Crystal structure of human prion protein bound to a therapeutic antibody.

Prion infection is characterized by the conversion of host cellular prion protein (PrP(C)) into disease-related conformers (PrP(Sc)) and can be arrested in vivo by passive immunization with anti-PrP monoclonal antibodies. Here, we show that the ability of an antibody to cure prion-infected cells correlates with its binding affinity for PrP(C) rather than PrP(Sc). We have visualized this interaction at the molecular level by determining the crystal structure of human PrP bound to the Fab fragment of monoclonal antibody ICSM 18, which has the highest affinity for PrP(C) and the highest therapeutic potency in vitro and in vivo. In this crystal structure, human PrP is observed in its native PrP(C) conformation. Interactions between neighboring PrP molecules in the crystal structure are mediated by close homotypic contacts between residues at position 129 that lead to the formation of a 4-strand intermolecular beta-sheet. The importance of this residue in mediating protein-protein contact could explain the genetic susceptibility and prion strain selection determined by polymorphic residue 129 in human prion disease, one of the strongest common susceptibility polymorphisms known in any human disease.

Antiganglioside antibodies in Guillain-Barré syndrome after a recent cytomegalovirus infection.

OBJECTIVE: To study the association between anti-ganglioside antibody responses and Guillan-Barré syndrome (GBS) after a recent cytomegalovirus (CMV) infection. METHODS: Enzyme linked immunosorbant assay (ELISA) was undertaken on serum samples from 14 patients with GBS with recent cytomegalovirus (CMV) infection (CMV+GBS) and 12 without (CMV-GBS), 17 patients with other neurological diseases (OND), 11 patients with a recent CMV infection but without neurological involvement, 11 patients with recent Epstein-Barr virus (EBV) infection but without neurological involvement, and 20 normal control (NC) subjects. RESULTS: IgM antibodies were found at 1:100 serum dilution to gangliosides GM2 (six of 14 patients), GM1 (four of 14), GD1a (three of 14) and GD1b (two of 14) in the serum samples of the CMV+GBS patients, but not in those of any of the CMV-GBS patients. IgM antibodies were also found to gangliosides GM1, GD1a, and GD1b in one of 11 OND patients, to ganglioside GM1 in one of 11 non- neurological CMV patients, and to ganglioside GD1b in one of 20 NC subjects. Some patients with EBV infection had IgM antibodies to gangliosides GM1 (five of 11), GM2 (three of 11), and GD1a (two of 11). However, the antibodies to ganglioside GM2 had a low titre, none being positive at 1:200 dilution, whereas five of the CMV+GBS serum samples remained positive at this dilution. CONCLUSION: Antibodies to ganglioside GM2 are often associated with GBS after CMV infection, but their relevance is not known. It is unlikely that CMV infection and anti-ganglioside GM2 antibodies are solely responsible and an additional factor is required to elicit GBS.

The distribution of CD1 molecules in inflammatory neuropathy.

The CD1 molecules have been shown to present non-protein antigens, such as complex lipids to Mycobacteria, and may be important in presenting glycolipids which are involved in inflammatory neuropathies. To study the expression of CD1 molecules in peripheral nerve, we examined nerve biopsies from two patients with acute inflammatory demyelinating polyradiculoneuropathy (AIDP), five with acute axonal neuropathy, six with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), nine with chronic axonal neuropathy, six with vasculitic neuropathy and three with no histological abnormality. Immunocytochemical studies showed strong labelling of CD1b on endoneurial macrophages (CD68+) and on myelinated nerve fibres in both AIDP patients, but it was rarely observed in the other patients. Weaker staining was seen on endoneurial macrophages and/or other endoneurial cells in some of the patients with other peripheral neuropathies, but none of the control nerves. CD1a had a weaker, but similar pattern. There was endoneurial infiltration of CD4+ and CD8+ T cells in the AIDP and CIDP nerves and sometimes in the other peripheral neuropathy nerves, but not in the normal nerves. Most T cells had alpha beta+ T cell receptors (TCR), but gamma delta+ TCR T cells were found in the nerves of both AIDP patients and sometimes in the nerves of other patients with peripheral neuropathy. Staining for mannose receptor was almost universal, being more intense in AIDP, chronic axonal neuropathy and vasculitis patients. We conclude that CD1 molecule expression is upregulated in peripheral neuropathy, especially in association with inflammation.

T cell receptor V beta gene usage in Guillain-Barré syndrome.

We set out to determine whether the T cell receptor (TCR) V beta gene usage in acute inflammatory demyelinating polyradiculoneuropathy (AIDP) is restricted. We separated activated from non-activated peripheral blood T cells with anti-IL2 receptor (anti-CD25) antibody-labelled magnetic beads from four AIDP patients and four normal control (NC) subjects. The TCR V beta gene usage of circulating activated and non-activated T cells was heterogeneous in all the patients and controls, but the activated T cells of all four of the AIDP patients showed a more limited usage of V beta genes and enhanced V beta 15 usage, as compared to the non-activated T cells. This was not seen in the healthy controls. The activated and non-activated T cells from a patient with acute motor and sensory axonal neuropathy (AMSAN) showed a similar V beta gene usage to that of the controls. From a further patient with AIDP, we studied the V beta gene usage of short-term T cell lines reactive to the peripheral nerve myelin proteins P2, P0 and the P0 peptide amino acid sequence 194-208. The V beta gene usage of the lines was heterogeneous, with enhanced usage of V beta 15 in the cell line responsive to the Pzero peptide. We conclude that T cells activated during the immune response associated with AIDP preferentially used V beta 15, which may indicate a restricted response to a common antigen, or a role for an as yet undefined superantigen in the pathogenesis of AIDP.

