Linda Clover

IgG1 antibodies to acetylcholine receptors in ‘seronegative’ myasthenia gravis

Only around 80% of patients with generalized myasthenia gravis (MG) have serum antibodies to acetylcholine receptor [AChR; acetylcholine receptor antibody positive myasthenia gravis (AChR-MG)] by the radioimmunoprecipitation assay used worldwide. Antibodies to muscle specific kinase [MuSK; MuSK antibody positive myasthenia gravis (MuSK-MG)] make up a variable proportion of the remaining 20%. The patients with neither AChR nor MuSK antibodies are often called seronegative (seronegative MG, SNMG). There is accumulating evidence that SNMG patients are similar to AChR-MG in clinical features and thymic pathology. We hypothesized that SNMG patients have low-affinity antibodies to AChR that cannot be detected in solution phase assays, but would be detected by binding to the AChRs on the cell membrane, particularly if they were clustered at the high density that is found at the neuromuscular junction. We expressed recombinant AChR subunits with the clustering protein, rapsyn, in human embryonic kidney cells and tested for binding of antibodies by immunofluorescence. To identify AChRs, we tagged either AChR or rapsyn with enhanced green fluorescence protein, and visualized human antibodies with Alexa Fluor-labelled secondary or tertiary antibodies, or by fluorescence-activated cell sorter (FACS). We correlated the results with the thymic pathology where available. We detected AChR antibodies to rapsyn-clustered AChR in 66% (25/38) of sera previously negative for binding to AChR in solution and confirmed the results with FACS. The antibodies were mainly IgG1 subclass and showed ability to activate complement. In addition, there was a correlation between serum binding to clustered AChR and complement deposition on myoid cells in patients’ thymus tissue. A similar approach was used to demonstrate that MuSK antibodies, although mainly IgG4, were partially IgG1 subclass and capable of activating complement when bound to MuSK on the cell surface. These observations throw new light on different forms of MG paving the way for improved diagnosis and management, and the approaches used have applicability to other antibody-mediated conditions.

Evidence of underdiagnosis of myasthenia gravis in older people

Background: Myasthenia gravis is a potentially serious but treatable muscle disease caused by autoantibodies directed at the acetylcholine receptor (AChR) on the postsynaptic membrane of the neuromuscular junction. There is anecdotal evidence that the diagnosis is sometimes missed in older patients. Objective: To examine the incidence and age distribution of positive AChR antibodies in samples referred to diagnostic laboratories in the UK, and the prevalence of positive AChR antibodies in samples from a cohort of older individuals. Methods: Positive AChR antibody tests were identified from all UK centres registered for the assay with the European quality assurance scheme (EQAS) during 1997–99, and the age and sex specific incidence was calculated, based on the UK population. The prevalence of AChR antibodies in sera from a sample of 2000 individuals aged ≥ 60 years was determined. Results: 3183 individuals had positive AChR antibody tests on routine screening during the years 1997 to 1999 in the UK, giving an annual incidence of 1.8/100 000. In both sexes, the age specific incidence rose steeply between the ages of 45 and 74, reaching 9.9/100 000 in men, and then fell, with a sharp decline above the age of 80. In the prevalence study, whereas only one serum from individuals aged 60–74 years was positive for AChR antibodies (0.12%), sera from eight individuals aged ≥ 75 years were positive (0.7%). Only one had a previous clinical diagnosis of myasthenia gravis but four others had histories of stroke or transient ischaemic attacks. Conclusions: The sharp fall in the incidence of clinically recognised myasthenia gravis in people over 80 years of age in our national AChR antibody incidence study, and the high prevalence of previously unrecognised positive AChR antibodies in those ≥ 75 years old, suggest that myasthenia gravis may be substantially underdiagnosed in older people.

