Agata Walczak

Transdermal application of myelin peptides in multiple sclerosis treatment.

IMPORTANCE Demonstration of efficacious antigen-specific therapy in multiple sclerosis. OBJECTIVE To assess the safety and efficacy of transdermally applied myelin peptides in patients with relapsing-remitting multiple sclerosis. DESIGN One-year double-blind, placebo-controlled cohort study. SETTING Referral center. PARTICIPANTS Thirty outpatients aged 18 to 55 years with relapsing-remitting multiple sclerosis. INTERVENTION Skin patch with a mixture of 3 myelin peptides, MBP85-99, MOG35-55, and PLP139-155. MAIN OUTCOMES AND MEASURES Cumulative number of active gadolinium-enhanced (Gd+) lesions per patient per scan, mean volume of Gd+ lesions, cumulative number of new T2 lesions, and T2 lesion and T1 lesion volume change from baseline to the end of the study. Total number of relapses during the year of the study per patient (annual relapse rate), proportion of relapse-free patients, and proportion of patients with 3 months of confirmed disability worsening on the Expanded Disability Status Scale at month 12. RESULTS All patients completed the study. Compared with placebo, treatment with a myelin peptide skin patch (1 mg) showed a 66.5% reduction in the cumulative number of Gd+ lesions (P = .02) during the 12 months of the study. The annual relapse rate in patients treated with a mixture of myelin peptides (1 mg) was significantly lower compared with the placebo group (0.43 vs 1.4; P = .007). Treatment with a myelin peptide skin patch was well tolerated and no serious adverse events were reported. CONCLUSIONS AND RELEVANCE In patients with relapsing-remitting multiple sclerosis, treatment with a myelin peptide skin patch significantly reduced both magnetic resonance imaging and clinically defined measures of disease activity and was safe and well tolerated.

Suppression of experimental autoimmune encephalomyelitis with a TNF binding protein (TNFbp) correlates with down-regulation of VCAM-1/VLA-4

The effect of a novel TNF binding protein (TNFbp), a polyethylene glycol‐linked form of the type I soluble receptor of TNF, on the expression of adhesion molecules has been investigated with a passive transfer model of experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. The expression of L‐selectin, VLA‐4 and LFA‐1 on spleen cells of EAE animals treated with TNFbp or saline was examined by FACS analysis. The expression of VCAM‐1 and ICAM‐1 was investigated by immunochemistry in spinal cord tissue of SJL/J mice with EAE. In animals sensitized for EAE and treated with TNFbp, the expression of VCAM‐1 in the central nervous system as well as VLA‐4 on spleen cells was clearly diminished. Reduction in VCAM‐1 staining and VLA‐4 expression corresponded to inhibition of inflammation in the spinal cord and to prevention of clinical signs of EAE. The results have also shown that myelin basic protein responses as well as non‐antigen‐specific responses were not diminished in animals treated with TNFbp. The findings suggest that TNFbp might prevent EAE development by modulating the expression of VCAM‐1 and VLA‐4.

Brain-Derived Heat Shock Protein 70-Peptide Complexes Induce NK Cell-Dependent Tolerance to Experimental Autoimmune Encephalomyelitis

Heat shock proteins (Hsp) are markedly up-regulated at sites of inflammation during autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). In this study, we show that Hsp70-peptide complexes (pc) isolated from brains of mice with EAE prevented the development of EAE clinically and pathologically when administered before proteolipid protein 139–151 (PLP139–151) immunization. In contrast, pure Hsp70 or Hsp70-pc derived from brains of healthy mice or other inflamed tissue did not modulate the expression of EAE. In animals in which EAE had been suppressed by Hsp70-pc, lymphocytes showed increased cell death in response to PLP139–151 that correlated with elevated IFN-γ and NO production. Coculture of spleen cells from Hsp70-pc immunized mice with spleen cells from untreated EAE mice, in addition to depletion experiments, showed that NK cells reduced reactivity to PLP139–151. Transfer of NK cells from Hsp70-pc-immunized mice to recipients sensitized for EAE abolished disease development. Thus, we propose that Hsp70 demonstrate the ability to bind to peptides generated during brain inflammation and to induce a regulatory NK cell population that is capable of preventing subsequent autoimmunization for EAE.

A heat shock protein gene (Hsp70.1) is critically involved in the generation of the immune response to myelin antigen

Protracted inflammation has been associated with the generation of autoimmune responses. In this respect, increase in the chaperonin, heat shock protein 70 (hsp70) is an outcome of prolonged inflammatory stress. Previous experiments have shown that overexpression of inducible hsp70 in vitro enhanced myelin autoantigen recognition. To prove the role of hsp70 in myelin‐directed responses in vivo, we applied a mouse deficient in the major gene encoding inducible hsp70, hsp70.1. Hsp70.1–/– mice sensitized for experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55, displayed almost complete resistance to the disease. This correlated with the loss of T cell proliferation and IFN‐γ production in response to MOG35–55. T cell transfer experiments as well as antigen presentation assays in vitro demonstrated that hsp70 deficiency was associated with dysfunction in the activation of autoreactive T cells. Moreover, T cell responses to ovalbumin (OVA) peptide 323–339 were altered and CD4+ T cells were more prone to TCR‐induced apoptosis, suggesting broader spectrum of T cell defect in hsp70.1–/– mice. These results provide compelling evidence for generalized effect mediated by inducible hsp70 in the recognition of myelin self and non‐self antigens that influences the cytokine profile of the immune response affecting autoimmune demyelination.