Antibody responses to P0 and P2 myelin proteins in Guillain-Barré syndrome and chronic idiopathic demyelinating polyradiculoneuropathy.

Immunisation with the peripheral nerve myelin proteins P0 or P2 induces inflammatory neuropathy in animals. We sought antibodies with an ELISA to these proteins in 38 patients with acute Guillain-Barré syndrome (GBS), 32 patients with chronic idiopathic demyelinating polyradiculoneuropathy (CIDP), 31 patients with other neuropathies (ONP) and 26 normal control (NC) subjects. We discovered IgM antibodies to human P0 protein in the sera of 18.5% of the patients with GBS, 15.6% with CIDP, 6.4% with ONP and 3.8% of NC subjects. Of the sera which reacted with P0, sera from 4/7 of GBS, 3/5 of CIDP, 1/2 of ONP patients and 0/1 of NC subjects reacted with a synthetic P0 peptide representing residues 150-169 from the cytoplasmic portion of the molecule. IgG antibodies to P0 were slightly less common than IgM antibodies, being present in only 7.9% of GBS, 0% of CIDP and 3% of ONP patients and 0% of NC subjects. We found antibodies to bovine P2 protein more commonly than antibodies to P0. IgM antibodies were present in 39.5% of GBS, 34.4% of CIDP, 16.1% of ONP patients and 15.4% of NC subjects. IgG antibodies were present in 18.4% of GBS, 12.5% of CIDP, 3.2% of ONP patients and 7.6% of NCs. Of the sera which contained antibodies to P2 protein, only a few reacted with P2 peptides 14-25 or 58-81, but without any consistent pattern of reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

T cell responses to myelin proteins in Guillain-Barré syndrome.

We have investigated the hypothesis that the pathogenesis of Guillain-Barré syndrome (GBS) involves an autoimmune T cell response to P0 and P2 proteins of peripheral nerve myelin. The proliferative responses of blood mononuclear cells (MNC) to myelin proteins and synthetic peptides derived from them were determined in patients with GBS and chronic idiopathic demyelinating polyradiculoneuropathy (CIDP), normal controls (NC) and patients with other neuropathies (ONP). Twelve out of 19 GBS patients responded to P0 or P2, 6 to P0 and its peptides only, 3 to P2 and its peptides only, and 3 to both P0 and P2 antigens. Responses to at least one of the antigens were also found in 6/13 of CIDP patients, but in only 4/17 NC and 2/6 ONP. Immune responses in GBS are heterogeneous. The early T cell responses to P0 protein, described here for the first time, may be important in the pathogenesis of some cases.

Immunoelectron microscopical labelling of a glycolipid in the envelopes of brain cell-derived budding viruses, Semliki Forest, influenza and measles, using a monoclonal antibody directed chiefly against galactocerebroside resulting from Semliki Forest virus infection.

Neurotropic RNA budding viruses such as Semliki Forest virus (SFV), influenza and measles were each grown in identical mouse brain cell cultures. Positive immunoelectron microscopical labelling with gold was seen in the envelope of these viruses using an anti-SFV derived glycolipid monoclonal antibody (MAb), 373 shown to be directed chiefly against galactocerebroside. The results indicate that each enveloped virus grown from the same cell type contains the same glycolipid in its envelope. The presence of common glycolipids derived from the host cell in the envelopes of various enveloped budding viruses may play a significant role in the pathogenesis of virus induced, immune mediated CNS autoimmunity and demyelination, particularly in multiple sclerosis (MS).

Immunocytochemical evidence for Semliki Forest virus antigen persistence in mouse brain.

Semliki Forest virus (SFV) is neurotropic in mice. Mature virulent virus (strain L10) can be identified within the CNS by electron microscopy in adult mice. Inspite of high virus titres, avirulent SFV A7(74) cannot be visualised in the brain of adult mice. Immunocytochemical studies using monoclonal antibodies (MAbs) to A7(74) E1 and E2 proteins and viral envelope glycolipids, showed viral E1 to be labelled in the cerebral capillaries, the E2 and the putative envelope glycolipids were labelled in the cytoplasm of neurons, particularly in the hippocampal areas and glia in the cerebellum. By double labelling the presence of viral antigens in astrocytes and oligodendrocytes was demonstrated. Viral antigens were identified in the brain up to 183 days after infection. Paraffin sections from Bouin-fixed tissue were found to be the most suitable material for immunocytochemistry of SFV. The presence of life-long anti SFV antibody in the sera of animals after SFV infection, could be due to the persistence of viral antigens acting as constant stimuli to the immune system.

Immunological relationship between a demyelinating RNA enveloped budding virus (Semliki Forest) and brain glycolipids.

A monoclonal antibody (MAb) raised against central nervous system (CNS) myelin (212) and a MAb (308) raised against brain with Semliki Forest virus (SFV) were both found to react against the same CNS glycolipids. Both these MAbs were also found to react strongly with SFV and against certain brain glycolipid fractions in an immunosorbent assay (ELISA). This demonstrates the presence of common glycolipid antigens in the viral envelope and CNS myelin. MAb 212 had no SFV neutralising capacity and that of MAb 308 was not significant. However, MAb 212 inhibited the neutralisation of the virus by the MAbs (302, 307) specific to SFV proteins. The implications of these findings in relation to the viral induced CNS autoimmunity and persistence of virus in the CNS is discussed.