Rapid eye movement sleep behavior disorder and potassium channel antibody–associated limbic encephalitis

Of six patients registered in our center with nonparaneoplastic limbic encephalitis associated with antibodies to voltage‐gated potassium channels, the five men had rapid eye movement sleep behavior disorder (RBD) coincident with voltage‐gated potassium channel antibody–associated limbic encephalitis onset. In three patients, immunosuppression resulted in resolution of RBD in parallel with remission of the limbic syndrome. RBD persisted in two patients with partial resolution of the limbic syndrome. Our findings suggest that RBD is frequent in the setting of voltage‐gated potassium channel antibody–associated limbic encephalitis and can be related to autoimmune‐mediated mechanisms. In addition, these observations suggest that impairment of the limbic system may play a role in the pathogenesis of RBD. Ann neurol 2006

Voltage‐gated potassium channel antibodies in limbic encephalitis

We found voltage‐gated potassium channel (VGKC) antibodies in 4 of 15 patients with limbic encephalitis (LE). Two patients with idiopathic LE had high VGKC antibody levels (>800pM; controls <100pM), that fell in parallel with a clinical response to immunotherapy. Two patients with lower VGKC antibodies (170pM, 300pM) had lung cancer (radiological evidence only in one) and the LE improved with immunotherapy in one. The other 11 patients without VGKC antibodies had paraneoplastic LE and eight onconeural antibodies (Hu in 6; Ma2 in 2). VGKC antibodies do not unambiguously discriminate between idiopathic or paraneoplastic LE but probably indicate a good response to immunotherapy. Ann Neurol 2003;54:530‐533

Localization of tumour necrosis factor production in cells at the materno/fetal interface in human pregnancy

Biologically active tumour necrosis factor (TNF) was detected in medium conditioned by incubation with cxplants of human pregnancy decidua or fetal chorionic villous tissue, taken in the first trimester and at term. Addition of endotoxin increased TNF release in most cases. ELISA assays gave similar results for TNF–α and also demonstrated low levels of TNF–β. Using cell populations purified by flow cytometry, secretion of biologically active TNF was shown to be localized to the macrophages. Cytotrophoblast purified from term amniochorion produced no TNF. Both decidual and chorionic villous tissue at term contained mRNA for TNF–α and TNF–β. TNF–α mRNA was confined to decidual macrophages in first trimester tissue, and was not present in chorionic cytolrophoblast. TNF–β mRNA, in contrast, was detected in both macrophage and no‐acrophage populations in term dccidua.

Antigenic heterogeneity of human cytotrophoblast and evidence for the transient expression of MHC class I antigens distinct from HLA-G

Expression of MHC class I antigens on trophoblast populations in first trimester human chorionic villous tissue was assessed by immunohistology. Antibodies used were W6/32 which recognizes a non-polymorphic framework determinant of HLA- A, -B, -C, MHM5 specific for HLA-B, C and 4E and B23.1 which are specific for HLA-B. Syncytiotrophoblast and villous cytotrophoblast were negative with all the anti (HLA class I) antibodies tested. Interstitial trophoblast cells within the maternal decidua were identified with a new antibody, NDOG5, which is specific for extravillous cytotrophoblast. Double labelling showed that they bind W6/32 but not 4E, MHM5 or B23.1; consistent with the expression of the monomorphic HLA-G. In contrast the cytotrophoblast cells of the cell islands and cytotrophoblast shell, which also express the NDOG5 antigen, were positive with W6/32, 4E, MHM5 and B23.1. Cell column cytotrophoblast cells were negative with all four MHC class I antibodies. These results suggest that differentiation of cytotrophoblast from noninvasive to invasive forms is associated with transient expression of class I antigens other than HLA-G on cytotrophoblast shell and cell island cytotrophoblast.

