP-LR in the down-regulation of T cells, we conclude that peptide affinity plays a critical role in this multi-modal approach of T cell modulation.

A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: a basis for peptide-specific therapy after onset of clinical disease.

The development of autoimmune disease is accompanied by the acquired recognition of new self-determinants, a process commonly referred to as determinant spreading. In this study, we addressed the question of whether determinant spreading is pathogenic for progression of chronic-relapsing experimental autoimmune encephalomyelitis (EAE), a disease with many similarities to multiple sclerosis (MS). Our approach involved a systematic epitope mapping of responses to myelin proteolipid protein (PLP) as well as assaying responses to known encephalitogenic determinants of myelin basic protein (MBP 87-89) and myelin oligodendrocyte glycoprotein (MOG 92-106) at various times after induction of EAE in (SWR X SJL)F1 mice immunized with PLP 139-151. We found that the order in which new determinants are recognized during the course of disease follows a predictable sequential pattern. At monthly intervals after immunization with p139-151, responses to PLP 249-273, MBP 87-99, and PLP 137-198 were sequentially accumulated in al mice examined. Three lines of evidence showed that determinant spreading is pathogenic for disease progression: (a) spreading determinants mediate passive transfer of acute EAE in naive (SWR X SJL)F1 recipients; (b) an invariant relationship exists between the development of relapse/progression and the spreading of recognition to new immunodominant encephalitogenic determinants; and (c) after EAE onset, the induction of peptide-specific tolerance to spreading but not to nonspreading encephalitogenic determinants prevents subsequent progression of EAE. Thus, the predictability of acquired self-determinant recognition provides a basis for sequential determinant-specific therapeutic intervention after onset of the autoimmune disease process.