Fedor Heidenreich

The spectrum of mutations causing end-plate acetylcholinesterase deficiency.

The end‐plate species of acetylcholinesterase (AChE) is an asymmetric enzyme consisting of a collagenic tail subunit composed of three collagenic strands (ColQ), each attached to a tetramer of the T isoform of the catalytic subunit (AChET) via a proline‐rich attachment domain. The principal function of the tail subunit is to anchor asymmetric AChE in the synaptic basal lamina. Human end‐plate AChE deficiency was recently shown to be caused by mutations in COLQ. We here report nine novel COLQ mutations in 7 patients with end‐plate AChE deficiency. We examine the effects of the mutations on the assembly of asymmetric AChE by coexpressing each genetically engineered COLQ mutant with ACHET in COS cells. We classify the newly recognized and previously reported COLQ mutations into four classes according to their position in ColQ and their effect on AChE expression. We find that missense mutations in the proline‐rich attachment domain abrogate attachment of catalytic subunits, that truncation mutations in the ColQ collagen domain prevent the assembly of asymmetric AChE, that hydrophobic missense residues in the C‐terminal domain prevent triple helical assembly of the ColQ collagen domain, and that other mutations in the C‐terminal region produce asymmetric species of AChE that are likely insertion incompetent. Ann Neurol 2000;47:162–170.

Serum and CSF levels of soluble intercellular adhesion molecule‐1 (ICAM‐1) in inflammatory neurologic diseases

Intercellular adhesion molecule-1 (ICAM-1), a cell surface receptor important for cellular interactions in immune responses, especially leukocyte trafficking into inflamed tissue, is released in a soluble form (sICAM-1) into the extracellular space. In this study, we measured sICAM-1 in paired serum and CSF samples from patients with inflammatory diseases of the nervous system (IND) and calculated a sICAM-1 index as a measure of the intrathecal release of ICAM-1. In comparison with noninflammatory neurologic disease (NIND) controls, we found increased sICAM-1 index levels in viral meningoencephalitis, bacterial meningitis and, to a lesser degree, multiple sclerosis but not in Guillain-Barre syndrome. Serial examination of viral meningoencephalitis patients in most cases showed a decrease of sICAM-1 index in parallel with falling cell counts and clinical improvement. Except for those in bacterial meningitis, sICAM-1 serum levels of IND patients were not significantly different from those of NIND controls. The increased intrathecal release of sICAM-1 in viral meningoencephalitis and bacterial meningitis most likely reflects activation of macrophages and lymphocytes and provides evidence for a strong local immune response that itself, in addition to the infectious agent, may damage nervous tissue.

Antibody heterogeneity and specificity in myasthenia gravis.

Anti-AChR is heterogeneous within individuals and between individuals. Anti-AChR idiotypes are not shared to any large extent. Ten monoclonal antibodies raised against human AChR: (a) bind to five partially overlapping regions; (b) are not idiotypically identical even within a region; (c) do not all bind to the main immunogenic region; (d) four distinguish between normal and denervated human AChR; (e) can be used to define the antigenic determinants in MG. Antigenic specificities vary in different clinical groups. Antigenic specificities can change during the course of the disease, but some remain relatively constant. Thymus cultures make antibodies with the same specificity as those present in the serum of the individual. All monoclonal antibodies bind to myoid cells of normal and MG thymus. We find no convincing evidence of naturally occurring antiidiotype antibodies in MG sera.

Glatiramer acetate (GA) induces IL-13/IL-5 secretion in naive T cells

In order to define possible mechanisms of immunomodulation by glatiramer acetate (GA), we investigated the primary in vitro cytokine response of peripheral blood mononuclear cells (PBMCs) and T-cell subpopulations. In PBMCs from healthy subjects and untreated patients with multiple sclerosis (MS) GA-induced T-cell proliferation and mRNA expression/cytokine, secretion of IL-13 and IL-5 but not of IL-10, TGF-beta or IL-12, IL-4 was detected at the mRNA level only. IFN-gamma was induced in a few subjects at very low concentrations. The response to GA was driven by the CD4(+)/CD45RA(+) T-cell subpopulation and was mediated by T-cell receptor (TCR) engagement as determined by anti-TCR blocking antibodies. The findings are compatible with the hypothesis that GA functions as partial or weak TCR-agonist activating naive T cells to produce the Th2 cytokines IL-13 and IL-5.

Anti‐acetylcholine receptor antibody specificities in serum and in thymic cell culture supernatants from myasthenia gravis patients

We investigated the role of the thymus in myasthenia gravis by comparing the antigenic specificities of antiacetylcholine receptor antibodies (anti-AChR), defined by competition with mouse monoclonal antibodies that bind to five different regions on human muscle AChR, in thymic culture supernatants and in serum pre- and post-thymectomy. Anti-AChR specificities present in the serum were broadly unchanged in 16 non-thymoma and six thymoma patients 7-30 months after thymectomy compared with an initial sample, although total anti-AChR frequently fell. The fine specificities of the anti-AChR synthesized in vitro by cultured lymphocytes from the thymus of ten patients (without thymoma) correlated significantly with that of the anti-AChR in the serum at the same time. We conclude that AChR-specific B cells in the thymus are representative of the total AChR-specific repertoire, and that thymectomy does not selectively deplete particular B cell clones.

Interferon-β increases the stimulatory capacity of monocyte-derived dendritic cells to induce IL-13, IL-5 and IL-10 in autologous T-cells

Dendritic cells (DCs) are key regulators of immune responses and have been associated with autoimmunity in animal models and human disease. The effects of interferon beta (IFN-beta), an immunomodulatory cytokine used in multiple sclerosis (MS) therapy, on DCs are not well understood. Monocyte-derived DCs at different stages of maturation were stimulated with IFN-beta and DC-phenotype and stimulatory function were measured. IFN-beta inhibited the development of DCs at early stages but enhanced DC maturation. Moreover, IFN-beta enhanced the capacity of DCs to stimulate autologous T-cells to secrete IL-13, IL-10 and IL-5. Thus, IFN-beta has both immunostimulatory and immunosuppressive effects on DCs depending on the stage of maturation.