Naked DNA vaccination differentially modulates autoimmune responses in experimental autoimmune encephalomyelitis

Vaccination with naked DNA represents a therapeutic strategy currently under consideration in multiple sclerosis (MS). In this study, we tested the potential therapeutic effect of vaccination with a naked DNA construct encoding proteolipid protein (pRc/CMV-PLP) upon the outcome of subsequent sensitization for experimental autoimmune encephalomyelitis (EAE) actively-induced in SJL mice with PLP139-151 peptide in adjuvant. Intramuscular vaccination with the naked DNA pRc/CMV-PLP construct led to PLP expression in local muscle tissue that persisted for about 8 weeks. Early sensitization for EAE (4 weeks after DNA vaccination) caused recipient mice to develop a severe, exacerbated form of disease (in comparison to control mice), while late sensitization (>10 weeks) resulted in a milder, ameliorated form. In the groups sensitized <10 weeks post-DNA vaccination with pRc/CMV-PLP induction of a Th1-type cytokine response was noted. In contrast, sensitization >10 weeks post-DNA vaccination led to peripheral tolerance as evidenced by a decrease in T cell proliferation and cytotoxic T cell response, no Th2 response, and no increase in apoptosis. These data are novel in that they demonstrate a differential effect of DNA vaccination and have important implications for its use as a mechanism to enhance or modulate immune reactivity.

Shedding of TNF receptors in multiple sclerosis patients.

OBJECTIVE To evaluate the rate of shedding of tumor necrosis factor (TNF) receptors (TNFRs) in MS patients. BACKGROUND It was previously suggested that TNF might play a significant role in the immunopathologic mechanism of MS. TNF mediates its biologic effects by interacting with two distinct receptors: TNFR-p55 and TNFR-p75. Both of these receptors exist in soluble and membrane-bound forms. Soluble receptors have been shown to influence TNF activity in vitro and in vivo and maintain balance between active, free TNF and inactive form of this cytokine bound to its soluble receptors. METHODS In the current study, the authors measured shedding of TNFRs from cell surface of peripheral blood mononuclear cells, peripheral blood lymphocytes, and monocytes in three groups of MS patients: relapsing-remitting in relapse, relapsing-remitting in remission, and chronic progressive. RESULTS The authors observed a significant distortion in generation of both soluble TNF receptors. Whereas the TNFR-p55 was shed at lower rate compared with healthy volunteers, the shedding of TNFR-p75 was significantly higher in MS patients. CONCLUSION Disturbance in TNFR shedding might contribute to the distortion of a fine balance between circulating TNF and its natural inhibitors in MS.

Differential prevention of experimental autoimmune encephalomyelitis with antigen-specific DNA vaccination

We compared the potential therapeutic effect of vaccination with DNA constructs encoding two encephalitogenic proteins, PLP and MOG, on the outcome of subsequent sensitization of EAE induced in SJL/J and C57/B6 mice. Early sensitization for EAE (4 weeks after DNA vaccination) caused recipient animals to develop enhanced disease with DNA-encoding PLP but not with DNA-encoding MOG. Late sensitization (more than 10 weeks) resulted in an amelioration of EAE in animals vaccinated with both PLP and MOG DNA constructs. These results, confirming the DNA-mediated ameliorating effect on EAE, also indicate significant differences in the kinetics of development of EAE tolerance in response to vaccination with different DNA-encoding myelin antigens. Since PLP and MOG require different MHC presentation and induce different EAE models, the results point to potential differences in immune system requirements for efficient DNA-induced amelioration of the autoimmune response.

Effect of Hsp70-peptide complexes generated in vivo on modulation EAE

Heat shock proteins (Hs;ps) are a family of proteins that are constitutively expressed by eukaryotic cells. They act as molecular chaperones, participating in intracellular translocation of proteins and enabling proper folding of polypeptide chains. It is postulated that unique chaperoning properties of Hsps might contribute to generating specific immune responses against peptides bound to theme’2. Many studies have demonstrated that Hsps isolated from cancer or viral infected cells elicit antitumor or antiviral immune response. However, Hsps derived from normal cells did not shown such effect3’4.It has been suggested that peptides associated with Hsps are responsible for this specific immunogenecity5.

Protective effect of naked DNA immunization in experimental autoimmune encephalitis

581 Reactive Changes of Thymocytes and Retieuloepithelium during Autotransplantation of the Thymus A.A. Stadnikov, O.Y. Nickolaeva. StateMedJcalAcademy, Drenbur~ Russia 584 Systemic lntedeukin-12 Displays Antitumor Activity in the Mouse Central Nervous System K. Shim i ~ , H. Kishim a, Osaka UniversityMedicalSchool, Japan, Y. Miyao, Suita Municipal Hospital, Japan, K. Tamurn, Osaka University Medical School, Japan, M. Tamura, Minoh MunicipaI Hospital, Japan, M. Sasaki, Osaka University Medical School, Japan

Down-regulation of the Th-2 associated GATA-3 transcription factor in peripheral blood mononuclear cells in multiple sclerosis

4 2 5 Cerebrospinal Fluid (CSF) Indices of Inflammatory Markers Aernss the Clinical Spectrum of Multiple Sderosis (MS) S.A. McMillan. G.V. McDonnell, J.P. Douglas, S.A. Hawldns,RoyalGroupof Hospitals, Belfast, Ireland 4 2 8 Increased Spontaneous and Inducible Susceptibility toApoptosis of Peripheral Lymphocytes in Multiple Sclerosis M.P. Myeko. A. Walczak, MedicalAcademy of Lodz, Poland, L. Pokoca, Military MedicaIAcademy, Lodz, Poland, K. Selm aj, MedicalAeademy of Lodz, Poland