Dominique Bernard

Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis

Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4+ T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedly in vitro expressed a unique channel phenotype (“chronically activated”) with large numbers of Kv1.3 voltage-gated channels (≈1500 per cell) and small numbers of IKCa1 Ca2+-activated K+ channels (≈50–120 per cell). In contrast, resting T cells displayed 0–10 Kv1.3 and 10–20 IKCa1 channels per cell (“quiescent” phenotype), whereas T cells stimulated once or twice expressed ≈200 Kv1.3 and ≈350 IKCa1 channels per cell (“acutely activated” phenotype). Consistent with their channel phenotype, [3H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap22) engineered to be highly specific for Kv1.3, but not by a selective IKCa1 blocker (TRAM-34). The combination of ShK-Dap22 and TRAM-34 enhanced the suppression of MBP-stimulated T cell proliferation. Based on these in vitro results, we assessed the efficacy of K+ channel blockers in AT-EAE. Specific and simultaneous blockade of the T cell channels by ShK or by a combination of ShK-Dap22 plus TRAM-34 prevented lethal AT-EAE. Blockade of Kv1.3 alone with ShK-Dap22, but not of IKCa1 with TRAM-34, was also effective. When administered after the onset of symptoms, ShK or the combination of ShK-Dap22 plus TRAM-34 greatly ameliorated the clinical course of both moderate and severe AT-EAE. We conclude that selective targeting of Kv1.3, alone or with IKCa1, may provide an effective new mode of therapy for multiple sclerosis.

Selective Blocking of Voltage-Gated K+ Channels Improves Experimental Autoimmune Encephalomyelitis and Inhibits T Cell Activation

Kaliotoxin (KTX), a blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. First, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Second, Kv1.1 is found in paranodal regions of axons in the central nervous system. Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission. Therefore, we investigated the therapeutic properties of KTX via its immunosuppressive and symptomatic neurological effects, using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The T line cells used to induce adoptive EAE were myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by KTX. Activation is a crucial step for MBP T cells to become encephalitogenic. The addition of KTX during Ag-T cell activation led to a great reduction in the MBP T cell proliferative response, in the production of IL-2 and TNF, and in Ca2+ influx. Furthermore, the addition of KTX during T cell activation in vitro led a decreased encephalitogenicity of MBP T cells. Moreover, KTX injected into Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this blocker of neuronal and lymphocyte channels to Lewis rats improved the symptoms of EAE. We conclude that KTX is a potent immunosuppressive agent with beneficial effects on the neurological symptoms of EAE.

Cryptogenic Partial Epilepsies with Anti-GM1 Antibodies: A New Form of Immune-Mediated Epilepsy?

Summary: Purpose: We wished to study immune system dysfunction which has been proposed as a potential cause of epilepsy. Epileptogenic action of antibodies directed against GM1 gangliosides was demonstrated in rats, but the potential role of anti‐GMI antibodies in human epilepsy has not yet been studied.

Quantification of Immunoglobulin Free Light Chains in CerebroSpinal Fluid by Nephelometry

Oligoclonal free light chains (FLC) banding has been described in multiple sclerosis (MS) and should be correlated with disease activity. However, discrepancies between studies have been reported because of differences in methods. A new quantitative, rapid, and automated method using nephelometry is now available. Our objective was to investigate the interest of this method for the diagnosis and prognosis of MS. For this purpose, FLC index was determined in paired samples of CSF and serum from consecutive and unselected patients from the same department of neurology. We enrolled 89 patients (33 MS, 15 “possible MS”, and 41 controls) and correlated with IgG index, IgG oligoclonal banding, and clinical MS progression criteria. The main results were (1) FLC kappa index was more sensitive but less specific than IgG index for the diagnosis of MS, (2) two MS patients were negative for oligoclonal banding but exhibited a positive kappa index, (3) no relation between FLC kappa indices, MS clinical criteria, and disease progression was found. In conclusion, FLC kappa index should be considered as a useful complementary test for MS diagnosis. Its pronostic interest remains to be determined on a larger cohort of possible MS patients.

Appearance of neurofilament subunit epitopes correlates with electrophysiological maturation in cortical embryonic neurons cocultured with mature astrocytes.

E14 rat cortical neurons which have almost no glial progenitors were cocultured with a homogeneous population of mature type 1 astrocytes at a 4/1 ratio in serum free medium. Maturation of neurons was evaluated using a set of well characterized antibodies and two new monoclonal antibodies (MN2E4 and MN3H6) raised against various neurofilament subunits and whole-cell patch clamp experiments. We observed that this coculture method leads to a well-timed and very homogeneous neuronal maturation and that sequential appearance of neurofilament subunits in developing neurons correlates with the electrophysiological maturation. This sequence, early expression of the 68 kDa neurofilament subunit and late appearance of the 200 kDa neurofilament subunit, occurs in normal brain development, which validates this culture model as a useful tool for studying neuronal maturation and differentiation. MN2E4 staining (non-phosphorylated 200 kDa cytoskeletal protein antibody) appeared just before the neurons became excitable. It could thus be used as a functional neuronal marker. MN3H6 staining (phosphorylated 160-200 kDa neurofilament subunit antibody) appeared just after the neurons made synaptic contacts and generated synaptically driven spike bursts. This finding indicated that some phosphorylated epitopes of 160-200 kDa neurofilament followed synaptogenesis. These processes may play a key role in stabilizing the synapses to achieve a functional neuronal network.