Serum levels of soluble E‐selectin (ELAM‐1) in immune‐mediated neuropathies

Adhesion molecules are critically involved in inflammatory responses. We studied serum concentrations of the soluble form of E-selectin (endothelial-leukocyte adhesion molecule-1, ELAM-1) in 187 patients with neuropathies of diverse etiology, 54 patients with other noninflammatory, nondemyelinating neurologic disorders, and 15 healthy controls. Serum E-selectin levels, quantitated by a two-site enzyme-linked immunosorbent assay, were significantly increased in 126 patients with Guillain-Barré syndrome (GBS) (mean ± SD, 45.1 ± 16.3 ng/ml) and 13 patients with vasculitic neuropathies (47.1 ± 19.1 ng/ml) compared with patients with other neurologic diseases (19.8 ± 7.4 ng/ml) and healthy controls (21.9 ± 8.1 ng/ml). In GBS, E-selectin levels were temporally related to disease activity. Cytokine-mediated upregulation of E-selectin may be important in homing and attachment of leukocytes to en-doneurial endothelial cells. Raised E-selectin concentrations probably reflect endothelial cell activation occurring early in the sequence of immunopathologic events culminating in peripheral nerve damage.

Human polymorphonuclear neutrophils express a B7-1-like molecule.

Polymorphonuclear neutrophils (PMN) are part of the innate immune system and are first‐line effector cells in acute inflammatory responses. On activation PMNs secrete cytokines and oxygen metabolites that might be involved in the regulation of the acquired immune response. We show here that peripheral blood PMNs constitutively express a B7‐1‐like molecule as detected by immunostaining with several B7‐1 antibodies. Reverse transcriptase‐polymerase chain reaction using three sets of primers spanning different regions of B7‐1 indicate dissimilarities at the mRNA level. B7‐1 mRNA is expressed in bone marrow cells and lipopolysaccharide (LPS)‐stimulated but not in unstimulated PMNs. The B7‐1‐like molecule is localized to the cytoplasmic granules and translocated to the cell surface after stimulation with LPS or interleukin‐12 in some donors. Binding of CTLA4‐Ig suggests that the B7‐1‐like molecule can interact with functional B7 ligand and might be important in the immunobiology of PMNs. J. Leukoc. Biol. 66: 945–952; 1999.

Costimulatory molecules B7-1 and B7-2 on CSF cells in multiple sclerosis and optic neuritis.

The aberrant expression of B7 costimulatory molecules is involved in the pathogenesis of autoimmune diseases and overexpression of B7-1 was found in inflammatory multiple sclerosis (MS) lesions. We here report that costimulatory molecules B7-1 and B7-2 are expressed on cerebrospinal fluid (CSF) monocytes and B-lymphocytes from patients with MS, optic neuritis (ON) and other inflammatory central nervous system (CNS) diseases. In patients with ON but not MS, increased expression of B7-2 was detected as compared to non-inflammatory controls. The expression of B7-1 in MS and ON patients correlates with disease duration but not with relapses in patients with MS indicating a role in early disease but not as a reliable marker of disease activity at later stages of MS.

Treatment de-escalation after mitoxantrone therapy: results of a phase IV, multicentre, open-label, randomized study of subcutaneous interferon beta-1a in patients with relapsing multiple sclerosis

Objective: The objective of this study was to assess the effect of treatment with interferon (IFN) β-1a, 44 µg subcutaneously (sc) three times weekly (tiw), on clinical and magnetic resonance imaging (MRI) outcomes in patients with relapsing multiple sclerosis (MS) following mitoxantrone therapy. Methods: This was an open-label, randomized, multicentre, rater-blinded, 96-week observational study conducted in Germany. Clinically stable patients with relapsing forms of MS, who had discontinued mitoxantrone treatment 1–6 months before study entry, were randomized to IFN β-1a sc 44 µg tiw, or no treatment. The primary endpoint was time to first relapse. Secondary endpoints included the number of relapse-free patients, disease activity assessed by MRI and time to 3-month confirmed Expanded Disability Status Scale (EDSS) progression, all at week 96. Results: A total of 30 patients were randomized (intent-to-treat population: 14 IFN β-1a, 15 untreated; one patient from the safety population discontinued the study after 25 days owing to an adverse event and without providing any postbaseline efficacy data, and was thus excluded from the intent-to-treat population). Overall, 71.4% (10/14) of patients in the IFN β-1a group remained relapse free over 96 weeks, versus 46.7% (7/15) in the untreated group (p = 0.26). IFN β-1a delayed the time to first relapse versus no treatment (p = 0.14); time to first relapse (25th percentile) was 95.4 (IFN β-1a) versus 46.0 weeks (no treatment). Confirmed EDSS progression was observed in five patients in each treatment group. Mean change in EDSS score was 0.3 in both groups (p = 0.79). Changes in the number or volume of T1 and T2 lesions at week 96 were not significantly different between treatment groups (p > 0.05). There were no new or unexpected adverse events related to IFN β-1a treatment. Conclusions: Several endpoints appeared to show a benefit of IFN β-1a treatment, but no significant differences could be detected owing to the small sample. Therefore, these data only permit, at best, tentative conclusions about the disease course in patients with MS after de-escalation from mitoxantrone and continuation with or without IFN β-1a. Larger confirmatory studies are required.