CSF findings in patients with voltage gated potassium channel antibody associated limbic encephalitis

UNLABELLED Recently, a new subtype of limbic encephalitis (LE) has been described, serologically characterized by the presence of antibodies against voltage gated potassium channels (VGKC, to be called VGKC-LE). Only little is known about CSF findings in this new disorder. Here we report the results of 29 lumbar punctures in 17 patients with VGKC-LE. Slight pleocytosis, mainly consisting of lymphocytes and monocytes, and elevated total protein concentrations were present in 41 and 47%, respectively. Intrathecal immunoglobulin (Ig) synthesis as defined by the presence of CSF-specific oligoclonal IgG bands, an increased IgG index, or an elevated IgG, IgA, or IgM ratio, was not detected in any of the patients, but dysfunction of the blood-CSF barrier was found in 35%. CSF findings were normal in 23%. CONCLUSIONS Unlike paraneoplastic LE, VGKC-LE is not frequently associated with intrathecal Ig production or markedly elevated white cell counts. Thus, normal CSF findings do not preclude the disease. VGKC-Ab should, therefore, be determined whenever LE is clinically suspected, irrespective of the presence or absence of inflammatory CSF changes.

Clustering of perinatal markers of birth asphyxia and outcome at age five years

Objectives In a cohort of term infants with cerebral depression at delivery, to investigate the association of perinatal signs of birth asphyxia, particularly abnormal fetal heart rate patterns in labour, acidaemia, and serious neonatal encephalopathy, with neurodevelopmental outcome at age five years.

Effect of sera from AChR-antibody negative myasthenia gravis patients on AChR and MuSK in cell cultures.

A proportion of patients with myasthenia gravis (MG) do not have antibodies to the acetylcholine receptor (AChR). Some of these patients have antibodies to muscle specific kinase (MuSK), whereas others have neither antibody (seronegative MG, SNMG). Both MuSK antibody positive MG (MuSK-MG) and SNMG are antibody-mediated diseases but how they cause neuromuscular junction failure is not clear. One possibility is that they reduce the clustering and expression of AChRs. We looked at the effects of MuSK-MG and SNMG sera/IgG on surface AChR distribution and expression, and AChR subunit and MuSK mRNA by quantitative RT-PCR, in TE671 and C2C12 myotubes. In TE671 cells MuSK-MG sera reduced AChR expression by about 20%, but had no effect on AChR subunit or MuSK mRNA expression. In C2C12 myotubes, MuSK-MG sera caused a reduction in the number of agrin-induced clusters, but the clusters became larger and there was no significant effect on total surface AChR numbers or AChR subunit or MuSK mRNA. By contrast, SNMG sera not only reduced AChR numbers by about 20% in TE671 cells, but modestly upregulated AChR gamma subunit expression in TE671 cells and both AChR gamma subunit and MuSK expression in C2C12 myotubes. Thus, although these results have, disappointingly, demonstrated little effect of MuSK antibodies on AChR expression, they do imply that SNMG antibodies act on AChR-associated pathways.

A role for autoantibodies in some cases of acquired non-paraneoplastic gut dysmotility.

Background : Antibody-mediated autoimmunity underlies a diverse range of disorders, particularly in the nervous system where domains of ion channels and receptors are potential targets. The aetiology of many adult-onset conditions of severe gut dysmotility is not known. We looked for antibodies to neuronal antigens in patients with severe (slow-transit-type) constipation (STC). Methods : Eleven sera from adultonset STC patients and 18 from childhood onset cases were tested by routine immunoprecipitation assays for antibodies against neuronal antigens including voltage-gated potassium channels (VGKCs), calcium channels (VGCCs), both muscle and neuronal acetylcholine receptor and glutamic acid decarboxylase (GAD). Results were compared with positive and negative control populations. Results : Two of the 11 sera from patients with adult-onset STC showed highly positive anti-VGKC antibodies. Both had onset of symptoms de novo in adulthood without evidence of autoimmune, neoplastic or neurological disease. One of these patients, and one other, had anti-GAD antibodies. None of the childhood-onset STC had evidence of anti-neuronal antibodies. Conclusions : Anti-neuronal antibodies are found in some patients with a condition of severe acquired gut dysmotility of previously unknown aetiology. Future studies may demonstrate an autoimmune role for such antibodies.